A reversible allosteric inhibitor of GlyT2 for neuropathic pain without on-target side effects

Domain: neuroscience
Published: 2026-02-16
Editorial outcome: major_revision

Abstract
Chronic neuropathic pain, caused by nerve damage or disease, is increasing in prevalence, but current treatments are ineffective and over-reliant on opioids. The neuronal glycine transporter, GlyT2, regulates inhibitory glycinergic neurotransmission and represents a promising target for new analgesics. However, most GlyT2 inhibitors cause significant side effects, in part due to irreversible inhibition at analgesic doses. Here we develop a reversible inhibitor of GlyT2, RPI-GLYT2-82, and identify its binding site by determining cryo-EM structures of human GlyT2. We capture three fundamental conformational states of GlyT2 in the substrate-free state, and bound to either glycine, RPI-GLYT2-82 or the pseudo-irreversible inhibitor ORG25543. We demonstrate that RPI-GLYT2-82 dissociates from GlyT2 faster than ORG25543, providing analgesia in mouse neuropathic pain models without on-target side-effects or addiction liability. Our data provide a mechanistic understanding of allosteric inhibition of glycine transport, enabling structure-based design of non-opioid analgesics.

Introduction
Chronic neuropathic pain affects 7–10% of the population yet only one in four of these patients respond to the gold standard treatment, pregabalin. Furthermore, because the efficacy of pregabalin is limited and accompanied by dose-dependent side effects, there is an over-reliance on opioid medications, exacerbating opioid use disorder and its associated mortality1,2. The rising incidence of neuropathic pain has therefore created an urgent need for the development of safe and effective analgesics that circumvent the opioid mechanism by targeting new pathways2.

A key feature of neuropathic pain is a reduction of glycinergic neurotransmission in the dorsal horn of the spinal cord – an adaptation that impairs our capacity to distinguish between painful and harmless stimuli3. Glycine is an inhibitory neurotransmitter that hyperpolarises postsynaptic neurons by binding to and opening Cl–-permeable glycine receptors. In the central nervous system, the concentration of extracellular glycine is tightly regulated by glycine transporters 1 and 2 (GlyT1 and GlyT2). GlyT1 is expressed at both excitatory and inhibitory synapses, whereas GlyT2 is exclusively localized to the presynaptic neurons of inhibitory synapses, primarily in the spinal cord4. GlyT2 thus plays a key role in regulating glycinergic neurotransmission by controlling glycine receptor activity on postsynaptic neurons and by recycling glycine for subsequent release from presynaptic neurons5–8.

GlyT2 (encoded by SLC6A5) belongs to the solute carrier 6 (SLC6) subfamily of secondary active neurotransmitter/sodium symporters (NSSs)9,10. NSSs are membrane proteins that operate via an alternating access mechanism, whereby transmembrane helices undergo conformational rearrangements during the transport cycle that alternately expose the substrate-binding site to each side of the membrane. In eukaryotic NSSs, binding of extracellular substrate and initiation of the transport cycle is dependent on a Na+ and Cl− gradient, such that GlyT2 co-transports three Na+ ions and one Cl− ion with each glycine11–13. Upon ion and substrate binding, access to the extracellular environment is occluded as the transporter transitions from an outward-facing to an inward-facing conformation, followed by release of solutes.

Several competitive and non-competitive SLC6 inhibitors have been developed that target either outward or inward-facing conformations of these transporters14–25. At glycinergic synapses, competitive GlyT2 inhibitors are likely to be ineffective due to the high concentrations of glycine in the cleft during synaptic transmission, whereas non-competitive inhibitors that target allosteric sites will be more effective8. Allosteric inhibitors are also more likely to result in subtype selectivity as substrate binding sites show little amino acid divergence across the NSS superfamily. Furthermore, because glycine is the only known substrate transported by GlyT2, the chemical space that can be explored for orthosteric inhibition is restricted26. Interestingly, ORG25543 (4-(benzyloxy)-N-{[1-(dimethylamino)cyclopentyl]methyl}−3,5-dimethoxybenzamide) is a potent, pseudo-irreversible, and non-competitive27 inhibitor of GlyT2 that provides analgesia in animal models of neuropathic pain28–31, without affecting acute or inflammatory pain thresholds28. In rat spinal cord slices, ORG25543 enhances tonic glycinergic neurotransmission and prolongs the decay time course of both miniature and evoked glycinergic inhibitory postsynaptic currents, suggesting that its effects are likely due to elevated extracellular glycine concentrations32. However, the compound did not progress to clinical use due to its narrow therapeutic index and significant on-target side effects, including tremors, seizures, and mortality at the maximal analgesic dose. These effects, which resemble the phenotype of the mouse GlyT2-/- knockout, are likely due to the high affinity and slow dissociation rate of ORG2554328,29. Indeed, siRNA knockdown of GlyT2 to 30% of wild-type (WT) levels results in prolonged analgesia without confounding side effects in mouse models of neuropathic pain33. In addition, the endogenous bioactive lipid N-arachidonoyl glycine and synthetic derivatives such as oleoyl-D-lysine cause partial inhibition of GlyT2 comparable to knockdown studies and provide analgesia without side effects. However, the poor pharmacokinetics of these bioactive lipids prevented progression to clinical trials28,34,35. Recently, an ORG25543-based benzamide, opiranserin (VVZ-149), reached a confirmatory phase 3 trial for the treatment of post-colectomy pain36,37, validating the importance of GlyT2 as a target for new pain therapeutics. Opiranserin, however, only shows moderately potent inhibitory activity on GlyT238.

Here we synthesize a potency-tuned analogue of ORG25543, RPI-GLYT2-82 (1-benzoyl-N-((4-(dimethylamino)tetrahydro-2H-pyran-4-yl)methyl)indoline-5-carboxamide) and determine its suitability for development as a treatment for neuropathic pain. We determine the structures of human GlyT2 (hGlyT2) – without substrate (inward-open conformation) and bound to either glycine (inward-occluded state), ORG25543 (outward-open state), or RPI-GLYT2-82 (outward-open state) – and confirm the allosteric action of both inhibitors. We further show that RPI-GLYT2-82 is efficacious at alleviating allodynia in both chronic constriction injury (CCI) and partial sciatic nerve ligation (PSNL) mouse models, without any side effects at the maximal analgesic dose. Our study thus reveals the molecular mechanism of allosteric inhibition of glycine transport, providing a framework by which new analgesics can be developed.

Results
RPI-GLYT2-82 is a non-competitive GlyT2 inhibitor
The narrow therapeutic index of ORG25543 has been attributed to its slow dissociation rate, suggesting that ORG25543-based compounds with improved reversibility may decrease on-target side effects and toxicity29,39,40. To overcome the liabilities associated with ORG25543 while simultaneously introducing chemical novelty into the GlyT2 inhibitor scaffold, we pursued the design of rigidified benzamide derivatives with reduced conformational flexibility, a strategy anticipated to enhance passive permeability and improve overall drug-like properties. Among the structural variants examined, ring-constrained heterocycles, most notably indolines, emerged as effective and structurally innovative isosteric replacements for the tri-alkoxybenzamide motif of ORG25543, maintaining both GlyT2 inhibitory potency and reversible binding kinetics. A subsequent integrated drug discovery effort focused on these conformationally restricted indoline scaffolds ultimately yielded the allosteric GlyT2 inhibitor RPI-GLYT2-82 (Fig. 1a, b, and Supplementary methods).

We first characterized the inhibitory action of RPI-GLYT2-82 by expressing WT hGlyT2 (hGlyT2WT) in Xenopus oocytes and using the two-electrode voltage clamp technique to record glycine-induced transport currents in the presence of different concentrations of the compound. RPI-GLYT2-82 behaved as a non-competitive inhibitor (Fig. 1c, and Supplementary Table 1) with a half-maximal inhibitory concentration (IC50) of 554 nM (Fig. 1d). This confirmed the lower inhibition potency of RPI-GLYT2-82 for hGlyT2WT than ORG25543, which displayed an IC50 of 11.4 nM (Fig. 1e). Glycine-induced transport currents recovered to pre-inhibition levels within five minutes of RPI-GLYT2-82 removal, indicating a markedly faster transport recovery rate than ORG25543, which required 30 minutes for currents to be restored to only 12.5% of pre-inhibitor levels (Fig. 1f, g, and Supplementary Fig. 1). Together, these data show that RPI-GLYT-82 is a non-competitive GlyT2 inhibitor with reduced potency compared to ORG25543. Like ORG25543, RPI-GLYT2-82 is selective for GlyT2 over GlyT1 (Fig. 1h). Interestingly, while the inhibition of transport by both compounds appears to be Na⁺-independent, RPI-GLYT2-82 exhibits slightly increased potency under lower Na⁺ concentrations, whereas inhibition by ORG25543 remains unaffected (Fig. 1i, j).

hGlyT2 adopts a LeuT fold
To understand the mechanism by which ORG25543 and RPI-GLYT-82 inhibit GlyT2, we sought to determine the structures of hGlyT2 in the absence and presence of these inhibitors and the substrate glycine. Because the N-terminus of hGlyT2 is predicted to be largely unstructured and its deletion does not affect trafficking to the plasma membrane or glycine transport41, we created a construct lacking residues 2-185 (hGlyT2Δ185) to facilitate expression and purification. hGlyT2Δ185 exhibited a similar half-maximal effective concentration (EC50) for glycine transport, and similar IC50 values for both ORG25543 and RPI-GLYT2-82, to full-length hGlyT2WT (Fig. 1d, e, k).

We purified hGlyT2Δ185 in mixed micelles containing lauryl maltose neopentyl glycol (LMNG), glyco-diosgenin (GDN), and cholesteryl hemisuccinate (CHS) at a ratio of 1.0:1.0:0.1 to generate monodisperse and homogenous hGlyT2Δ185 (Supplementary Fig. 2) for cryo-electron microscopy (cryo-EM) studies. This enabled structures of hGlyT2Δ185 in the absence of substrate and bound to glycine, ORG25543, or RPI-GLYT2-82 to be determined at 2.97 Å, 3.02 Å, 2.49 Å, and 2.79 Å resolution, respectively (Fig. 2a–h, Supplementary Fig. 3–6, and Supplementary Table 2). The high-quality of the resulting density maps enabled unambiguous modelling of all 12 transmembrane (TM) helices, revealing unwinding of TMs 1 and 6 approximately halfway across the membrane – a characteristic feature of the substrate binding site in SLC6 transporters (Fig. 2, and Supplementary Fig. 7–10). We were also able to model the intracellular and extracellular loops, except parts of extracellular loop 2 (EL2; residues 302–393) due to its high degree of flexibility (Fig. 2, and Supplementary Fig. 7-10), as well as the position of bound ligands and ions. The conserved disulfide bridge between C311 and C320 in EL2 is present in the inhibitor-bound and substrate-free structures (Supplementary Fig. 7, 9–11). However, although we observed clear densities for C311 and C320 in glycine-bound hGlyT2Δ185, we did not detect a distinct density for the disulfide bridge, suggesting that the cysteine residues may be reduced in the substrate-bound state (Supplementary Fig. 8). The overall architecture adopted by hGlyT2 is characteristic of SLC6 transporters: an inverted pseudo-twofold symmetric organisation of TMs 1–5 relative to TMs 6–10 (Fig. 2, and Supplementary Fig. 7-10), known as the LeuT fold due to its identification in the bacterial amino acid importer LeuT42.

GlyT2 and related SLC6 transporters are modulated by cholesterol31,43–46. Accordingly, we observed several cholesterol-like features in our hGlyT2Δ185 density maps, which were of sufficient resolution to be modelled in our substrate-free and inhibitor-bound structures (Supplementary Fig. 12). In these three structures, we observed a density consistent with a cholesterol (CHOL) molecule in an inner leaflet pocket formed by TM2, TM7, and TM11, equivalent to the previously reported CHOL2 site17,20,21. We identified an additional neighbouring density where we modelled a CHS molecule (CHOL5). These two molecules form hydrogen bonds with N674 (IL5). Interestingly, while the cholesterol at the CHOL2 site appears to be conserved, this asparagine residue is not conserved among NSS family members. In the Drosophila dopamine transporter (dDAT) and human serotonin transporter (hSERT) it is a glycine residue, in human DAT (hDAT) a serine, and in hGlyT1 and LeuT a proline (Supplementary Figs. 11, 12). We modelled a second CHS molecule adjacent to, but distinct from, the CHOL1 site (CHOL6)19,21. This CHOL6 site lies parallel to and interacts with TM7. Above CHOL6, we modelled a cholesterol molecule in another site, CHOL7, formed by TM5, TM7, and EL4 (Supplementary Fig. 12). Two lipid densities were observed in CHOL6 and CHOL7 sites in a recent hDAT structure20. We further modelled a cholesterol molecule into a site that partially overlaps with CHOL3, and CHOL5 site proposed in molecular dynamics simulations of dDAT (named CHOL3’ in GlyT2)17,18,47. This cholesterol is embedded within the protein, at a methionine-rich site formed by TMs 3, 9, 10 and 12 (Supplementary Fig. 12), in contrast to its alignment with the membrane in other SLC6 structures.

We further examined the stability of sterols in the inhibitor-bound GlyT2 structures and found that most remained stably bound throughout the simulations (Supplementary Fig. 12d–l). However, the cholesterol molecule at the CHOL3’ site in the RPI-GLYT2-82-bound structure sampled multiple conformations while maintaining contact with the site, suggesting local flexibility in this region. Because this feature, along with other sterol molecules we modelled, is consistently present in all hGlyT2Δ185 structures in three different conformational states, we suggest that these sterols act primarily as important structural stabilisers of hGlyT2 rather than as regulatory elements. We did not observe any clear cholesterol-like densities at CHOL1 or CHOL4 sites17.

Substrate-free hGlyT2 is in an inward-open state
Despite inclusion of the GlyT2 inhibitor oleoyl-D-lysine throughout purification, we did not observe any density corresponding to the inhibitor. The low solubility of the compound likely prevented it from remaining in sufficient concentration in solution to bind effectively. We therefore present this structure as the substrate-free state of hGlyT2Δ185 (Fig. 2, and Supplementary Fig. 3, 7).

We captured substrate-free hGlyT2Δ185 in an inward-open conformation in which the extracellular gate is closed due to formation of a conserved salt bridge between residues R216 (TM1b) and D633 (TM10) (Cα-Cα distance of 9.8 Å). Solvation of TM5 and protrusion of TM1 into the micelle creates a large open cavity facing the intracellular milieu. The intracellular part of TM5 is unwound, just before the helix-breaking G419(X9)P429 (Gly(X9)Pro) motif, allowing water from the intracellular space to access the second Na+-binding site (Na2)48. The N-terminal segment of TM1 (TM1a) is bent away from the core of the protein, opening the pathway for solute to be released into the intracellular side. This splaying of TM1 disrupts the interaction between TM1a and the hydrophobic patch on the intracellular part of hGlyT2Δ185 that is present in other outward-facing or inward-occluded NSS structures17,49,50 as well as our outward-facing conformations of hGlyT2Δ185 (Fig. 2e, and Supplementary Fig. 13).

We observed a clear density for Cl− in the substrate-free cryo-EM map of hGlyT2Δ185. This Cl– is coordinated by conserved residues Y233 (TM2), Q473 (TM6a), S477 (unwound region of TM6a), N509 (TM7), and S513 (TM7) at a mean coordination distance of 3.1 Å ± 0.4 (Fig. 3). We did not, however, observe any densities for Na+ at Na1, Na2, or the putative Na3 site, consistent with an inward-open conformational state of the transporter16,50 (Fig. 3).

Glycine-bound hGlyT2 is in an inward-occluded state
Glycine binds within a cavity at the centre of hGlyT2 formed by residues from TMs 1, 3, 6, and 8 (Figs. 2 and 4a, b). The glycine-binding pocket is midway through the membrane bilayer and inaccessible from both extracellular and intracellular sides, indicative of an occluded state of the transporter. The extracellular gate residues, R216 and D633, form a salt bridge (Cα-Cα distance of 9.6 Å), which precludes access from the extracellular milieu. The guanidinium of R216 forms a cation-π interaction with the phenyl group of conserved F476 (TM6a), which sterically blocks the extracellular pathway. The Cα of F476 is 11.3 Å from the Cα of conserved Y287 (TM3), which together cap the glycine binding site (Fig. 4b). The intracellular permeation pathway is occluded by TM1a, which has a more perpendicular arrangement to the membrane than in the substrate-free state (Fig. 2b, f, and Supplementary Fig. 13). The distorted density observed in the intracellular half of TM5 (residues 415–419) (Supplementary Fig. 8) suggests an unwound region preceding the conserved helix-breaking Gly(X9)Pro motif, creating a solvent pathway from the intracellular milieu to Na247, indicative of an inward-occluded state of glycine-bound hGlyT2.

The carboxylate group of glycine forms hydrogen bonds with the backbone nitrogen atoms of G210 and G212 (TM1), the hydroxyl group of T578 (TM8), and more distantly, a weak hydrogen bond with the hydroxyl group of Y287 (TM3). Although the binding mode of glycine’s carboxylate group resembles that of other α-amino acid transporters, including closely related GlyT1, the positioning of the amino group differs25. In hGlyT2, the amino group of glycine appears to form a hydrogen bond with the side-chain hydroxyl of S479 (unwound region of TM6) and is further coordinated by the backbone carbonyl oxygens of Y207 and A208 on TM1, whereas in GlyT1, it adopts a different orientation to interact with the backbone of TM6 residues (Fig. 4c). Residue 479 is a glycine in GlyT1, and the corresponding mutation in GlyT2 (S479G) markedly increases the EC50 for glycine transport, confirming the importance of this residue for substrate transport by hGlyT226,31. The binding pocket for glycine is further constrained by W482 in the conserved (G/A/C)ΦG motif (A481W482G483 in hGlyT2) on the non-helical region of TM6, which allows different sized ligands to be accommodated by the various SLC6 family members51. A conserved glutamate residue in TM2, E248, coordinates the AWG loop via a direct hydrogen bond with backbone nitrogen of A481 – an interaction that is conserved among SLC6 transporters51.

A density for Cl− is clearly apparent in the cryo-EM map of glycine-bound hGlyT2Δ185, coordinated by the same residues as the substrate-free state (Y233, Q473, S477, N509 and S513) at a mean coordination distance of 3.2 Å ± 0.5 (Fig. 3, and Supplementary Fig. 14). We also observed a prominent (13.5 r.m.s.d.) signal for Na+ at Na2, with distorted octahedral coordination by the side chain of D577 (TM8), the backbone carbonyl oxygens of G206 (TM1), Y207 (TM1), V209 (TM1), L574 (TM8), T578 (TM8), and a cytoplasmic water molecule (mean coordination distance of 2.9 Å ± 0.4), similar to previous inward-occluded NSS structures48 (Fig. 3c). We did not observe any densities for Na+ at Na1 or the putative Na3 site. The conserved S477 residue, equivalent to the Na+-coordinating S317 residue at Na1 in GlyT1, is rotated away from the substrate-binding site in glycine-bound hGlyT2, which may suggest that this inward-occluded conformation is in a transitional state.

ORG25543 and RPI-GLYT2-82 lock hGlyT2 in an outward-open state
In the presence of either ORG25543 or RPI-GLYT2-82, the central substrate-binding site of hGlyT2Δ185 is accessible to solvent via the extracellular vestibule, indicating that the inhibitors lock the transporter in an outward-open conformation. The Cα-Cα distance between the two extracellular gating residues, R216 and D633, is 13.9 Å in both ORG25543- and RPI-GLYT2-82-bound structures, consistent with previous outward-facing NSS structures17,18. The cytoplasmic pathway is closed by the location of TM1a within the protein core, allowing the conserved residue W194 to plug a hydrophobic pocket formed by residues on TMs 1, 5, 6, and 7 on the intracellular face of GlyT2 (Fig. 2, and Supplementary Fig. 11, 13).

In both inhibitor-bound structures, there is a clear density for a Cl−, which is coordinated by four of the same residues as those in both inward-facing confirmations (Y233, Q473, S477, and S513; mean coordination distance of 2.8 Å ± 0.3 and 2.9 Å ± 0.5 for ORG25543- and RPI-GLYT2-82 bound structures, respectively. However, Cl− is not directly coordinated by the fifth residue N509 and instead interacts with this residue via a water molecule (Fig. 3d, e). We did not observe any densities for Na+ at Na2 or the putative Na3 site12,13, consistent with ORG2554327 and likely RPI-GLYT2-82, binding being Na+-independent (Fig. 1i, j). We observed a prominent (9.7 r.m.s.d.) signal for a non-protein density in the Na1 site of both inhibitor-bound structures. We initially assigned this density as a water molecule because one of the main Na+ coordinating residues required for the stable octahedral ion coordination geometry, N213 (TM1b), had rotated away from Na1 to interact with the respective inhibitor (Fig. 3d, e). Modelling of the protein complex with water in the Na1 site using molecular dynamics (MD) simulations showed that extracellular Na+ spontaneously bound to this site in four out of six replicate simulations and the site remained occupied by Na+ throughout 50% of the 3000 ns combined replicate simulations (Supplementary Fig. 15, and Supplementary Table 3). As a result, we subsequently modelled Na+ at the Na1 site, coordinated by the backbone carbonyl oxygens of A208 (TM1) and S477 (TM6) as well as a water molecule, reminiscent of a loosely bound hydrated Na+ exposed to the extracellular space (Fig. 3 and Supplementary Fig. 15).

The increased potency of RPI-GLYT2-82 at lower Na⁺ concentrations likely reflects reduced competition of the compound with Na⁺ for N213 coordination, under partially depleted Na⁺ conditions. In contrast, the potency of ORG25543 remains unchanged, likely due to its inherently high potency that renders it insensitive to local Na⁺ occupancy at the Na1 site (Fig. 1i, j).

ORG25543 and RPI-GLYT2-82 occupy an allosteric site
The cryo-EM densities of ORG25543 and RPI-GLYT2-82 were of high quality allowing unambiguous assignment of the respective poses when bound to hGlyT2Δ185 (Fig. 4d, e). We modelled the inhibitors in an allosteric pocket sandwiched between TM1b, TM10, and EL4 that is lined with predominantly hydrophobic residues from TMs 1, 3, 5, 7, 8, 10 and EL4 (Fig. 4f, g). The scaffold of ORG25543 and RPI-GLYT2-82 has four key substituents (R1–R4) (Fig. 4h, i). In ORG25543 and RPI-GLYT2-82, respectively, these substituents are: the central benzamide phenyl ring and the central indoline (R1), benzyloxy, and benzoyl phenyl rings (R2), cyclopentyl and tetrahydropyran rings (R3), and the pendant ammonium in both compounds (R4). For both inhibitors, the central R1 group is stacked between W215 (TM1b) and P543 (EL4), and the distal R2 ring is caged by a network of residues including W215 (TM1b), I520 (TM7), Y550 (EL4), and F567 (TM8) as well as A546 (EL4) and M570 (TM8). The R3 rings are located in a relatively large, solvent-exposed pocket, and stabilized by the hydrophobic residues I290 (TM3), P543 (EL4), and F629 (TM10). The extracellular gate residues R216 and D633 engage in hydrogen bond and salt bridge interactions with both inhibitors (Fig. 4f, g), including a charged-reinforced hydrogen bond and a salt bridge between D633 and the R4 pendant ammoniums, and a hydrogen bond between the side chain of R216 and the carbonyl oxygen of R1. The allosteric pocket resembles that in MRS7292-bound DAT20, suggesting a common allosteric regulatory site in SLC6 transporters. However, this site is distinct from the vilazodone binding site in SERT22 (Supplementary Fig. 16).

MD-based r.m.s.d. analysis confirmed stable binding of ORG25543 to the allosteric site for 95.4% of combined simulation replicas. The central benzamide phenyl ring (R1) showed minimal fluctuation, due to stabilization by TM1b, EL4, and TM8 (Supplementary Fig. 17, 18, and Supplementary Table 4), however the distal benzyloxy phenyl (R2) and solvated pendant ammonium (R4) had greater mobility (Supplementary Fig. 19). R2 frequently interacted with its surrounding residues (Supplementary Table 4, Supplementary Fig. 18) and despite higher fluctuations, R3 remained in a hydrophobic cavity flanked by Y286 and Y287 on TM3 (Supplementary Fig. 19).

Hydrogen bond analysis indicated stable interactions between the benzamide of ORG25543 and L211 as well as intermittent interactions with R216. The protonated amine (R4) of ORG25543 intermittently engaged in a charged-reinforced hydrogen bond with D633, in agreement with our cryo-EM data (Supplementary Table 4, Supplementary Figs. 18, 19). The probability density map showing the relative likelihood of bound hGlyT2 ligand positions (Supplementary Fig. 19) demonstrated that ORG25543 remained in a deep energetic minimum at the allosteric binding site with an occupancy of 95%. In comparison, RPI-GLYT2-82, adopted a less stable binding pose than ORG25543, exhibiting higher positional fluctuations within the first 50 ns of simulations. It remained in the binding site for only 48% of the combined simulation replicas and moved towards the central vestibule of GlyT2 for the remaining time (Supplementary Fig. 19). Like ORG25543, the R2–R4 substituents of RPI-GLYT2-82 exhibited the greatest positional fluctuations, and the central indoline of R1 formed contacts with the same set of residues that coordinate ORG25543’s R1 moiety (Supplementary Table 4, and Supplementary Fig. 18, 19).

To analyse MD simulation trajectories of inhibitor-bound hGlyT2 complexes, we calculated the binding enthalpy over the first 50 ns of the replicate simulations, during which both inhibitors maintained their cryo-EM binding conformations in hGlyT2. This revealed the significant contribution of L211, W215, and D633 to ORG25543 binding and the 0.83–0.92-fold reduction in binding enthalpy for RPI-GLYT2-82 compared to ORG25543 (95% C.I.) (Supplementary Fig. 19). The per-residue energy decompositions further suggest the lower experimental IC50 for RPI-GLYT2-82 stems from an altered interaction enthalpy with L211, R216, and P543 (Supplementary Fig. 19). Together, these data show that both ORG25543 and RPI-GLYT2-82 occupy the same allosteric site, but that RPI-GLYT2-82 adopts a less stable binding pose.

To determine whether glycine can bind to inhibitor-bound hGlyT2 conformations obtained by cryo-EM, we first docked glycine into the inhibitor-bound structures and assessed the stability of the binding poses using MD simulations. The observed exchange of solvent and ions between the extracellular space and the substrate-binding site, together with glycine’s mobility within the substrate binding cavity, suggests that glycine can access the ORG25543- or RPI-GLYT2-82–bound hGlyT2Δ185 from the extracellular solvent. These findings support a mechanism in which allosteric inhibitors such as ORG25543 and RPI-GLYT2-82 inhibit glycine transport by preventing conformational cycling, a process independent of glycine binding (Supplementary Table 5, 6 and Supplementary Fig. 20).

RPI-GLYT2-82 binds reversibly to hGlyT2
Having characterized the allosteric pocket to which ORG25543 and RPI-GLYT2-82 bind, we sought to determine the features responsible for reversible binding of RPI-GLYT2-82. Likely due to the important role of W215 in accommodating the R1 and R2 substituents of both inhibitors, a conservative mutation to phenylalanine resulted in a detectable decrease in the potency of inhibition of transport by ORG25543 (~three-fold) and significant decrease in the potency of RPI-GLYT2-82 (IC50 > 30 μM) (Fig. 4j, l). Interestingly, although this W215F mutation had a relatively minor effect on potency, it significantly increased the rate of ORG25543 washout (Fig. 4k), similar to the effect of the mutation of the nearby Y550 (EL4) residue31. Likewise, mutation of the neighbouring D633 residue to glutamate significantly reduced the potency of inhibition for both ORG25543 (~14-fold) and RPI-GLYT2-82 (>18-fold) and accelerated ORG25543 washout (recovery from ORG25543 within five minutes), highlighting the critical role of this interaction for the potency of the inhibitors (Fig. 4j–l). Notably, washout of RPI-GLYT2-82 from both GlyT2W215F and GlyT2D633E was almost immediate, consistent with the reversible binding mode of the compound (Fig. 4m).

The pendant phenyl rings (R2) of both ORG25543 and RPI-GLYT2-82 snorkel deep into the binding pocket to engage in edge-to-face π-π interactions with W215 and Y550 (Fig. 4f, g). The R2 groups of both inhibitors are positioned orthogonally to their R1 core, however, as a benzamide, the pendant benzoyl phenyl ring of RPI-GLYT2-82 is likely to adopt a lower energy coplanar orientation with the carbonyl to which it is attached, reducing conformational flexibility. However, in its binding pose in the allosteric pocket, this RPI-GLYT2-82 pendant phenyl ring must twist nearly 90 degrees to adopt an orthogonal orientation, which likely induces torsional strain and is therefore energetically unfavourable52. The indoline benzamide carbonyl oxygen of RPI-GLYT2-82 also points toward a non-polar pocket (Fig. 4g), further contributing to destabilizing contacts. The R3 substituent of ORG25543 is a small hydrophobic cyclopentane ring that easily fits into a hydrophobic pocket in the binding site (Fig. 4f), potentially displacing any resident waters and interacting via van der Waals bonds. However, RPI-GLYT2-82 has a tetrahydropyran R3 substituent that is larger and more polar than the ORG25543 cyclopentane ring and its oxygen atom is not engaged in any significant binding interactions within the hydrophobic R3 pocket of the binding site (Fig. 4g). The increased size of this tetrahydropyran ring coupled with the lack of significant binding interactions with the polar tetrahydropyran oxygen may enable solvent water to “dissolve” the ring out of hydrophobic binding. Thus, the conformational inflexibility of R2, together with the larger and more polar R3 substituent, are likely responsible for the lower potency and improved washout kinetics of RPI-GLYT2-82 compared to ORG25543.

RPI-GLYT2-82 provides analgesia in mouse models of neuropathic pain
Although ORG25543 provided analgesia in animal models of neuropathic pain, it caused significant side effects that were likely due to its high affinity and slow dissociation rate28. To evaluate the in vivo utility of our reversible GlyT2 inhibitor RPI-GLYT2-82, we performed plasma and central nervous system pharmacokinetic (PK) profiling in mice and calculated unbound drug concentrations in plasma (Cu,p) and brain (Cu,b) at 30- and 120-minutes post-administration (Supplementary Note 1, and Supplementary Fig. 21, Supplementary Table 7, 9–13,). We initially selected a 50 mg/kg i.p. dose to obtain a preliminary PK readout and quantify unbound exposures in both compartments. Notably, this dose produced unbound brain concentrations that approximated the in vitro IC50 of RPI-GLYT2-82, indicating that it would likely afford sufficient target engagement and thus serve as a rational starting point for assessing analgesic efficacy (Supplementary Table 14–15). Indeed, RPI-GLYT2-82 achieved a Cu,b of 597.89 nM at 30 minutes after dosing (Fig. 1d, and Supplementary Table 7), approximately equal to its hGlyT2 IC50, supporting the expectation of achieving pharmacologically meaningful GlyT2 occupancy in vivo. Encouraged by these data, we tested RPI-GLYT2-82 in the CCI mouse models of neuropathic pain by measuring mechanical and cold allodynia in mice upon i.p. administration. When measuring mechanical allodynia, we observed dose-dependent anti-allodynia at both 50 and 100 mg/kg, with maximum efficacy between 90 min and 3 hours post-administration (Fig. 5a). This is comparable to the effect of 30 mg/kg ORG25543, which peaked between 1 and 2 hours post-administration28. Similarly, when measuring response by cold allodynia, peak anti-allodynia effects were observed within 2 hours at both 50 and 100 mg/kg with comparable efficacy to the control compound gabapentin (Fig. 5b). We further tested RPI-GLYT2-82 in the PSNL model by measuring its effects on mechanical allodynia and observed similar effects to the CCI model (Fig. 5c).

Although ORG25543 reversed mechanical allodynia at a dose of 50 mg/kg, this coincided with near-complete loss of rotarod performance, seizures and in one case, lethality28. A 50 mg/kg analgesic dose of RPI-GLYT2-82 was not associated with adverse neuromotor effects or muscle weakness (Fig. 5d–f), and did not change respiration frequency, minute volume (MVb), or tidal volume (TVb) measured by whole-body plethysmography (Fig. 5g–i). A supramaximal 250 mg/kg dose (i.p.), which is five-fold higher than the maximally analgesic dose of RPI-GLYT2-82, produced an initial 61% reduction in baseline rotarod performance, which was restored over the 90-minute time course, and caused transient muscle weakness 30 min post-injection which recovered at the 60-min time point (Fig. 5d, e). Whilst the grip strength and rotarod experiments show mild, transient deficits in muscle strength and motor coordination at the supramaximal analgesic dose, they do not coincide with the anti-allodynic effects. Given the temporal separation of these motor side-effects from the anti-allodynia effects, they do not pose a confound in interpreting the motor response-dependent pain-like behaviors. The supramaximal 250 mg/kg dose of RPI-GLYT2-82 also caused transient changes in respiratory frequency and MVb in the first hour, and TVb in the first 30 min (Fig. 5g–i). These effects coincided with transient sedation, which was most apparent in the first 15–30 minutes and fully recovered by 90 minutes (Fig. 5d–f).

These in vivo data provide evidence that RPI-GLYT2-82 produces statistically significant dose dependent analgesia without the adverse neuromotor effects associated with ORG2554328,33. Interestingly, although the primary target of RPI-GLYT2-82 is GlyT2 (IC50 = 554 nM, Fig. 1d), it is also a less potent antagonist at the serotonin 2A (5-HT2A) receptor (IC50 = 1.9 μM, Supplementary Note 1, Supplementary Fig. 22, 23). This additional activity may contribute to its suppression of pain signals. Indeed, antagonism of 5-HT2A has previously been reported for other ORG25543-based GlyT2 inhibitors, including opiranserin37,38. RPI-GLYT2-82 also exhibits appreciable inhibitory activity at SERT (IC50 = 4.7 μM, Supplementary Fig. 23), but has no significant binding activity at the dopamine (hDAT) or norepinephrine (hNET) transporters (10 μM RPI-GLYT2-82 displaces ~20% of 3H-1-[1-(2-benzo[b]thiopheneyl)cyclohexyl]piperidine hydrochloride (3H-BTCP) from hDAT and <5% of 3H-nisoxetine from hNET, Supplementary Note 1, Supplementary Fig. 22). Additionally, it is inactive at other off-target sites covered by our Cerep panels screen that are known to promote sedation (γ-aminobutyric acid type A (GABAA), histamine (H1), adenosine, adrenergic, and muscarinic receptors) (Supplementary Fig. 22, and Supplementary Table 16, 17). Finally, because the rewarding properties of opioids limit their use in the clinic, we compared RPI-GLYT2-82 and morphine in a conditioned place preference (CPP) paradigm. Mice treated with morphine (10 mg/kg, i.p.) exhibited a significant increase in preference for the paired chamber (p = 0.0287) whereas mice treated with RPI-GLYT2-82 (50 and 150 mg/kg, i.p.) showed no significant preference (p = 0.78 and p = 0.15, respectively) (Fig. 5j). Collectively, our data show that RPI-GLYT2−82 provides a foundation for the development of a non-addictive medication for neuropathic pain.

Methods
Ethics statement
Xenopus laevis oocytes were supplied by Victor Chang Cardiac Research Institute and approved by the Garvin Institute/St Vincents Hospital Animal Ethics Committee (Animal Research Authority 23_11). Non-CPP behavioural experiments were performed under approval from the Animal Ethics Committee of the University of Sydney (AEC protocol numbers 2022/2229, 2018/1363 and 2022/2146). CPP behavioural experiments were conducted in accordance with the Guidelines of the Animal Care and Use Committee of the Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health (ASP protocol 23-NRB-43). Pharmacokinetics studies conducted by Eurofins Discovery Partner including housing, experimentation, and animal disposal were performed in general accordance with the “Guide for the Care and Use of Laboratory Animals: Eighth Edition” (National Academies Press, Washington, D.C., 2011) in the AAALAC-accredited laboratory animal facility. In addition, the animal care and use protocol was reviewed and approved by the IACUC at Pharmacology Discovery Services (PDS) Taiwan, Ltd.

Experimental model and subject details
DH5α (New England Biolabs), XL1-Blue Competent Cells (Agilent) and DH10EMBacVSV (Geneva Biotech) were cultured in lysogeny broth (LB) at 37°C to amplify plasmids. Expi293F cells (Thermo Fisher Scientific) were cultured in HE400AZ medium at 37°C and 8% CO2. Sf9 insect cells (Thermofisher Scientific) were cultured in Sf-900™ III SFM medium at 27°C.

Xenopus laevis oocytes were stored at 18°C in frog Ringer’s solution (96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 5 mM HEPES, pH 7.5) supplemented with 2.5 mM sodium pyruvate, 0.5 mM theophylline, 50 μg/mL gentamicin and 100 μg/mL tetracycline.

Animals
The CPP behavioural experiments were performed with adult male C57BL/6J mice, 8-12 weeks old, obtained from The Jackson Laboratory (Maine, USA). Mice were housed in groups of four and had free access to food and water. Housing rooms were temperature and humidity controlled and maintained on a 12 h light/dark cycle. Experiments were conducted during the light phase.

All other behavioural experiments undertaken with male and female C57BL/6 mice, sourced from the Animal Research Centre (WA, Australia) or Australian BioResources (NSW, Australia). Mice were 6 weeks of age upon arrival and housed in individually ventilated cages (IVC, Sealsafe Plus Green Line, Techniplast) with Smart Flow Air Handling Units (Techniplast), lined with corncob bedding. Mice were housed in groups of 4 in a holding room maintained on a 12-h light-dark cycle (0545 to 1745) maintained at 21 ± 2°C and relative humidity at 50 ± 5% with ad libitum access to food (Irradiated rat and mouse chow; Specialty Feeds, WA) and filtered water. Mice were given one week to habituate to the research facility and were then acclimatised to all aspects of the testing procedure including the observation room, handling and scuffing by the experimenter, and the relevant testing apparatus for at least three days prior to commencement of experiments. Animals were monitored at least twice a week for irregularities such as dehydration, skin conditions, and weight loss.

Pharmacokinetic studies were performed by Eurofins Discovery Partner with ICR mice. RPI-GLYT2-82 in-life plasma and BBB pharmacokinetic study was also conducted by Eurofins Discovery Partner with C57BL/6 mice. Male ICR mice weighing 30 ± 5 g and male C57BL/6 mice weighing 22 ± 2 g were provided by BioLasco Taiwan Co., Ltd. (under Charles River Laboratories Licensee). Animals were acclimated for 3 days prior to use and were confirmed to be in good health. The animals were housed in animal cages with a space allocation of 30×19×13 cm. All animals were maintained in a controlled temperature (20–24°C) and humidity (30%-70%) environment with 12-h light-dark cycles. Free access to standard lab diet [MFG (Oriental Yeast Co., Ltd., Japan)] and autoclaved tap water were granted.

GlyT2 construct
The human GlyT2 complementary DNA sequence was codon optimised and synthetised by Azenta for expression in mammalian cells. The GlyT2 construct contains a N-terminal deletion of residues 2 to 185 (hGlyT2Δ185). The sequence of hGlyT2Δ185 followed by a G(GGGS)3 linker, a HRV3C protease cleavage site, enhanced green fluorescent protein (eGFP), a twin Strep-Tag II tag and a decahistidine tag were cloned into a pCDNA3.1 vector for transient transfection in Expi293F human cells (Thermo Fisher Scientific). For glycine-bound hGlyT2Δ185, the same construct and tags were cloned into pACEMam1 from the MultiBacMam Kit (Geneva Biotech) for baculovirus production in Sf9 insect cells (Thermo Fisher Scientific). The Expi293F cell stock was authenticated by Thermo Fisher Scientific. The cell line was regularly tested for mycoplasma contamination and tested negative for contamination. No misidentified cell lines were used.

Chemical Synthesis of RPI-GLYT2-82
The general procedure for the synthesis of RPI-GLYT2-82 is described in Supplementary methods.

Expression of hGlyT2Δ185
The hGlyT2Δ185 protein was expressed in Expi293F cells cultured in HE400AZ expression medium (GMEP Cell Technologies, Japan) in 600 mL TubeSpin bioreactors, incubating in an orbital shaker at 37°C, 8% CO2 and 175 rpm in a humidified atmosphere. The Expi293F cells (Thermo Fisher Scientific; authenticated and free of mycoplasma contamination) were transfected at a density of 3.0–3.5 × 106 cells per mL and viability of above 97%. A 25 kDa linear polyethylenimine (LPEI) was used as the transfection reagent, at a GlyT2 DNA:LPEI ratio of 1:2 with a total plasmid concentration of 2.0 mg per mL of culture volume to transfect. The LPEI/DNA complexes were incubated in Opti-MEM™ I Reduced Serum Medium (Thermo Fisher Scientific) for 15 min before adding to the cells. At approximately 17 h post-transfection, cell cultures were supplemented with 10 mM sodium butyrate. The cells were harvested at approximately 44 h post-transfection at a viability of above 70% and stored at -70°C until purification.

For glycine-bound hGlyT2Δ185, baculovirus was generated by transfecting Sf9 insect cells. The bacmid was prepared by transforming the pACEMam1 vector containing the hGlyT2Δ185 construct into Escherichia coli Dh10Bac competent cells (Geneva Biotech). For transduction, 1.5 μg of purified bacmid was incubated in 100 μL of Sf-900™ III SFM media and separately 0.1 mg of Cellfectin™ II Reagent (Thermo Fisher Scientific) incubated in 100 μL of Sf-900™ III SFM media for 5 min at room temperature. Subsequently, the bacmid DNA and Cellfectin™ II mixtures were combined and incubated for a further 20 min. The mixture was added dropwise to a well in a 6-well plate of attached Sf9 insect cells at 0.45 × 106 cells per mL (2 mL final volume). Following 4 h of incubation at 27°C, media was exchanged for fresh Sf-900™ III SFM media supplemented with 2% heat-inactivated FBS (Gibco). P0 was harvested following 72-h incubation at 27°C by centrifuging at 650×g for 20 min and collecting the supernatant. P1 and P2 were subsequently generated by transducing Sf9 insect cells at 2.0 × 106 cells per mL in Sf-900™ III SFM media which was then supplements with 2% heat-inactivated FBS at time of harvest. For expression in Expi293F cells, hGlyT2Δ185 baculovirus was added at 1.5% (v/v) to the cells at a density of 3.0–3.5 × 106 cells per mL in HE400AZ expression medium. At time of transduction, cell cultures were supplemented with 10 mM sodium butyrate. The cells were harvested at approximately 44 h post-transduction at a viability of above 70% and stored at -70°C until purification.

Purification of GlyT2
The cell pellets were thawed on ice and resuspended in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 20% glycerol (v/v), 1 cOmplete™ Protease Inhibitor Cocktail tablet per 50 mL of buffer (Roche), 2 mg of DNAse I and incubated on rotation for 30 min at 4°C. The suspension was solubilised in 1% (w/v) n-dodecyl-β-D-maltopyranoside (DDM) supplemented with 0.1% cholesterol hemisuccinate (CHS) with 15–25 μM brain polar lipids extract (Avanti) for 45 min at 4°C. Cell debris was removed by centrifugation at 140,000g, 4°C for 20 min. To the cleared lysate, StrepTactin XT Sepharose chromatography resin (Cytiva) was added in batch and incubated overnight at 4°C, with stirring.

The resin was washed with 10 column volumes (CV) of buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% (w/v) GDN, 0.1% (w/v) LMNG, 0.02% (w/v) CHS and 15-25 μM brain polar lipids extract. The resin was subsequently washed with 20 CV of the same buffer supplemented with 10 mM MgCl2 and 2 mM adenosine 5′-triphosphate disodium salt hydrate (ATP). The resin was then washed with 10 CV of buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% (w/v) GDN, 0.05% (w/v) LMNG, 0.01% CHS, and 15-25 μM brain polar lipids extract. The protein was eluted in the same buffer supplemented with 20% glycerol (v/v) and 50 mM biotin (pH 8). The protein was then treated with HRV-3C protease (in-house) for 3 h at 10°C to cleave the eGFP–twin-Strep-Tag-His tag, while being dialysed in buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.003% (w/v) GDN, 0.003% (w/v) LMNG, 0.0006% (w/v) CHS and 20% glycerol (v/v). The eGFP–twin-Strep-Tag-His tag was removed by passing the sample through a nickel-nitrilotriacetic acid (Ni-NTA) HisTrap™ HP column (Cytiva) and impurities were removed by anion exchange chromatography with a pre-packed 1 mL HiTrap™ Q HP column (Cytiva). The sample was concentrated to above 2 mg per mL, using an Amicon Ultra-15 centrifugal filter unit (Merck) with a 100,000 dalton cut-off, flash frozen in liquid nitrogen and stored at -80°C.

On the day of grid preparation, the sample was further purified and buffer exchanged by size exclusion chromatography in buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.008% (w/v) GDN, 0.008% (w/v) LMNG, 0.0016% (w/v) CHS using a Shodex kW803 column connected to an Äkta Go purifier system. Fractions were concentrated to above 4 mg per mL and ultracentrifuged at 100,000g for 30 min at 4ºC.

For purification of the ORG25543-bound hGlyT2Δ185 sample, cell pellets were resuspended in the buffer supplemented with 100 μM ORG25543 (Tocris), and 10 μM ORG25543 was maintained in all the buffers throughout the purification. 1 mM glycine was maintained in all buffers throughout the purification of the glycine-bound hGlyT2Δ185 sample. For the substrate-free GlyT2Δ185 sample, resuspension buffer was supplemented with 100 μM oleoyl-D-lysine and subsequent buffers were supplemented with 10 μM oleoyl-D-lysine throughout the purification. For the RPI-GLYT2-82-bound hGlyT2Δ185 sample, the protein was purified without inhibitor and was incubated with 100 μM RPI-GLYT2-82 for 45 min on ice prior to SEC on the day of grid preparation. The SEC buffer was supplemented with 100 μM RPI-GLYT2-82.

Cryo-EM grid preparation and data collection
Immediately prior to grid preparation, the protein sample was supplemented with 0.01% (w/v) of fluorinated octyl maltoside (Anatrace). UltrAuFoil grids (R1.2/1.3 300 mesh, Quantifoil) were glow discharged (Leica EM ACE200) at 15 mA for 90 s. 3 μL of sample was applied to the grids, blotted for 5 s with a blot force of -3, or blotted for 4 s with a blot force of -4 before being plunge-frozen in liquid ethane using a Vitrobot mark IV (Thermo Fisher) at 100% humidity and 4°C in an environmental chamber. Movies were collected in counting mode at 165,000× magnification (300 kV), yielding a raw pixel size of 0.728 Å per pixel, using EPU 3.6 software (Thermo Fisher) on a Titan Krios G2 (Thermo Fisher) equipped with a Selectris X energy filter set to a slit width of 10 eV and a Falcon 4i direct electron detector (Thermo Fisher). A defocus value range of –0.6 to –1.8 μm and a dose rate of around 10.2 electrons (e) per pixel per second were used, giving a total dose of 60 e-/Å2 per movie (Supplementary Table 2).

Cryo-EM data processing
The cryo-EM data sets were processed using cryoSPARC v.4.5.359. A total of 12,374 movies were collected for the ORG25543-bound hGlyT2Δ185 sample, 16,094 for the RPI-GLYT2−82-bound hGlyT2Δ185 sample, 14,962 for glycine-bound hGlyT2Δ185, and 21,335 for the substrate-free hGlyT2Δ185 sample. The movies were motion corrected using Patch Motion Correction with default parameters. The contrast transfer function (CTF) parameters were estimated using Patch CTF Estimation and exposures with a CTF fit resolution of less than 3.6 Å were excluded during curation, yielding 11,378 exposures for ORG25543-bound hGlyT2Δ185, 14,629 for RPI-GLYT2-82-bound hGlyT2Δ185, 14,892 for glycine-bound hGlyT2Δ185, and 20,392 for the substrate-free state.

For the ORG25543-bound hGlyT2Δ185 sample, particles were picked by reference-free blob picker using a minimum and maximum particle diameter of 90 Å and 160 Å, respectively. A total of 2,807,000 particles were picked initially. Particles were then extracted with a box size of 300 pixels, Fourier cropped to 256 pixels and used to generate templates following a 2D classification, ab initio, heterogeneous refinement and non-uniform refinement60. Particles were then picked with the newly generated templates and extracted with a box size of 300 pixels (Supplementary Fig. 5). Following a 2D classification, a multi class ab initio and subsequent heterogeneous refinement were performed. A further heterogeneous refinement was performed using the selected particles. Suboptimal particles were removed by 3D classification and 2D classification. Selected particles were used for a one-class ab initio followed by a non-uniform refinement with initial low-pass resolution of 12 Å, deactivated dynamic mask start resolution and with the minimize over per-particle scale parameter on. Per-particle movement trajectories and empirical dose weights were estimated using reference-based motion correction followed by a final non-uniform refinement resulting in a 3D reconstruction of ORG25543-bound hGlyT2Δ185 map at 2.49 Å resolution, based on a Fourier shell correlation (FSC) cutoff of 0.143 with 283,838 particles.

For the RPI-GLYT2-82-bound hGlyT2Δ185 and substrate-free hGlyT2Δ185 sample, templates were generated from the ORG25543-bound hGlyT2Δ185 final map and used to pick particles. Subsequent data processing was similar to the ORG25543-bound hGlyT2Δ185 data processing pipeline (Supplementary Figs. 3, 6). Final non-uniform refinement for RPI-GLYT2-82-bound hGlyT2Δ185 and substrate-free hGlyT2Δ185 resulted in 3D reconstruction maps at 2.79 Å and 2.97 Å resolution with 201,519, and 180,698 particles, respectively (Supplementary Figs. 3, 6).

For glycine-bound hGlyT2Δ185, particles were picked by reference-free blob picker using a minimum and maximum particle diameter of 90 Å and 160 Å, respectively. A total of 3,363,288 particles were picked initially. Particles were then extracted with a box size of 300 pixels, Fourier cropped to 256 pixels and used to generate templates following a 2D classification, ab initio and heterogeneous refinement (using ORG25543-bound hGlyT2Δ185 map with decoy classes). Particles were then picked with the newly generated templates and extracted with a box size of 300 pixels and Fourier cropped to 256 pixels. Following a 2D classification, the set of 1,140,447 particles was split into for random batches. The map used to generate templates was further refined by several rounds of heterogeneous refinement, ab initio, 3D classification and non-uniform refinement that generated a 3.51 Å map from 267,885 particles. These 267,885 particles were used and combined individually with the four sets of particles and used to generate 2 class ab initio models. The four sets were combined to two sets by combining particles from the best ab initio class and removing duplicates by 2D classification. This was repeated to result in one combined set of particles that was used to generate a 4.01 Å map from 473,491 particles following several rounds of heterogeneous refinement and ab initio and non-uniform refinement. A molmap focus mask was generated using the substrate-free hGlyT2Δ185 model and used in a 3-class 3D classification with a filter resolution of 3 Å and a 5000 O-EM batch size (per class). The particles generating the best class were re-extracted at 300 pixels. A 2-class ab initio was followed by several rounds of non-uniform refinement with deactivated dynamic mask start resolution and with the minimize over per-particle scale parameter on and heterogeneous refinement. Reference-based motion correction was then followed by a final non-uniform refinement and finally a local refinement resulting in a 3D reconstruction of glycine-bound hGlyT2Δ185 map at 3.02 Å resolution, based on a Fourier shell correlation (FSC) cutoff of 0.143 with 162,255 particles (Supplementary Fig. 4).

Model building and refinement
The initial hGlyT2Δ185 structure model was derived using AlphaFold2 (AF-Q9Y345-F1)61. The predicted model was manually fitted into the cryo-EM map in UCSF ChimeraX v1.862 and was initially refined using Namdinator63. The models were refined using Phenix v1.21.1-528664 followed by visual examination and manual rebuilding in Coot65 v0.9.8.6 and ISOLDE v1.666. The initial Ramachandran outliers were corrected by manual rebuilding and local real-space refinement in Coot. The model was refined in real space using Phenix with the following settings: secondary structure, Ramachandran restraints, global minimization, group atomic displacement factors (ADP) refinement, 5 macrocycles. ISOLDE was then used to simulate the entire model at the start allowing the energy minimiser to alleviate clashes. The remaining Ramachandran and rotamer outliers were corrected using more localised simulations where real-time validation in ISOLDE enabled us to investigate the results of every change directly on the model. Coordinates were written out for a final refinement in Phenix using the input model restraints for the real space refinement. The ligand-restraint files for refinement were generated by phenix.elbow. Figures were prepared using ChimeraX v1.862.

The final models of ORG25543-bound hGlyT2Δ185 and RPI-GLYT2-82-bound hGlyT2Δ185 lack the initial methionine residue, residues 335–364 in EL2, and the last 13 residues of the C terminus. The final model of substrate-free hGlyT2Δ185 lacks residues 185–194 from the N terminus, residues 330–364 in EL2, and the last 12 residues of the C terminus. The final model of glycine-bound hGlyT2Δ185 lacks residues 185–194 from the N terminus, residues 327–365 in EL2, and the last 14 residues of the C terminus. Refinement and validation statistics are presented in Supplementary Table 2.

Expression and functional analysis of GlyT2 WT, GlyT2Δ185, and mutant GlyT2 in Xenopus laevis oocytes
Stock solutions of 50 mM ORG25543, and 20 mM RPI-GLYT2-82 were dissolved in dimethyl sulfoxide (DMSO) and diluted in frog Ringer’s solution (96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 5 mM HEPES, pH 7.5) to desired concentrations. Final solutions contained less than 0.001% DMSO, a concentration that had no effect on transporter function.

Human GlyT2a wild type (herein referred to as GlyT2) and codon optimised hGlyT2Δ185 complementary DNA were subcloned into the plasmid oocyte transcription vector (pOTV). Site-directed mutagenesis was performed using traditional PCR techniques and sequenced by the Australian Genome Research Facility (Sydney, Australia).

Purified plasmid DNAs were linearised with SpeI (New England Biolabs, Cat# R3133S). Complementary RNA was transcribed by the T7 RNA polymerase using the mMESAGE mMACHINE T7 kit (Ambion, Cat# AM1344).

Stage V Xenopus laevis oocytes were detached from the lobe by digestion with 2 mg/mL collagenase A (Boehringer, Mannheim, Germany). Defolliculated stage V oocytes were injected with 20 ng of cRNA encoding hGlyT2WT, hGlyT2Δ185, or mutant GlyT2 (Drummond Nanoinject, Drummond Scientific Co., Broomall, PA, USA). The oocytes were stored at 18°C in frog Ringer’s solution, which was supplemented with 2.5 mM sodium pyruvate, 0.5 mM theophylline, 50 μg/mL gentamicin and 100 μg/mL tetracycline for 3-6 days, until transporter expression was adequate for measurement using the two-electrode voltage clamp technique. Experiments were performed with at least n=5, from cells precured from at least two individual Xenopus laevis. Whole-cell glycine transport currents were recorded at -60 mV using a Geneclamp 500 amplifier (Axon Instruments, Foster City, CA, USA) with a Powerlab 2/25 chart recorder (ADInstruments, Sydney, Australia). LabChart version 8 software (ADInstruments, Sydney, Australia) was used to process and visualise current traces.

Glycine concentration dependent transport currents were measured for GlyT2WT and hGlyT2Δ185 and the mutant GlyT2s (n = 5) and the mutant GlyT2s (n = 5) and fit to the modified Michaelis Menten equation using GraphPad Prism (version 4.0, GraphPad Software, San Diego, USA):

I = Imax * [Gly] / (EC50 + [Gly])

where, I is the transport current generated by each glycine concentration (1-300 μM), Imax is the calculated maximal current and EC50 is the concentration of glycine that generates a half maximal current.

The potency of inhibition of hGlyT2WT, hGlyT2Δ185, or mutant GlyT2 was measured by applying the EC50 glycine concentration for each transporter and then in the presence of the EC50 for glycine with increasing concentrations of inhibitors ORG25543 (0.3–300 nM) and RPI-GLYT2-82 (0.01–10 μM). Currents were fit to the equation:

I = I_EC50 = Bottom + (Top - Bottom) / (1 + 10^(x - logIC50))

where I is current (nA) generated by EC50 for glycine in the presence of the indicated concentration of the inhibitor, and I_EC50 is the current generated by the EC50 (μM) glycine concentration in the absence of inhibitor. Top and Bottom are the maximal and minimal (≥0) plateau responses respectively, x is the concentration of inhibitor (nM), and IC50 is the concentration that causes 50% maximal inhibition.

The sodium dependence of inhibition by ORG25543 and RPI-GLYT2-82 was measured as described above but with 300 μM glycine to ensure measurable transport currents were obtained at low sodium concentrations. Choline chloride was used as a substitute salt when altering sodium concentrations.

The mechanism of inhibition of transport was determined by measuring glycine concentration-dependent transport currents with the co-application of RPI-GLYT2-82 at concentrations <3-fold IC50, IC50, and >3-fold IC50.

Reversibility from inhibition was determined by using the previously determined IC50 of ORG25543 or RPI-GLYT2-82 (or a maximal dose of 10 μM in cases where the IC50 is greater than 10 μM). The glycine EC50 for hGlyT2WT, hGlyT2Δ185, or mutant hGlyT2 was applied and then the IC50 of the inhibitor was co-applied until the current plateaued. The cell was then continuously perfused with frog Ringer’s solution to wash out the inhibitor from the bath solution. Every 5 min, glycine (EC50) was re-applied for 1 min, and repeated for a total wash period of 30 min or until complete transporter recovery. Washout kinetics were fitted using the equation below:

Recovery = (Y0 - Plateau) * e^(-Kt) + Plateau

Where recovery is the amount of transport-dependent current restored as a percentage of pre inhibitor levels (%), Y0 is the point which crosses the Y axis (≥0) and increases to the Plateau (≤100) in one phase, K is the rate constant (1/time), and t is time (mins).

Drug preparation
Pregabalin (50 mg/kg) and RPI-GLYT2-82 were dissolved in a vehicle of 5% Solutol in PBS. For PSNL experiments, a vehicle of 10% Solutol and 1% DMSO in 0.9% saline was used. For CCI acetone experiments, gabapentain (100 mg/kg) and RPI-GLYT2-82 were dissolved in a vehicle of 10% Cremophor and 5% Tween 80 in 0.9% saline. All solutions were prepared on the morning of experimentation in 3-5 mL glass v-vials immediately following baseline behavioural measurements. Mice were randomly assigned to a drug group by a second researcher using randomisation software (random.org), and the experimenter was blind to group allocations until after data collection. Compounds were administered via a single 200 μL i.p. injection into the lower right quadrant of the abdomen using 29-G insulin needles.

Chronic constriction injury and peripheral sciatic nerve ligation models of neuropathic pain
Neuropathic pain models were established via nerve injury to the left sciatic nerve via chronic constriction injury (CCI)67 via 2 mm polyethylene cuff or two 6-0 chromic gut ligatures placed 2 mm apart or partial sciatic nerve ligation (PSNL) via silk suture tightly constricting 1/3–1/2 of the nerve proximal to the trifurcation68. Mice were allowed to recover for 14 days after surgery prior to behavioural testing.

Mechanical allodynia assessment
Changes in sensitivity to a non-noxious mechanical stimulus was assessed by using a modified von Frey test. A single calibre of nylon filament that elicited approximately 15% response rate in naïve animals (0.4 g force) was applied to the plantar surface of the left hind paw until it just bent. Responses were defined as brisk withdrawal, shaking, or licking of the hind paw. Mechanical allodynia was assessed prior to surgery (baseline) and on the day of testing prior to drug administration (t=0) and at 0.5, 1, 1.5, 2, 3, 4, and 6 hours post injection. Exclusion criteria were response rates >30% at baseline or <70% on the test day at t=0. Data are presented as the percentage of responses out of 10 applications of the filament.

Cold allodynia assessment
Changes in sensitivity to a cold stimulus was assessed by using the acetone test69. Cold allodynia was assessed by counting the number of pain-like responses (rapid flinching, shaking or licking of the hind paw) to evaporative cooling induced application of acetone (20 μl) on the plantar surface of the operated hind paw over a 30 second period. Cold allodynia was assessed prior to surgery (baseline) and on the day of testing prior to drug administration (t=0) and at 0.5, 1, 1.5, 2, 4, and 6 hours post injection. Data are presented as the percentage of maximal possible responses. Data from n=1 mouse was exclude as it did not display cold allodynia compared to pre-CCI in baseline testing.

Rotarod
Motor coordination was assessed in naïve mice using an accelerating rotarod, using large cylinders (69.5-mm diameter, 99-mm width, 27-cm drop to landing platform) with speed increasing from 4 rpm to 20 rpm over 180 seconds. Mice underwent 3 days of training with 3 training sessions per day at 30-minute intervals. Mice were allowed 5 minutes of acclimatisation on cylinders at the start of each training day. On test day, mice were tested at t=0 and then 30, 60, 90, 120, and 180 minutes after drug administration. Data were normalised to % baseline.

Grip strength
Grip strength was assessed using a grip strength meter (Columbus Instruments; Columbus, Ohio) consisting of a force transducer with a triangular horizontal grip bar. Forelimb strength was assessed by gently holding mice by the base of the tail and lowering the animal toward the grip bar so that the forelimbs gripped the horizontal bar. The peak tension as the mouse was pulled away from the grip bar was recorded. The measurement was repeated 5 times over 2 minutes, and the average of 5 recordings was normalised to the animal’s body weight, expressed as kilogram-force per kilogram (KGF/kg).

Side effect scoring
Throughout any given behavioural assessment, all animals were monitored for adverse clinical events using a numerical rating scale. The measures assessed included hypoxia (blanched ears, abnormal colouration), sedation (unconsciousness), activity (unresponsive, inactive), hypothermia (piloerection or shivering), pain/discomfort (arching back, vocalisation, paw withdrawal, piloerection, respiratory depression, orbital tightening), hydration (sunken face), tone (weakness, poor coat), convulsions (clonic seizures, rearing), and grimace/discomfort (orbital tightening). Adverse events were recorded at each timepoint, observed when placed in the testing apparatus, and scored on a scale of 0–3 where 0=none, 1=mild, 2=moderate, and 3=severe. Scores were averaged for each treatment group.

Respiratory depression assessment
Respiratory function was assessed in naïve, uninjured mice via a whole-body plethysmography apparatus consisting of two parallel chambers connected to a pressure transducer (Buxco, DSI Instruments, USA). Mice were acclimatised to the chambers for 15 minutes on the two days preceding the experiment. Chambers were covered with a lab coat and divided with an opaque barrier to prevent any unwanted visual stimulation. On the day of experimentation, mice were acclimatised for 20 minutes, the last 5 of which were taken as the baseline. Mice were briefly removed from the chamber for injection, before being returned to the chamber. Respiratory function was then measured for a period of 5 minutes at 30, 60, 90, 120, and 180 minutes post-injection. Respiratory frequency, tidal volume (TVb) and minute volume (MVb) were recorded on the accompanying computer software (Buxco, FinePointe, DSI Instruments).

Conditioned place preference (CPP)
The CPP apparatus (Stoelting Co.) consisted of one middle (10 cm × 20 cm) and two outer chambers (18 cm × 20 cm). The middle chamber had bare walls with a smooth floor. One outer chamber had stripes on the walls with a smooth floor and the other had circles on the wall with a textured floor. CPP consisted of three stages: pre-testing, conditioning, and testing. Pretesting occurred on the first two days. Mice were placed in the middle chamber and allowed to freely explore the entire apparatus for 20 minutes. Time spent in each chamber was recorded using ANY-maze software (Stoelting Co.) and averaged across the two pretest days to determine baseline preferences. For the next eight days mice received intraperitoneal injection of test drugs (morphine, RPI-GLYT2-82) or vehicle on alternate days. Conditioning sessions lasted 40 minutes during which mice only had access to one of the outer chambers. Drugs were administered in the animals least preferred chamber identified during pretesting as this biased approach has been shown to produce reliable CPP with morphine in mice70. The CPP test was performed on day eleven using the same conditions as pretesting. Preference score was calculated as the percent change in time spent in the drug-paired chamber on the test day compared to pretesting.

Statistical analyses
To compare the glycine EC50 values and the inhibitor IC50 values between hGlyT2WT and hGlyT2Δ185, an unpaired two-tailed T-test was conducted. An unpaired two-tailed T-test was also conducted to compare inhibition by RPI-GLYT2-82 between hGlyT2WT and hGlyT1WT. To compare EC50 values and I300 values at different RPI-GLYT2-82 concentrations, Brown-Forsythe ANOVA with Dunnett’s T3 multiple comparisons test and a one-way ANOVA with Dunnett’s multiple comparisons test was conducted respectively. To compare inhibitor sodium dependence at 20, 40, and 100 mM, a two-way ANOVA with Tukey’s multiple comparisons test was conducted. CCI mechanical allodynia, PSNL mechanical allodynia and CCI cold allodynia were analysed by two-way ANOVA with Dunnett’s multiple comparisons test. Side effect scoring and grip strength data were also analysed A repeated measures two-way ANOVA with Greisser-Greenhouse correction and Tukey’s multiple comparisons was conducted to determine p values. Respiratory frequency, tidal volume (TVb) and minute volume (MVb) data were averaged and normalised to percentage (%) of baseline and analysed via two-way ANOVA with Dunnett’s multiple comparisons test. To determine CPP p values, a ROUT test (Q=10%) was used to remove outliers before performing an ordinary one-way ANOVA with Dunnett’s multiple comparisons test to analyse differences between vehicle and drug treated groups.

Molecular dynamics simulations
Molecular dynamics simulations of membrane-embedded GlyT2 inhibitors were initiated from the GlyT2 structures with bound inhibitors using the GROMACS 2023 MD package in conjunction with the GROMOS 54a7 force field71. The GROMOS 54a7 force field was chosen as it has been specifically parameterised to reproduce the experimental solvation free enthalpy and partition coefficients between polar and nonpolar environments for a range of chemical compounds and is ideally suited for lipid and membrane protein simulations. As the cryo-EM model contained a discontinuity between 330 and 364, acetyl and NH2 caps were applied to avoid the introduction of inappropriate charges. The disulfide bond (C311 and C320 (EL2)) was explicitly included. The protonation state of the side chains of ionizable residues was assigned according to the pKa predicted using PROPKa version 3.5.172,73.

Parameters for cholesterol hemisuccinate, ORG25543 and RPI-GLYT2-82 were generated using the Automated Topology Builder 3.069 (CHS molid 1736730, ORG25543 molid 1727769, RPI-GLYT2-82 molid 1732579)74. The pKa of the ORG25543 and RPI-GLYT2-82 amide groups were estimated with molgpka75 (ORG25543 pKa 8.4, RPI-GLYT2-82 pKa 8.4), and the compounds were simulated with the pendant ammonium protonated.

Proper dihedrals of benzamide groups were refined using a tool that will be included in the next release of the ATB74 (ORG25543 R1–R3 benzamide kΦ=18.31 kJ/mol/deg, RPI-GLYT2-82 R1–R2 benzamide kΦ=22.28 kJ/mol/deg, RPI-GLYT2-82 R1–R3 benzamide = 21.75 kJ/mol/deg). Benzamide dihedrals were fitted to the difference between the energy obtained by DFT (wB97X/6-31Gd) and energy minimisation with the ATB force field for dihedral angles constrained to 25 values between -180 and 180 degrees.

Two simulation systems were prepared, one using the GlyT2/ORG25543 cryo-EM complex, and a second using the GlyT2/RPI-GLYT2-82 complex. The composition of all MD simulation systems is given in Supplementary Table 8. The cryo-EM structures of each GlyT2 complex, containing ligand, bound chloride and bound sterols, was embedded in an 80% POPC and 20% cholesterol lipid bilayer created using the MemGen web server76. Each system was solvated with simple point charge (SPC) water, chosen for compatibility with the GROMOS 54a7 force field, and 150 mM NaCl for physiological relevance77. Counterions were added to ensure overall charge neutrality. All simulations were carried out under periodic boundary conditions. Systems were energy minimized using the steepest descent algorithm then equilibrated with decreasing harmonic restraints on the protein, bound ligand, and bound sterols in five sequential 2 ns simulations with a 2 fs timestep (1000 kJ/mol/nm2, 500 kJ/mol/nm2, 100 kJ/mol/nm2, 50 kJ/mol/nm2, 10 kJ/mol/nm2). Solute hydrogen masses were repartitioned to access longer simulation timesteps78 then the repartitioned systems were equilibrated with an additional 2 ns simulation using a 4 fs timestep, with 10 kJ mol–1 nm–2 harmonic restraints on the protein, ligand, and bound sterols.

The coordinates from the final frame of each equilibrated system were used as the starting configuration for 500 ns production simulations, using a 4 fs timestep. To increase the statistical sampling of the GlyT2/ligand conformation space and ensure convergence of the docked compounds, non-biased production simulations of each system were performed multiple replicates simulations (GlyT2/ORG25543 n=6, GlyT2/RPI-GLYT2-82 n=3). Random starting velocities were assigned at the start of each replicate simulation according to a Boltzmann distribution. The temperature was maintained at 310 K using the Bussi-Donadio-Parrinello velocity rescale thermostat and a coupling constant of 0.1 ps79. The pressure was maintained at 1 bar using semi-isotropic pressure coupling with the Parrinello--Rahman barostat80 using a 5 ps coupling constant and an isothermal compressibility of 4.5 × 10-5 bar-1. SETTLE81 was used to constrain the geometry of water molecules and LINCS82 was used to constrain the covalent bond lengths of the solute. The electrostatic interactions were calculated using the Particle Mesh Ewald summation, and non-covalent interactions were determined via the Verlet scheme with a 1.4 nm cut-off.

Simulations were visualized using VMD v1.9.4a557583. R.m.s.d. and r.m.s.f. analysis was performed on frames collected at 1 ns intervals, using gromacs tools. Energy landscapes were calculated on frames collected at 0.1 ns intervals with PLUMED version 2.984,85. Per residue contacts and hydrogen bonding frequency analyses were performed on frames collected at 1 ns with VMD v1.9.4a557583. Sodium density was performed on frames collected at 1 ns with MDAnalysis version 2.7.086. Molecular Mechanics-Poisson Boltzmann/Surface Area (MM-PB/SA) was used for binding enthalpy decomposition87,88. Per ligand enthalpic contributions to binding were calculated with g_mmpbsa89,90 on the first 50 ns of each replicate simulation, with frames spaced by 1 ns.

Analysis of MM-PBSA binding enthalpy
MM-PBSA calculations were performed using the one-trajectory method87,88,91 on the first 50 ns of MD simulation time for GlyT2/ORG5543 and GlyT2/RPI-GLYT2-82 systems, where the ligand remained in its experimentally determined pose. Binding entropy changes were not calculated. Frames were extracted at 1 ns intervals, yielding 50 frames per simulation for analysis.

The g_mmpbsa89 module of GROMACS was used to compute binding enthalpies with a grid spacing of 0.5 Å, a solvent probe radius of 1.4 Å, γ=0.03 kJ/mol/Å2, and an external solute (saline) dielectric constant of 80. To address variations in internal solute dielectric across the full protein, the analysis was restricted to the ligand (experimental pose) and its binding site residues: 211, 212, 213, 215, 216, 286, 287, 290, 433, 520, 541, 542, 543, 545, 546, 547, 550, 567, 570, 571, 574, 629, and 633. Focusing on the binding site enabled MM-PBSA calculations using a consistent internal solute dielectric value, eliminating contributions from distal residues to the binding site with a different solute dielectric value.

Electrostatic analysis using the APBS web server92 revealed binding site surface electric potentials ranging from approximately -15 to -30 kT/e for most of the binding site. This variation in surface electric potential afforded a solute dielectric range of 10-20 across the GlyT2 binding site. The conversion from surface electric potential to solute dielectric was done using Eq. 193,94. Here, ϕ is the surface electrostatic potential (in units of kT/e) from the APBS web server92, q is the charge of an electron, ξ0 is the vacuum permittivity constant, ξs is the internal solute dielectric for MM-PBSA, and r is set to the interaction cut-off distance from the MD simulations (r=1 nm). Specifically, ξ0 is scaled by ξs, such that ξ0ξs accounts for the overall permittivity of the medium. Equation 1 is reduced to the relationship from Coulomb’s law when ξs = 1.

|ϕ| = |q| / (4πξ0ξs r) (1)

We chose ξs=10 for two reasons: A) Eq. 1 afforded values of ξsolute between ~10-20; and, B) for ξs values between 10-20, the resultant MM-PBSA calculation with ξsolute=10 yielded a ratio of RPI-GLYT2-82/ORG25543 binding enthalpy (0.83-0.92, 95% C.I.) that was the most consistent with the experimental log(IC50) ratio of RPI-GLYT2-82/ORG25543 (0.79). Specifically, ξsolute=10 gave the best qualitative agreement with experiment. Binding enthalpies and per-residue enthalpy contributions were reported as mean values ± the 95% confidence interval in energy (Supplementary Fig. 19). We did not report per-residue binding energy benefits/penalties < 2 kT (5.15 kJ/mol).

Substrate docking
Docking was performed on the GlyT2 structure obtained from cryo-EM, with the inhibitor (ORG25543 or RPI-GLYT2-82), the cryogenic sodium ion, and the cryogenic chloride ion placed in their respective binding sites. Zwitterionic glycine was docked into the GlyT2/inhibitor/ions complex using autodock vina95. This structure was placed in a 80% POPC/20% CHOL bilayer, with cryogenic cholesterol and cholesterol hemisuccinate placed in their cryo-EM binding sites. The membrane embedded complex was solvated with SPC water containing 150 mM NaCl, with additional ions added to neutralize the overall system charge. To evaluate the possibility of glycine binding to the cryo-EM complexes, these solvated systems were used for a series of five replicate simulations.

Each replicate was equilibrated separately through a series of 2 ns simulations with decreasing position restraints on the protein backbone, cryogenic ions, cryogenic sterols, and docked glycine (fc 1000, 500, 100, 50, 10) with a 2 fs timestep. Following position restrained EQ, solute hydrogen masses were repartitioned to access longer simulation timesteps, then the repartitioned systems were equilibrated with an unrestrained 2 ns simulation using a 4 fs timestep. Coordinates and velocities of the equilibrated systems were used for 50 ns production simulations performed under the conditions described above.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_2": "In the light of the on-going opioid crisis, analgesic targets different from opioid receptor agonists are intensively searched for. Facilitation of glycinergic inhibition in the CNS is such an alternative analgesic strategy. This can either be accomplished with positive allosteric modulators of glycine receptors or, alternatively, be inhibitors of the glycine re-uptake transporters GlyT1 and GlyT2. While GlyT1 is expressed widely in the CNS, GlyT2 is restricted to areas receiving glycinergic input. However, glycine reuptake via GlyT2 not only regulates extracellular glycine concentrations but is also required for the recycling of glycine into presynaptic vesicles. (Quasi) irreversible inhibitors of GlyT2 therefore carry the risk of reducing rather than increasing extracellular glycine concentration and glycinergic inhibition.\n\nIn their current manuscript the authors address this problem. They characterize a new reversible inhibitor of GlyT2 with moderate analgesic efficacy in two mouse models of neuropathic pain and less pronounced undesired effects. The authors focus in their work largely on structural aspects of binding and inhibition of GlyT2. These parts appear very informative and import but they are outside my area of expertise. I therefore can only comment on the pharmacological experiments.\n\nMajor points:\n1. he authors convincingly demonstrate that their new molecule, RPI-GLYT2-82, is a reversible GlyT2 inhibitor. To link this effect to the observed analgesic efficacy, I recommend that the authors demonstrate that RPI-GLYT2-82 enhances glycinergic inhibition, e.g. in experiments in spinal cord slices, either through strengthening glycinergic postsynaptic currents or through increased tonic glycinergic currents. This link is currently missing.\n2. An important open question is whether the analgesic effect of RPI-GLYT2-82 indeed occurs via the inhibition of GlyT2 or via its interaction with other proteins, e.g. serotonin receptors. I agree that this question is not easily addressed. However, for publication in Nature Communications, the authors should make efforts to address this issue. A comparison of in vivo (CNS) receptor occupancies by RPI-GLYT2-82 at GlyT2 and at alternative targets (5HT2A receptors) and correlating these data with analgesic activity might yield relevant insights. The authors should check whether suitable radioligands are available for GlyT2 and 5HT2A receptors.\n3. When assessing analgesic efficacy, the authors exclusively rely on withdrawal responses. This is a potential issue, as an enhancement of glycinergic inhibition may interfere with withdrawal responses e.g. through decreased muscle strength. I like to encourage the authors to also employ withdrawal response independent readouts, such as the mouse grimace scale or conditioned place preference. The latter model has already been used by the authors to exclude reinforcing properties of RPI-GLYT2-82. It should therefore by relatively straight forward to use this model also for the assessment of analgesic efficacy.\n4. The authors test analgesic effects of RPI-GLYT2-82 only against mechanical allodynia. In order to obtain a more complete picture, I suggest testing also effects on heat hyperalgesia and, very important, against cold allodynia, a frequent and very unpleasant symptom of neuropathic pain.\n\nMinor points:\n5. An important mechanism of neuropathic pain sensitization appears to be a reversal of the chloride gradient in spinal cord neurons leading to depolarizing effects of glycine and GABA though the downregulation of KCC2. The authors should discuss their finding in context with these earlier results.\n6. The effects of RPI-GLYT2-82 on reinforcing properties in comparison with morphine (fig. 5H9) are not very convincing. There seems to be an effect that did not reach statistical significance. Was the experiment sufficiently powered to exclude reinforcing properties? I also suggest to provide the actual p values also in cases where no significant result was obtained.\n7. The time course of the analgesic effect is very different from that of side effects. Analgesia peaks at about 2 – 3 hours after drug administration, respiratory depression and impaired rotarod performance already at 0.5 hours. Can the authors come up with a reasonable explanation.\n8. Relative to pregabalin, the gold standard treatment, the analgesic efficacy of RPI-GLYT2-82 is moderate. I like to suggest testing additional (higher) doses of RPI-GLYT2-82 to determine the maximal analgesia achievable with a saturating dose of RPI-GLYT2-82.\n9. The authors emphasize that a facilitation of glycinergic inhibition is analgesic in case of neuropathic pain but does not work for inflammatory pain. The literature on this question is controversial. I suggest that the authors test their compound also in an inflammatory model and discuss the relevant literature.", "reviewer_3": "The work solved the high resolution cryo-EM structures of human GlyT2 in substrate-free, and glycine, reversible inhibitor RPI-GLYT2-82, pseudo-irreversible inhibitor ORG25543 bound states, along with MD simulation and transport assay, providing prospects for non-opioid analgesics development. The analgesia effect of RPI-GLYT2-82 to mouse model of neuropathic pain is inspiring. It is surely a progress in the understanding of GlyTs substrate recognition and inhibition mechanism. Still some problems need to be addressed as described in the followings:\n\nMajor problems:\n1. The authors stated that“these data show that RPI-GLYT-82 is a non-competitive GlyT2 inhibitor that binds with lower affinity than ORG25543.”It is not a proper interpretation of the available experiment results. The electrophysiological assay based on oocyte is not a propriate method for affinity definition. In vitro assay based on purified protein is needed for binding affinity determination of RPI-GLYT-82 and ORG25543 to GlyT2.\n2. More evidence is needed to identify cholesterol from cholesterol hemisuccinate in the model building. As the molecules are structurally similar, and the cryo-EM map density surrounding the transmembrane helixes is relatively with more chaos, the cryo-EM map itself is not a robust evidence in distinguishing cholesterol from cholesterol hemisuccinate. Besides, there is no obvious evidence supporting the binding affinity differences between the different cholesterol or CHS binding sites. It is not reasonable that while some sites (like CHOL3’and CHOL7) bind cholesterol, some other sites (CHOL5 and CHOL6) bind CHS.\n3. The modulation function of cholesterol to GlyT2 is also need validation, especially CHOL3’. Is there possible cross-talk exist between cholesterol and chemical modulators like ORG25543 or RPI-GLYT2-82?\n4. The binding of sodium is different from GlyT1 structure. And the sodium ion binding in GlyT2 is also altered in different structures. What is the sodium ion function in the glycine transport by GlyT2? Transport assay in buffer depleted of sodium is suggested to address the problem. Besides, while there is still sodium binding in Na1 site (as supported by MD simulation results in Supplementary figure 13), it is not reasonable to state that “consistent with ORG25543, and likely RPI-GLYT2-82, binding being Na+-independent”. On the contrary, sodium could participate in the inhibition process. More experiments are needed to address this question.\n5. In MD simulation results, RPI-GLYT2-82 has higher possibility to slip in the central vestibule of GlyT2. Is there additional non-protein density appear in this site? Moreover, transport activity measurements of different mutations in the allosteric site and central vestibule are suggested to verify roles of the residues in RPI-GLYT2-82 inhibition.\n6. The transport process or mechanism of GlyT2 need to be investigated in detail so that the inhibitory effect upon RPI-GLYT2-82 and ORG25543 binding would be easier to understand. Obviously, glycine binding cannot trigger the conformational change from inward to outward. What are the possible factors that trigger conformational changes physiologically?\n\nSome minor problems:\n1. The rationale for Cl- binding is needed. Structural alignment with GlyT1 would make it more persuasive to build Cl-. What is the possible function of Cl- binding? Is it a substrate or regulatory factor for GlyT2?\n2. In glycine-bound structure, although the current glycine conformation fits well to the density, the cryo-EM map is insufficient for determination of the amino group orientation. The related statements of “…whereas in GlyT1, it adopts a different orientation to interact with the backbone of TM6 residues.”should be deemphasized.\n3. What could be the possible reasons for the current recovery larger than 100% of D633E in fig. 4k?\n4. The glycine binding site is far from the disulfide bond in EL2. What is the possible relation of disulfide bond reduction and glycine binding?\n5. The selectivity of RPI-GLYT2-82 to GlyT2 need more validation. What could be the possible reasons for absence of other SLC6s in the Off-target selectivity screening? Comparison with other SLC6 family proteins is suggested.", "reviewer_5": "In their work, Cantwell Chater and colleagues develop a reversible inhibitor of the neuronal glycine transporter GlyT2 that provides analgesia in mouse models of neuropathic pain. To elucidate its mechanism of action, the authors resolved the structure of human GlyT2 for the first time alone and in complex with glycine and two different inhibitors, including the newly synthetized one.\n\nElectrophysiology and structural insights, aided by MD simulations, showed that the newly synthetized compound acts as a reversible allosteric inhibitor that traps the transporter in an outward facing conformation. Because of its relatively low affinity, the inhibitor shows no on-target side effects, making it a very promising therapeutical target to treat chronic neuropathic pain.\n\nI believe this study to be highly relevant for the field of neurotransmitter transporters. First, by capturing GlyT2 at different conformations, including the one bound to its endogenous ligand, the authors set the ground for further mechanistic understanding of its transport cycle. More importantly, the rational design of a new promising inhibitor of GlyT2 may lay the foundation for further development of non-opioid pain therapeutics.\n\nThe conclusions of the study are sound and supported by a vast array of different methods, including structural and computational techniques, electrophysiology and animal models.\n\nThe manuscript is clear and well written; I found it quite easy to follow through the results although most techniques used are outside my field of expertise.\n\nComments/suggestions:\n- Modulation of GlyT2 by cholesterol seems to be an important aspect of its biological function. Although the authors briefly describe the sterol densities found in their structures and even assess their stability and interactions with MD simulations (Supplementary Fig. 11), this is, in my opinion, not emphasized enough in the main text. I would suggest to include a couple of sentences in the second section of the results summarizing the behavior of sterols at the proposed sites in MD simulations (Supplementary Fig. 11d-i). I would even suggest changing the title of the second section of the results to “hGlyT2 adopts a LeuT fold stabilized by lipids”.\n- In Supplementary Fig. 15, RMSD values show that the backbone of the protein takes some time to equilibrate. RMSD of heavy atoms of the inhibitors also show fluctuations throughout the simulations. However, the authors perform MM-PBSA only on the first 50 ns of the trajectories, where the protein has clearly not fully equilibrated. To me, it is not surprising that the MM-PBSA results agree with the structural data if the initial structure did not have the time to relax. I strongly recommend to perform the same MM-PBSA analysis using the full 500 ns trajectories. In case this drastically changes the results, more analysis might be needed, as this could suggest the existence of several different binding modes of the ligands in the pocket.\n\nMinor comments:\n- At the beginning of page 17 in the discussion section, when they mention the efficacy of pregabalin, the authors refer to Figure 4, but I believe they mean Figure 5.\n- In the MD Methods section (page 26), change “ionizable residue” to “ionizable residues”.\n- In the MD Methods section (page 26), it is not clear to me what the authors mean by “ORGH”, as this abbreviation has not been explained in the main text. I suggest changing “GlyT2-ORGH” by either “Glyt2-inhibitor” or by “Glyt2- ORG25543 and Glyt2-RPI-GlyT2-82” for clarity.\n- The text in Supp. Fig 17 a-b g-h is not very well readable. I would suggest to increase the font size in these plots.\n\nPublishing suggestion:\n- Publish with revisions", "reviewer_6": "“I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.”\n\nTo the authors\nThe manuscript focuses on GlyT2 and a new allosteric inhibitor RPI-GLYT2-82 with peculiar characteristics; the structural information collected by CryEM is consistent, and the results agree with previous data about other members of the SLC6 family, with a few novelties about the determinants involved in the interaction with the old and new inhibitor and the cholesterol binding site. However, the pharmacological investigation is insufficient and limited if the aim, apparently declared, is to present a new potential therapy for pain. The conclusion of the abstract and the end of the introduction agree with a “structural goal” of the manuscript, but in other points, the focus seems the importance of RPI-GLYT2-82 as possible new drugs.\n\nTo better explain the point, it is important to ask what is the significance of the manuscript for the research community?\nIf the goal of the paper is to show the structure of the transporters in three different conformations, identifying the Na, Cl, Gly binding site and the possible site of interaction of cholesterol and the of two allosteric inhibitors, and a possible new “inhibithors” that can represent the basis of the future work, some new experiments are required but the manuscript can be considered for publication after important revision. Indeed, if it is the case, this manuscript reports interesting data as in other recently published research about transporters of the same family inquiring and defining important binding sites, interactions and poses.\n\nInstead, if the goal of the authors is to show that the molecule RPI-GLYT2-82 is a potential new pain therapy, the manuscript needs to be completely reorganized. The study in vivo and in vitro must be part of the main manuscript. Details of the experimental plan and the investigation methods must be better explained, and the studies have to focus not only on GLYT2 but also on the other potential target of the PRI-GLYT2-82.\n\nFrom here, my comments will consider the “structural” aim to show the structure of the transporter and the binding site of a molecule that can serve as a future blueprint for potential new inhibitors.\n\nIn this case, first of all, to frame the results in the context of identifying a molecule suitable to act on GlyT2 specifically and without side effects in a way that may support future development of a therapeutic, the manuscript should include some new experiments to demonstrate the specificity and the action of RPI-GLYT2_82\nFirst, the data collected about GlyT2 in Figure 1 must also be collected for GLYT1. The absence of effect, SERT, DAT and NET must be shown. These last two (DAT and NET) are to be tested because usually any compound interacting with SERT has an action also on the other monoamine transporters (even if the data about the binding seems to exclude this possibility). This is important as the data reported in the table of the Off-Target Selectivity Screening of RPI-GLYT2-82 showed that serotonin receptor and transport seem to be targets of RPI-GLYT2-82.\nSimilarly, a fast screening of the effect on the 5-HTA receptors could better highlight the selectivity.\n\nRPI-GLYT2-82 action was tested in vitro in Xenopus laevis oocytes, defining it as a non-competitive inhibitor.\nHow was the maximal current recorded (Imax), does it change by the presence of RPI-GLYT2-82?\nThese data in a table will be useful\nAbout Figure 1, most importantly, the data are not supported by statistical analysis. The effect of the inhibitor and the similarity between the two GLYT2 (wt and truncated) need to be statistically verified.\nIf the n=5 refers to the number of oocytes, from how many batches are they? In the figure capture, it is important to report both the number of oocytes and the number of batches.\nA dot plot showing the experimental points will clarify better how the experiment was planned and conducted.\nSimilarly, to better understand the action of the old and new molecules, representative traces of the recording in the presence and in the absence of the two inhibitors (old ORG and new RPI) alone and in the presence of glycine must be shown at least in the supplementary material.\n\nOther points\nThe methods are very long and difficult to follow, and lack of statistical information about the statistical analysis\nIn the docking with glycine, looking at the Cysteine density. Chater et al. says that “However, although we observed clear densities for C311 and C320 in glycine-bound hGlyT2d185, we did not detect a distinct density for the disulfide bridge, suggesting that the cysteine residues may be reduced in the substrate-bound state”. Does this mean that the C311 and C320 are involved in glycine binding or are essential in conformational changes induced by the binding of Gly?\nA docking of the RPI-GLYT2-82and ORG25543 together with the glycine, both binding GLY2 is missing.\nSince finding a specific inhibitor for GlyTs is important, it will be essential to verify if RPI-GLYT2-82 could interact with GlyT1 even by docking\n\nMinor:\nGlyT2Δ185 was expressed in HE400AZ expression medium.. something is missing in the sentence\nThe definition of Imax is not correct and not clear, as in general the reported description of electrophysiological data analysis\nWhat does it “biological samples” mean? In the case of oocytes, is it the number of oocytes or batches?\nFigure 1d: the fitting line for the wt is missing\nFigure 4b, the circle described in the capture representing Na+ and Cl- are not visible.", "reviewer_7": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "- Reviewers comments\n- Our response\n- From the manuscript – Revised version (from the manuscript)\nReviewer #2 (Remarks to the Author):\nIn the light of the on-going opioid crisis, analgesic targets different from opioid receptor agonists are\nintensively searched for. Facilitation of glycinergic inhibition in the CNS is such an alternative\nanalgesic strategy. This can either be accomplished with positive allosteric modulators of glycine\nreceptors or, alternatively, be inhibitors of the glycine re-uptake transporters GlyT1 and GlyT2.\nWhile GlyT1 is expressed widely in the CNS, GlyT2 is restricted to areas receiving glycinergic\ninput. However, glycine reuptake via GlyT2 not only regulates extracellular glycine concentrations but is\nalso required for the recycling of glycine into presynaptic vesicles. (Quasi) irreversible inhibitors of\nGlyT2 therefore carry the risk of reducing rather than increasing extracellular glycine concentration\nand glycinergic inhibition.\nIn their current manuscript the authors address this problem. They characterize a new reversible\ninhibitor of GlyT2 with moderate analgesic efficacy in two mouse models of neuropathic pain and\nless pronounced undesired effects. The authors focus in their work largely on structural aspects of\nbinding and inhibition of GlyT2. These parts appear very informative and import but they are outside\nmy area of expertise. I therefore can only comment on the pharmacological experiments.\nMajor points:\n1. The authors convincingly demonstrate that their new molecule, RPI-GLYT2-82, is a reversible\nGlyT2 inhibitor. To link this effect to the observed analgesic efficacy, I recommend that the authors\ndemonstrate that RPI-GLYT2-82 enhances glycinergic inhibition, e.g. in experiments in spinal cord\nslices, either through strengthening glycinergic postsynaptic currents or through increased tonic\nglycinergic currents. This link is currently missing.\nWe thank the reviewer for this suggestion. The effects of ORG25543 on glycine neurotransmission\nhave been extensively studied by others. In particular, Bradaia et al. (2004, J Physiol, 559.1, 169-\n186) have demonstrated that ORG25543 enhances tonic glycine neurotransmission and also\nprolongs the time course of glycinergic mIPSCs and eIPSCs. These effects can be mimicked by\nperfusion of low concentrations of glycine (1-3 µM), which suggests that the effects of ORG25543\nare due to elevation of glycine concentrations. Furthermore, ORG25543 has no effect on GABAergic\nneurotransmission, a small effect on NMDA-R mediated eEPSC and no effect on AMPA-R mediated\neEPSC. We have modified the introduction to explicitly reference this work: “In rat spinal cord slices,\nORG25543 enhances tonic glycinergic neurotransmission and prolongs the decay time course of\nboth miniature and evoked glycinergic inhibitory postsynaptic currents, suggesting that its effects\nare likely due to elevated extracellular glycine concentrations32.” We fully expect that RPI-GLYT2-\n82 (at higher concentrations) will have similar effects as ORG25543 and given that the focus of this\nstudy, as the reviewer also correctly points out, was to establish how the structure of GlyT2 can be\nused to design analogues of ORG25543, we feel that the repeating the work on glycine\nneurotransmission for RPI-GLYT2-82 is beyond the scope of the current study. Our findings of\nanalgesic efficacy with RPI-GLYT2-82 are fully consistent with this established mechanism. We\nacknowledge that this is an important next step, and in the future, we will actively pursue\nelectrophysiological studies to directly confirm that the effects of optimised RPI-GLYT2-82 analogues\n– designed using the structural insights obtained here – on glycinergic inhibition, will be similar to\nthose established for ORG25543.\n2. An important open question is whether the analgesic effect of RPI-GLYT2-82 indeed occurs via\nthe inhibition of GlyT2 or via its interaction with other proteins, e.g. serotonin receptors. I agree that\nthis question is not easily addressed. However, for publication in Nature Communications, the\nauthors should make efforts to address this issue. A comparison of in vivo (CNS) receptor\noccupancies by RPI-GLYT2-82 at GlyT2 and at alternative targets (5HT2A receptors) and correlating\n1\nthese data with analgesic activity might yield relevant insights. The authors should check whether\nsuitable radioligands are available for GlyT2 and 5HT2A receptors.\nWe agree with the reviewer that this is an interesting point and have already addressed it on the final\npage of the Results section in the manuscript. We have revised the results section to clearly indicate\nwhere the relevant data are presented in the Supplementary Information.\nThese in vivo data provide evidence that RPI-GLYT2-82 produces statistically significant dose\ndependent analgesia without the adverse neuromotor effects associated with ORG2554328,33.\nInterestingly, although the primary target of RPI-GLYT2-82 is GlyT2 (IC = 554 nM, Fig. 1b),\n50\nit is also a less potent antagonist at the serotonin 2A (5-HT2A) receptor (IC = 1.9 µM,\npain signals. Indeed, antagonism of 5-HT2A has previously been reported for other ORG25543-\nbased GlyT2 inhibitors, including opiranserin37,38.\nBased on our selectivity screen (Supplementary data 1), we assume that a minor part of RPI-GLYT2-\n82 achieving analgesia is through the serotonin (5-HT2A) receptor. Furthermore, the mild transient\nsedative effect of 5 x the analgesic dose of RPI-GLYT2-82 (Fig. 5f) may be due to interactions with\n5HT2A receptors and/or SERT. Since there are currently no radioligands commercially available for\nthe allosteric binding site of GlyT2, we are unable to perform in vivo occupancy measurement for\nRPI-GLYT2-82 at GlyT2. In the future, we plan to develop a radioligand for GlyT2 to characterise\noptimised inhibitors (6 months to a year time scale).\nWe have however, partly addressed this concern by conducting plasma and central nervous system\npharmacokinetic (PK) profiling in mice to estimate the CNS concentration of RPI-GLYT2-82\navailable. We have:\n“calculated unbound drug concentrations in plasma (C ) and brain (C ) at 30- and 120-minutes\nu,p u,b\npost-injection (Supplementary data 1). RPI-GLYT2-82 achieved a C of 597.89 nM at 30\nu,b\nminutes following a 50 mg/kg intraperitoneal (i.p.) dose (Supplementary Table 7) –\napproximately equal to its IC on hGlyT2, suggesting the compound is likely to achieve\n50\nmeaningful hGlyT2 occupancy in vivo (Fig. 1b, Supplementary Table 7).”\nWe note that here we describe the unbound fraction of drug and emphasize that we are explicitly\naddressing the free drug hypothesis. Specifically:\n1- We measured how much of compound RPI-GLYT2-82 entered the CNS, at the site of the\ntarget (spinal cord).\n2- Of the total CNS drug concentration, we then calculated the fraction that was free and\nunbound.\nOnce in the CNS, drugs can bind non-specifically to membrane lipids, myelin proteins, cytosolic\nstructural proteins, and to the small amount of albumin in CSF. The fraction not bound to these\ncomponents is the pharmacologically active pool that can interact with GlyT2 and induce the desired\neffect.\nTo quantify this, we conducted mouse in vivo studies to measure total drug concentrations in plasma\nand brain at two post-dose timepoints. The concentrations of drug in the brain are an approximation\nof total CNS drug concentration.\nIn addition, we ran two in vitro binding studies with RPI-GLYT2-82:\n- Mouse Plasma protein binding (% bound): gives the fraction of RPI-GLYT2-82 unbound in\nplasma.\n- Mouse Brain tissue homogenate binding (% bound): provides the fraction of unbound RPI-\nGLYT2-82 in CNS tissue. Even though it is done with brain tissue, this is an approximation\nof unbound fraction in the entire CNS\nunbound, free concentrations of drug in plasma and brain. Importantly, at the 30-minute timepoint,\nthe unbound concentration of RPI-GLYT2-82 in brain was approximately equal to the IC₅₀ value,\nsuggesting that the compound has achieved meaningful GlyT2 target coverage in vivo.\nWe have clarified these points in the revised manuscript and improved referencing to the studies\ndescribed in the Supplementary data 1 (pages 35 – 57 in the revised supplementary data document),\nand supplementary table 7 to ensure that our findings are more clearly presented and easily\naccessible to the reader.\n3. When assessing analgesic efficacy, the authors exclusively rely on withdrawal responses. This is\na potential issue, as an enhancement of glycinergic inhibition may interfere with withdrawal\nresponses e.g. through decreased muscle strength. I like to encourage the authors to also employ\nwithdrawal response independent readouts, such as the mouse grimace scale or conditioned place\npreference. The latter model has already been used by the authors to exclude reinforcing properties\nof RPI-GLYT2-82. It should therefore by relatively straight forward to use this model also for the\nassessment of analgesic efficacy.\nWe thank the reviewer for this comment. We have complemented our anti-allodynia testing with the\nrotarod test to identify any motor deficits induced by the drugs and observed none at analgesic doses\nor at time points when analgesia was present. To further address the concern that drug-induced\nmuscle weakness could cause decreased withdrawal responses, we have conducted additional grip\nstrength measurement testing. We used a “five-fold higher than the maximally analgesic dose of\nRPI-GLYT2-82” of 250 mg/kg to assess neuromotor side effects:\nA supramaximal 250 mg/kg dose (i.p.), which is five-fold higher than the maximally analgesic dose\nof RPI-GLYT2-82, produced an initial 61% reduction in baseline rotarod performance, which was\nrestored over the 90-minute time course, and caused transient muscle weakness 30 min post-injection\nwhich recovered at the 60-minute time point (Fig 5d, e). Whilst the grip strength and rotarod\nexperiments show mild, transient deficits in muscle strength and motor coordination at the\nsupramaximal analgesic dose, they do not coincide with the anti-allodynic effects. Given the temporal\nseparation of these motor side-effects from the anti-allodynia effects, they do not pose a confound in\ninterpreting the motor response-dependent pain-like behaviors.\nFig. 5e. RPI-GLYT2-82 produced transient deficits in the grip-strength test,\nindicating muscle weakness at 250 mg/kg, but not at 50 mg/kg, 30 min post-\ninjection which was recovered at the 60-minute time point.\nWe have added the corresponding experimental details to the Methods section:\nGrip strength\nGrip strength was assessed using a grip strength meter (Columbus Instruments; Columbus, Ohio)\nconsisting of a force transducer with a triangular horizontal grip bar. Forelimb strength was\nassessed by gently holding mice by the base of the tail and lowering the animal toward the grip\nbar so that the forelimbs gripped the horizontal bar. The peak tension as the mouse was pulled\naway from the grip bar was recorded. The measurement was repeated 5 times over 2 minutes, and\n3\nthe average of 5 recordings was normalised to the animal’s body weight, expressed as kilogram-\nforce per kilogram (KGF/kg).\nRegarding the grimace test or conditioned place preference (CPP), we would like to elaborate:\nThe use of the grimace scale has been shown to be robust and reliable in “acute” pain (e.g. post\noperative) and “chronic trigeminal nerve” pain (trigeminal neuralgia; Akintola et al, 2017), but it does\nnot detect and is “not reliable for chronic neuropathic pain” in the sciatic nerve injury models such\nas the ones we have used in our study (CCI, SNI; Langford et al., 2010).\nFurthermore, while CPP can in some cases be used for analgesic assessment, its interpretability is\nlimited. It is most effective for analgesics with strong rewarding/euphoric effect (e.g. opioids) in\naddition to alleviating allodynia or sensory pain. Since our inhibitor lacks rewarding effects, we do\nnot expect it to cause robust place preference. Although CPP is well-suited for evaluating opioid\nanalgesics, where addiction is a factor, its use in this context would introduce ambiguity, and the\nresults are unlikely to be definitive or yield a clear, interpretable outcome. Furthermore, chronic pain\nhas been shown to alter the reinforcing properties of compounds in an inconsistent manner as\ndemonstrated by opioid intravenous self-administration (IVSA) studies: some studies report\nenhanced reinforcement, while others find no difference (e.g., references 1 – 6, below). This\nambiguity represents a key limitation, as an increase in CPP may not necessarily reflect genuine\nanalgesia but could instead arise from pain-induced alterations in reinforcement, making it\nimpossible to clearly disentangle the two effects.\nMoreover, CPP is not routinely performed for analgesia testing in early drug discovery/drug\nscreening, as different analgesic drug classes generate different responses in the CPP (e.g. NSAIDs\ndo not generate a place preference in inflammatory pain (Sufka, 1994)). As such, CPP testing for\nanalgesic effects is out of scope for the early-stage drug development in this manuscript.\nFor these reasons, we have focused on the sensory allodynia tests, and tests that rely on motor\nresponses, in this study, which are robust, sensitive, and well validated for all known analgesic drug\nclasses and mechanisms of action. These tests reflect a predominant symptom of clinical chronic\nneuropathic pain (allodynia) and therefore provide the most relevant and interpretable outcomes for\nthis study.\n1- Reiner DJ, et al. Lack of effect of different pain-related manipulations on opioid self-administration, reinstatement of\nopioid seeking, and opioid choice in rats. Psychopharmacology (Berl). 2021 Jul;238(7):1885-1897.\n2- Barattini AE, Gilpin NW, Pahng AR. Chronic inflammatory pain reduces fentanyl intake during early acquisition of fentanyl\nself-administration, but does not change motivation to take fentanyl in male and female rats. Pharmacol Biochem Behav.\n3- Burgess GE, Traynor JR, Jutkiewicz EM. The effects of chronic neuropathic pain on the self-administration of highly\npotent MOR agonist, fentanyl. bioRxiv [Preprint]. 2024 Nov 21:2024.11.19.624389.\n4- Barattini AE, et al. Chronic inflammatory pain promotes place preference for fentanyl in male rats but does not change\nfentanyl self-administration in male and female rats. Neuropharmacology. 2023 Jun 15;231:109512.\n5- Hipólito L, et al. Inflammatory Pain Promotes Increased Opioid Self-Administration: Role of Dysregulated Ventral\nTegmental Area μ Opioid Receptors. J Neurosci. 2015 Sep 2;35(35):12217-31.\n6- Sarka BC, et al. Neuropathic pain has sex-specific effects on oxycodone-seeking and non-drug-seeking ensemble\nneurons in the dorsomedial prefrontal cortex of mice. Addict Biol. 2024 Aug;29(8):e13430.\n4. The authors test analgesic effects of RPI-GLYT2-82 only against mechanical allodynia. In order\nto obtain a more complete picture, I suggest testing also effects on heat hyperalgesia and, very\nimportant, against cold allodynia, a frequent and very unpleasant symptom of neuropathic pain.\nWe agree with the reviewer that cold allodynia is a frequent and very unpleasant symptom of\nneuropathic pain and testing the compound against cold allodynia will strengthen our study. We have\nadded these experiments, and the result is very promising, showing clear efficacy of the compound\nRPI-GLYT2-82 against cold allodynia: “Encouraged by these data, we tested RPI-GLYT2-82 in the\nCCI mouse models of neuropathic pain by measuring mechanical and cold allodynia in mice upon\n4\ni.p. administration. When measuring mechanical allodynia, we observed dose-dependent anti-\nallodynia at both 50 and 100 mg/kg, with maximum efficacy between 90 minutes and 3 hours\npost-administration (Fig. 5a). This is comparable to the effect of 30 mg/kg ORG25543, which\npeaked between 1 and 2 hours post-administration28. Similarly, when measuring response by cold\nallodynia, peak anti-allodynia effects were observed within 2 hours at both 50 and 100 mg/kg\nwith comparable efficacy to the control compound gabapentin (Fig. 5b). We further tested RPI-\nGLYT2-82 in the PSNL model by measuring its effects on mechanical allodynia and observed\nsimilar effects to the CCI model (Fig. 5c).” (Note: Gabapentin was used as a control instead of\npregabalin due to unavailability of pregabalin in Australia during the revision period. Gabapentin is\nanother established alternative and a gold-standard treatment comparable to pregabalin).\nFig. 5b RPI-GLYT2-82 reduced response rate to acetone stimulus in CCI mice. Two-\nway ANOVA with Dunnett’s multiple comparison post hoc test was conducted\nbetween CCI model mice administered vehicle and every other group. ANOVA\nshowed significant main effects of time (p < 0.0001; F (4, 93) = 25.8) and treatment\n(p < 0.0001; F (3, 26) = 21.8, along with an interaction effect (p < 0.0001; F (11, 93)\n= 4.4). Both 50 and 100 mg/kg (i.p.) doses demonstrated efficacy in reducing cold\nallodynia, significant between 0.5 and 6-hours postinjection compared to vehicle (p\n< 0.05-0.001). Gabapentin was also significant at 1-6 hours (p<0.05-0.001). All\ntreatments peaked by the 2-hour time point, with 59%, 62% and 75% reductions,\nrespectively.\nMinor points:\n5. An important mechanism of neuropathic pain sensitization appears to be a reversal of the chloride\ngradient in spinal cord neurons leading to depolarizing effects of glycine and GABA though the\ndownregulation of KCC2. The authors should discuss their finding in context with these earlier\nresults.\nWe thank the reviewer for this comment and do agree that the downregulation of KCC2 and changes\nin chloride gradient in spinal cord neurons is one of the important pathological features of neuropathic\npain1. The shift in the anion gradient in lamina I of the dorsal cord results in an overall shift from\ninhibitory neurotransmission towards excitatory neurotransmission. The increase intracellular\nchloride changes the dynamics and leads to GABA and glycine receptor activation depolarising the\npost-synaptic neuron2. However, we do not anticipate that GlyT2 inhibitors would have a marked\neffect on the KCC2 associated pathophysiological changes in neuropathic pain as GABAergic\nneurotransmission is dominant in lamina I of the dorsal horn where GlyT2 expression is relatively\nlow3.\nConsidering the vast array of pathological changes in neuropathic pain, which include the chloride\ndysregulation attributed to KCC2, we have added the following statement to the Discussion:\nNeuropathic pain is caused by a wide variety of insults and encompasses a large range of distinct\npathological phenotypes. As such it is likely that a range of therapeutics will be required to\nalleviate the debilitating symptoms. The development of safer and more efficacious GlyT2\ninhibitors has the potential to add to the array of therapeutic options as a stand-alone treatment or\nin combination with other analgesics.\n1- Mapplebeck JCS, Lorenzo LE, Lee KY, Gauthier C, Muley MM, De Koninck Y, Prescott SA, Salter MW. Chloride\nDysregulation through Downregulation of KCC2 Mediates Neuropathic Pain in Both Sexes. Cell Rep. 2019 Jul\n16;28(3):590-596.e4. doi: 10.1016/j.celrep.2019.06.059. PMID: 31315039.\n2- Cheung DL, Toda T, Narushima M, Eto K, Takayama C, Ooba T, Wake H, Moorhouse AJ, Nabekura J. KCC2\ndownregulation after sciatic nerve injury enhances motor function recovery. Sci Rep. 2023 May 15;13(1):7871. doi:\n10.1038/s41598-023-34701-y. PMID: 37188694; PMCID: PMC10185507.\n3- Spike RC, Watt C, Zafra F, Todd AJ. An ultrastructural study of the glycine transporter GLYT2 and its association with\nglycine in the superficial laminae of the rat spinal dorsal horn. Neuroscience. 1997 Mar;77(2):543-51. doi: 10.1016/s0306-\n4522(96)00501-5. PMID: 9472410.\n5\n6. The effects of RPI-GLYT2-82 on reinforcing properties in comparison with morphine (fig. 5H9) are\nnot very convincing. There seems to be an effect that did not reach statistical significance. Was the\nexperiment sufficiently powered to exclude reinforcing properties? I also suggest to provide the\nactual p values also in cases where no significant result was obtained.\nWe thank the reviewer for this comment. We performed the assay within the range of previously\npublished morphine CPP assays that have been sufficiently powered to show the reinforcing effects\nof morphine, utilizing group sizes ranging from 6-12. We have replicated the known effect of\nmorphine, and demonstrated comparable levels of variation to previous studies (Please see the\nfollowing references as examples: Badshah et al. 2024; Hou et al. 2023; Koek 2016; Sakae and\nMartin 2019; Tahsili-Fahadan et al., 2006). We therefore see no shortcomings in the data provided\nhere to make our conclusions.\nWe have amended the results to include non-significant p values:\n“Finally, because the rewarding properties of opioids limit their use in the clinic, we compared\nRPI-GLYT2-82 and morphine in a conditioned place preference (CPP) paradigm. Mice treated\nwith morphine (10 mg/kg, i.p.) exhibited a significant increase in preference for the paired\nchamber (p = 0.0287) whereas mice treated with RPI-GLYT2-82 (50 and 150 mg/kg, i.p.) showed\nno significant preference (p = 0.78 and p = 0.15, respectively) (Fig. 5j). Collectively, our data\nshow that RPI-GLYT2-82 provides a foundation for the development of a non-addictive\nmedication for neuropathic pain.”\n1- Badshah, Ismail, Neelum Gul Qazi, Maira Anwar, Bushra Shaukat, Muhammad Imran Khan, and Babar Murtaza. 2024.\nCarveol mitigates the development of the morphine anti-nociceptive tolerance, physical dependence, and conditioned\nplace preference in mice, Heliyon, 10, e27809.\n2- Hou, Xue-Fei, Ya-Bo Zhao, Yue-Xiong Yang, Jing Zhu, Li-Su Zhu, Lin Xu, and Qi-Xin Zhou. 2023. A morphine reward\ngeneralization mouse model based on conditioned place preference and aversion, Brain and Behavior, 13: e2970.\n3- Koek, W. 2016. Morphine-induced conditioned place preference and effects of morphine pre-exposure in adolescent\nand adult male C57BL/6J mice, Psychopharmacology (Berl), 233: 2015-24.\n4- Sakae, D. Y., and S. J. Martin. 2019. Formation of a morphine-conditioned place preference does not change the size\nof evoked potentials in the ventral hippocampus–nucleus accumbens projection, Scientific Reports, 9: 5206.\n5-Tahsili-Fahadan, Pouya, Noushin Yahyavi-Firouz-Abadi, Mohammad Ali Khoshnoodi, Rouzbeh Motiei-Langroudi, Seyed\nAmir Tahaei, Mohammad Hossein Ghahremani, and Ahmad Reza Dehpour. 2006. Agmatine Potentiates Morphine-Induced\nConditioned Place Preference in Mice: Modulation by Alpha(2)-Adrenoceptors, Neuropsychopharmacology, 31: 1722-32.\n7. The time course of the analgesic effect is very different from that of side effects. Analgesia peaks\nat about 2 – 3 hours after drug administration, respiratory depression and impaired rotarod\nperformance already at 0.5 hours. Can the authors come up with a reasonable explanation.\nWe thank the reviewer for this interesting comment. Our study demonstrates analgesic efficacy of\nRPI-GLYT2-82 that peaked 2-3 h after systemic administration (which was maximal at 50 mg/kg with\nsimilar analgesia between 50 mg/kg and 100 mg/kg, and a higher dose of 150 mg/kg produced no\nfurther analgesia). RPI-GLYT2-82 produced no side effects at our maximal analgesic dose of 100\nmg/kg tested in anti-allodynia mice models. We pushed the side effect testing to 2.5x this dose, at\n250 mg/kg, and observed side effects of sedation, decreased motor performance, and respiratory\ndepression. The side effects all coincide with each other at the 30 min timepoint, and all quickly\nrecover with a return to baseline by 60-90 min. This suggests that an acute disruption to the\nglycinergic system occurs with the administration of the high concentration (250 mg/kg) bolus of\nGlyT2 inhibitor, but that the effects of this disruption have dissipated by the timepoints that we see\nanalgesia (and are, importantly, not seen at analgesic doses of 50-100 mg/kg).\nWe theorise that, in chronic pain states, there is long-term disrupted function of the glycinergic\nsystem and that GlyT2 inhibitors such as RPI-GLYT2-82 produce their analgesic effects by\n“rebalancing” glycinergic function at the spinal cord. This takes on the order of 2+ hours to manifest\nas analgesia, as glycinergic function is returned to a more normal state.\nthe balance of glycine in brainstem regions that are important for sedation, motor control, and\nrespiration. This is likely to be via an indirect effect, resulting from GlyT2 inhibition in the brainstem\n– where GlyT2 is abundantly expressed – which elevates the local extracellular glycine levels and\nthereby enhances glycine receptor activation to suppress these functions.\nAn alternate explanation is that at the supramaximal dose of 250 mg/kg, 5HT2A receptors are\ntransiently inhibited, which causes the observed sedation in the 30–60-minute time frame. As the\neffective concentration diminishes with time, 5HT2A receptor occupancy and the sedative effects are\nreduced.\n8. Relative to pregabalin, the gold standard treatment, the analgesic efficacy of RPI-GLYT2-82 is\nmoderate. I like to suggest testing additional (higher) doses of RPI-GLYT2-82 to determine the\nmaximal analgesia achievable with a saturating dose of RPI-GLYT2-82.\nWe thank the reviewer for this comment. We observed that the 100 mg/kg dose of RPI-GLYT2-82\ngenerated very similar analgesia as 50 mg/kg which suggests that we have reached maximal\ninhibition at 50 mg/kg.\n9. The authors emphasize that a facilitation of glycinergic inhibition is analgesic in case of\nneuropathic pain but does not work for inflammatory pain. The literature on this question is\ncontroversial. I suggest that the authors test their compound also in an inflammatory model and\ndiscuss the relevant literature.\nWe thank the reviewer for pointing this out. We have previously performed this experiment for two\ninhibitors of GlyT2 that are more potent than RPI-GLYT2-82, ORG25543 (same binding site and\nmechanism of inhibition as presented in the manuscript) and oleoyl-D-lysine. They both had no\neffects on inflammatory pain or acute nociceptive pain thresholds (Wilson B., et al., 2022). This can\nbe explained by the fact that pathological changes in glycine neurotransmission induced in the\nneuropathic pain model do not manifest until at least two weeks post injury, whereas in the\ninflammatory pain model, testing is conducted on day 3. In both acute and inflammatory pain, there\nis insufficient time for the pathological features to develop, which accounts for the lack of efficacy of\nGlyT2 inhibitors in these models. We have more clearly addressed these points in the discussion\nsection of the revised manuscript to make the rational for our choice of pain models clearer for the\nreader.\nFurther, the GlyT2 inhibitors ORG25543 and oleoyl-D-lysine have shown no effects on\ninflammatory and acute nociceptive pain thresholds and therefore the observed analgesic effects\nare likely limited to neuropathic pain28.\nReviewer #3 (Remarks to the Author):\nThe work solved the high resolution cryo-EM structures of human GlyT2 in substrate-free, and\nglycine, reversible inhibitor RPI-GLYT2-82, pseudo-irreversible inhibitor ORG25543 bound states,\nalong with MD simulation and transport assay, providing prospects for non-opioid analgesics\ndevelopment. The analgesia effect of RPI-GLYT2-82 to mouse model of neuropathic pain is\ninspiring. It is surely a progress in the understanding of GlyTs substrate recognition and inhibition\nmechanism. Still some problems need to be addressed as described in the followings:\nMajor problems:\n1. The authors stated that “these data show that RPI-GLYT-82 is a non-competitive GlyT2 inhibitor\nthat binds with lower affinity than ORG25543. It is not a proper interpretation of the available\nexperiment results. The electrophysiological assay based on oocyte is not a propriate method for\n7\naffinity definition. In vitro assay based on purified protein is needed for binding affinity determination\nof RPI-GLYT-82 and ORG25543 to GlyT2.\nWe thank the reviewer for this comment and agree that the description “lower affinity” is not\nappropriate. We have revised the text to clarify that our data demonstrate lower potency of\ninhibition by RPI-GLYT2-82 compared with ORG25543, and not lower affinity.\nThis has been corrected throughout the text, for example here:\nTogether, these data show that RPI-GLYT-82 is a non-competitive GlyT2 inhibitor that binds with\nreduced potency compared to ORG25543.\nWe have tried to measure binding affinities of RPI-GLYT-82 and ORG25543 to purified GlyT2 using\nseveral biophysical methods including nanoDSF, Thermofluor assay, ITC, and GCI. Despite our best\nefforts we could not observe any significant difference between the apo and inhibitor bound protein.\nWe therefore modified the text to correctly reflect the potency difference of the two compounds. We\nhave included the negative results below:\nAffinity measurements of GlyT2 with inhibitors:\nnanoDSF (No stabilization effect is observed upon the addition of four different ligands)\nFigure 1. Thermal denaturation profiles of GlyT2 recorded by NanoDSF, Figure 2. First derivative of the NanoDSF 330/350 nm fluorescence ratio as\nmonitoring the fluorescence intensity ratio at 330/350 nm as a function of a function of temperature. No detectable shift in melting temperature (Tm)\ntemperature. No stabilization effect is observed upon the addition of four is observed between GlyT2 alone and samples containing four different\ndifferent ligands (100 μM). Fluorescence-based melting curves were ligands (100 μM). Derivative curves were plotted using the MoltenProt tool\nplotted using the MoltenProt tool (spc.embl-hamburg.de). (spc.embl-hamburg.de).\nLabelFree MST\nMedium MST power\nFigure 3. Normalized thermophoresis traces of GlyT2 (200nM, intrinsic Figure 4. Duplicated data showing concentration dependent change in signal\nfluorescence) incubated with increasing concentrations of control (AU) between GlyT2 (200 nM, intrinsic fluorescence) and control compound\ncompound (7.6 nM to 250 μM). Buffer contains 2% DMSO. Data was (7.6 nM to 250 μM). Buffer contains 2% DMSO, indicating detectable binding\nanalyzed and plotted using the ThermoAffinity tool (spc.embl-hamburg.de). under these conditions. Reported Kds are 42.6 μM and 66.8 μM. Control\ncompound is not soluble on buffer above 500 μM. These traces were done at\nmedium MST power, 15% excitation. Data was analyzed and plotted using\nthe ThermoAffinity too (spc.embl-hamburg.de).\nMedium MST power High MST power\nFigure 5. Normalized thermophoresis traces of GlyT2 (100 nM, labeled with Strep-Tactin XT DY-649) incubated with increasing concentrations of ORG25543 (3.1 nM\nto 100 μM). Data were analyzed and plotted using the ThermoAffinity tool (spc.embl-hamburg.de). No concentration-dependent change in fluorescence or\nthermophoretic behavior was observed, indicating the absence of detectable binding under these conditions. These traces were done at medium and high MST power.\nLabelled MST - Cys-based\nMedium MST power\nFigure 6. Normalized thermophoresis traces of GlyT2 (100 nM, labeled with Cys-\nbased label) incubated with increasing concentrations of ORG25543 (3.1 nM to 100\nμM). Data were analyzed and plotted using the ThermoAffinity tool (spc.embl-\nhamburg.de). No concentration-dependent change in fluorescence or thermophoretic\nbehavior was observed, indicating the absence of detectable binding under these\nconditions.\nGCI - TwinStrep Tag with GFP\nRapidWAVE GCI sensorgrams after reference subtraction (reference channel immobilized with GFP-\nTwinStrep). Red curves correspond to individual injections; I marks the start of analyte injection\n(association phase) and R marks the beginning of the dissociation phase. The black trace represents\nthe kinetic fit, which could not be reliably performed due to the absence of a measurable binding\nresponse.\nFigure 7. GlyT2-GFP-TwinStrep was immobilized on the sensor surface and Figure 8. RapidWAVE GCI sensorgrams after reference subtraction (reference\nsubjected to increasing pulse durations of 10 μM ORG25543. No specific binding channel immobilized with GFPTwinStrep). GlyT2-GFP-TwinStrep was immobilized\nresponse was detected beyond the bulk refractive index effect. on the sensor surface and subjected to increasing pulse durations of 10 μM RPI-\nGLYT2-82. No specific binding response was detected beyond the bulk refractive\nindex effect.\nchannel immobilized with GFPTwinStrep). GlyT2-GFP-TwinStrep was channel immobilized with GFPTwinStrep). GlyT2-GFP-TwinStrep was\nimmobilized on the sensor surface and subjected to increasing pulse durations immobilized on the sensor surface and subjected to increasing pulse durations\nof 10 μM control compound. No specific binding response was detected beyond of 200 μM ORG25582. The black trace represents the kinetic fit, which could\nthe bulk refractive index effect. not be reliably performed due to the absence of a measurable binding response.\nNo specific binding response was detected beyond the bulk refractive index\neffect.\nFigure 11. RapidWAVE GCI sensorgrams after reference subtraction\n(reference channel immobilized with GFPTwinStrep). GlyT2-GFP-TwinStrep\nwas immobilized on the sensor surface and subjected to increasing pulse\ndurations of 200 μM control compounds. The black trace represents the kinetic\nfit, which could not be reliably performed due to the absence of a measurable\nbinding response. No specific binding response was detected beyond the bulk\nrefractive index effect.\nClassic GCI sensorgrams after reference subtraction (reference channel immobilized with GFP-\nTwinStrep). GlyT2-GFP-TwinStrep was immobilized on the sensor surface and exposed to\nincreasing concentrations of ORG25543 Ranging from 312.5 nM to 2.5 μM. Red curves correspond\nto individual injections; I mark the start of analyte injection (association phase) and R marks the\nbeginning of the dissociation phase. The black trace represents the kinetic fit, which could not be\nreliably performed due to the absence of a measurable binding response.\nFigure 12. No specific binding response was detected beyond\nthe bulk refractive index effect.\nIsothermal Titration Calorimetry\nFigure 13. ITC thermograms for GlyT2 titrated into buffer (blue),\n500 nM ORG25543 (red), and 50 μM ORG25543 (yellow). The\nheat signals are comparable across all three conditions, indicating\nno detectable binding under these experimental settings. The\nobserved thermal effects likely originate from dilution or buffer\nmismatch rather than ligand interaction. Protein concentration\nwas 21 μM in all titrations. The buffer composition was 50 mM Tris\npH 7.5, 150 mM NaCl, 0.006% LMNG, 0.006% GDN, 0.0012%\nCHS, and 1% DMSO.\nGlyT2 (in GDN/LMNG/CHS) for the inhibitors. Despite extensive efforts, we were unable to obtain\nconclusive Kd values, except for a weak affinity detected with a control compound using MST\nLabelFree technology. Both compounds investigated in this study, ORG25543 and RPI-GLYT2-82,\nexhibit intrinsic fluorescence and strong absorption around 300 nm, which interfered with signal\ndetection in both MST LabelFree and nanoDSF assays. Furthermore, any potential stabilising effects\nof the ligands in nanoDSF might have been masked, most likely due to the stabilising effect of\nLMNG/GDN micelle composition surrounding the transporter.\nA positive control using glycine as the ligand was also tested with MST LabelFree technology;\nhowever, the resulting curves were noisy and indicated only weak binding. Given the small size of\nglycine (75 Da), MST may not be the most suitable technique for this control.\nWe also explored alternative methods, including GCI and ITC, but did not obtain measurable binding\nsignals. The detergent micelle environment likely masks or dampens the detectable interaction\nbetween GlyT2 and its ligands in these biophysical assays and limits our ability to detect GlyT2–\nligand interactions with these methods.\n2. More evidence is needed to identify cholesterol from cholesterol hemisuccinate in the model\nbuilding. As the molecules are structurally similar, and the cryo-EM map density surrounding the\ntransmembrane helixes is relatively with more chaos, the cryo-EM map itself is not a robust evidence\nin distinguishing cholesterol from cholesterol hemisuccinate. Besides, there is no obvious evidence\nsupporting the binding affinity differences between the different cholesterol or CHS binding sites. It\nis not reasonable that while some sites (like CHOL3’and CHOL7) bind cholesterol, some other sites\n(CHOL5 and CHOL6) bind CHS.\nWe appreciate the reviewer’s thoughtful comment and understand the concern regarding the\ndistinction between cholesterol and cholesterol hemisuccinate (CHS) in cryo-EM maps. While we\nrecognize the structural similarity between these molecules and the potential ambiguity in lower-\nresolution regions, we would like to clarify the basis for our assignments. Cholesterol is a sterol with\na hydroxyl group at C3, whereas CHS contains an additional hemisuccinate group linked via an ester\nbond to this hydroxyl. This extra group produces a distinct, well-resolved density in high-quality cryo-\nEM maps, making CHS distinguishable from cholesterol in favourable cases (please see the figure\nbelow). We have only modelled cholesterol or CHS molecules where the density unambiguously\nsupported the assignment (Exhibit 1). Furthermore, although sterol densities were present in all\nstructures, we only modelled them in higher-resolution maps with clear, unambiguous density.\nWe have re-evaluated the modelling of sterol molecules and decided to remove the CHOL5 (CHS)\nmodelled in substrate-free structure (marked with red box in Exhibit 1, below), however the same\nCHS is modelled in both inhibitor-bound states with high confidence (Exhibit 1, a and b). We have\naccordingly updated Figure 2 and Supplementary Figure 12.\ncorresponding non-proteinatious densities. a, ORG25543-bound hGlyT2Δ185 sterols (Chimera X level= 0.034).\nb, RPI-GLYT2-82-bound hGlyT2Δ185 sterols (Chimera X level= 0.024). c, Substrate-free hGlyT2Δ185 sterols\n(Chimera X level= 0.024).\nThis approach is consistent with many examples in the literature where CHS and cholesterol\nmolecules have been differentiated in membrane protein structures, particularly among SLC6\ntransporters. For instance, the latest hDAT study from the Eric Gouaux laboratory (Exhibit 2),\n(Srivastava, D.K., Navratna, V., Tosh, D.K. et al. Structure of the human dopamine transporter and\nmechanisms of inhibition. Nature 632, 672–677 (2024)) provides a clear precedent for this\ndistinction.\nExhibit 2 – Adopted from Srivastava, D.K., et al.\nStructure of the human dopamine transporter and\nmechanisms of inhibition. Nature 632, 672–677 (2024) –\nCHS and cholesterol molecules (as well as lipids) have\nbeen modelled in the density.\n[REDACTED]\nFurther, the differing occupancy of cholesterol and CHS at various sites is not unexpected. CHS is\na detergent-derived analogue present in our purification buffers, whereas cholesterol originates from\nthe cell membrane (gets extracted from the membrane together with the protein) and remains bound\nto the protein. We therefore assume that higher-affinity sites are preferentially occupied by\ncholesterol, which could not be displaced by CHS during purification, while lower-affinity or more\naccessible sites are more likely to be occupied by CHS, replacing cholesterol in the process.\n12\n3. The modulation function of cholesterol to GlyT2 is also need validation, especially CHOL3’. Is\nthere possible cross-talk exist between cholesterol and chemical modulators like ORG25543 or\nRPI-GLYT2-82?\nWe thank the reviewer for this question and would like to emphasise that we do not make any claims\nregarding the modulatory effects of the cholesterol or CHS molecules modelled in our structures.\nDetermining such effects would require a dedicated study involving, for example, targeted\nmutagenesis of sterol-binding residues, cholesterol depletion/repletion experiments, replacement\nwith cholesterol analogues, and functional assays to assess transporter activity. Our current\ninterpretation is based solely on structural observations: “these sterols are consistently present in all\nthree conformational states obtained here” (two inhibitor-bound outward-open states, a substrate-\nfree inward-open state, and a substrate-bound inward-occluded state), indicating that their role is\nlikely structural rather than regulatory and unlikely to modulate the inhibitory action of ORG25543 or\nRPI-GLYT2-82.\nWe have added a clearer statement in the revised manuscript indicating that we do not expect this\ncholesterol or other sterols to have modulatory functions (we have also elaborated on sterols\nbehaviour in MD simulations as requested by reviewer #5):\n“This cholesterol is embedded within the protein, at a methionine-rich site formed by TMs 3,\n9, 10 and 12 (Supplementary Fig. 12), in contrast to its alignment with the membrane in other SLC6\nstructures.\nWe further examined the stability of sterols in the inhibitor-bound GlyT2 structures and found\nthat most remained stably bound throughout the simulations (Supplementary Fig. 12d-l).\nHowever, the cholesterol molecule at the CHOL3’ site in the RPI-GLYT2-82-bound structure\nsampled multiple conformations while maintaining contact with the site, suggesting local\nflexibility in this region. Because this feature, along with other sterol molecules we modelled, is\nconsistently present in all hGlyT2Δ185 structures in three different conformational states, we\nsuggest that these sterols act primarily as important structural stabilisers of hGlyT2 rather than as\nregulatory elements. We did not observe any clear cholesterol-like densities at CHOL1 or CHOL4\nsites17.”\n4. The binding of sodium is different from GlyT1 structure. And the sodium ion binding in GlyT2 is\nalso altered in different structures. What is the sodium ion function in the glycine transport by GlyT2?\nTransport assay in buffer depleted of sodium is suggested to address the problem.\nBesides, while there is still sodium binding in Na1 site (as supported by MD simulation results in\nSupplementary figure 13), it is not reasonable to state that “consistent with ORG25543, and likely\nRPI-GLYT2-82, binding being Na+-independent”. On the contrary, sodium could participate in the\ninhibition process. More experiments are needed to address this question.\nWe thank the reviewer for this comment and recommended experiments. We would like to\nrespectfully clarify that the Na⁺ binding sites (Na1 and Na2) are conserved across all SLC6\ntransporters, including both GlyT1 and GlyT2. We have updated the sequence alignment of the SLC6\ntransporters where we have marked the residues coordinating the Na+ binding in these transporters\n(see supplementary Fig. 11, below) to clarify the conserved Na+ binding sites. The main distinction\nbetween GlyT1 and GlyT2 is that GlyT2, along with BGT1 and ATB⁰,⁺, contains a third Na⁺ binding\nsite that was not resolved in our density maps.\nhGlyT2 (UniProt Q9Y345), hGlyT1 (UniProt P48067), hATB0,+ (UniProt Q9UN76), hPROT\n(UniProt Q99884), hB0AT1 (UniProt Q695T7), hSERT (UniProt P31645) and hDAT (UniProt\nQ01959) were aligned. Transmembrane (TM) helices and extracellular loops (EL) 2 and 4 are\nindicated above the alignment. Indicated in black boxes are the residues contributing to sodium\nbinding in the conserved Na1 (circles) and Na2 (stars) sites. Side chain interactions are shown with a\nfilled circle/star, and backbone interactions are shown with an empty circle/star. Colouring was\ngenerated using Jalview 2.11.4.1.\nRegarding the different conformational states of GlyT2 and the Na⁺ binding sites, as also across all\nSLC6 transporters, we respectfully note that these conserved sites are maintained in both outward-\nand inward-facing conformations. What differs is their functional role: in the outward-facing state, the\nNa⁺ sites exhibit high affinity, allowing Na⁺ binding from the extracellular space to drive transport,\nwhereas in the inward-facing state, the site has a lower affinity (loose binding) to enable Na⁺ release\ninto the cytoplasm. Thus, weaker or “looser” binding in the inward-facing conformation is not\nunexpected but reflects the physiological transport cycle.\nRegarding the role of Na⁺ in glycine transport by GlyT2, we would like to clarify that GlyT2, like other\nSLC6 transporters, functions as a secondary active transporter that relies on the transmembrane\nNa⁺ and Cl⁻ gradients to drive substrate transport. Binding and occlusion of the extracellular\nsubstrate glycine, depends on the Na+ (and Cl− in eukaryotic NSS) gradient. Binding of Na+ and Cl–\n14\nions and substrate is followed by a rearrangement of the transporter to an inward-facing state and\nsubsequent intracellular opening and release of ions and substrate glycine. We elaborate on the role\nof Na+ and Cl– ions and the stoichiometry in the introduction. Please see:\nGlyT2 (encoded by SLC6A5) belongs to the solute carrier 6 (SLC6) subfamily of secondary active\nneurotransmitter/sodium symporters (NSSs) 9,10. NSSs are membrane proteins that operate via an\nalternating access mechanism, whereby transmembrane helices undergo conformational\nrearrangements during the transport cycle that alternately expose the substrate-binding site to\neach side of the membrane. In eukaryotic NSSs, binding of extracellular substrate and initiation\nof the transport cycle is dependent on a Na+ and Cl− gradient, such that GlyT2 co-transports three\nNa+ ions and one Cl− ion with each glycine11-13. Upon ion and substrate binding, access to the\nextracellular environment is occluded as the transporter transitions from an outward-facing to an\ninward-facing conformation, followed by release of solutes.\nConsidering the critical role of Na+ for transprot, we respectfully note that it is not possible to perform\na transport assay in Na⁺-“depleted buffer”, as Na⁺ is an essential driver of the transport and co-\ntransports with the substrate. However, inspired by this comment we have performed a sodium\ndependence experiment, where inhibitor dose responses were performed as described for the rest\nof electrophysiological experiments here but with higher (300 μM) glycine concentration to ensure\nmeasurable transport currents were obtained at low sodium concentrations. Choline chloride was\nused as a substitute salt when altering sodium concentrations. We present the results in the update\nfigure 1:\nFig. 1| RPI-GLYT2-82 is a non-competitive inhibitor of hGlyT2. a Glycine concentration-dependent\ncurrents in the presence of different concentrations of RPI-GLYT2-82 showing non-competitive inhibition.\nThere was no statistically significant difference in the glycine EC values across all four conditions (p = 0.49)\n50\nbut there was a statistically significant reduction in I between 0 nM RPI-GLYT2-82 (mean I ± SEM=\nmax max\n1.07 ± 0.01) and the I at 600 nM RPI-GLYT2-82 (mean I ± SEM= 0.76 ± 0.04) (p < 0.0001 ) and also\nmax max\nthe I at 1.8 µM RPI-GLYT2-82 (mean I ± SEM= 0.39 ± 0.02) (p < 0.0001). Data analysed using nonlinear\nmax max\nregression (see Methods). b RPI-GLYT2-82 concentration-dependent inhibition of glycine transport currents.\nCurrent-responses were normalised to currents generated by 30 µM glycine. The difference between the\nhGlyT2WT IC = 554 nM (95% CI: 412 to 747 nM) and the hGlyT2Δ185 IC = 683 nM (95% CI: 370 to 996\n50 50\nnM) is not statistically significant (p = 0.45) c ORG25543 concentration-dependent inhibition of glycine\ntransport normalised to currents generated by 30 µM glycine. The difference between the hGlyT2WT IC =\n50\n11.4 nM (95% CI: 8.0 to 16.4 nM) and the hGlyT2Δ185 IC = 13.1 nM (95% CI: 9.4 to 16.8 nM) is not\n50\nstatistically significantly (p = 0.60). d RPI-GLYT2-82 is reversible and washes out within 5-minutes. Data\nfitted to a one-phase decay curve (WT not fitted due to full reversal before the first time point). e ORG25543\nhas prolonged inhibitory effects with minimal restoration of glycine-induced currents 30-minutes post-\nORG25543. Results normalised to currents generated by 30 µM glycine. f RPI-GLYT2-82 inhibits GlyT2 with\nminimal effect on GlyT1 showing only 6% inhibition at 10 µM (p < 0.0001). Mean inhibition ± SEM from 10\nµM RPI-GLYT2-82 is plotted. g RPI-GLYT2-82 is more potent at lower Na+ concentrations. The IC at 20\n50\nmM Na+ (IC = 159 nM (95% CI: 81 to 238 nM)) is significantly lower than that at 100 mM Na+ (IC = 279\n50 50\nnM (95% CI: 182 to 376 nM)) (p = 0.04). h ORG25543 potency does not change with decreasing Na+\nconcentration. The IC at 20 mM Na+ (IC = 18 nM (95% CI: 6.8 to 29 nM)) is not significantly different\n50 50\nthan that at 100 mM Na+ (IC = 17.6 nM (95% CI: 5.5 to 30 nM)) (p > 0.9999). i Normalised glycine-\n50\ndependent transport currents in hGlyT2WT- and hGlyT2Δ185-expressing Xenopus laevis oocytes showing\ncomparable transport activity in the two constructs (p = 0.78). EC , IC , washout and competition\n50 50\nexperiments performed with n = 5 cells, from at least two different batches of oocytes. Error bars represent\nSD.\nWe have updated the text to include this experiment:\nInterestingly, while the inhibition of transport by both compounds appears to be Na⁺-independent,\nRPI-GLYT2-82 exhibits slightly increased potency under lower Na⁺ concentrations, whereas\ninhibition by ORG25543 remains unaffected (Fig. 1g,h).\nAnd later in the results where we describe the binding pockets, we elaborate on this observation:\nThe increased potency of RPI-GLYT2-82 at lower Na⁺ concentrations likely reflects reduced\ncompetition of the compound with Na⁺ for N213 coordination, under partially depleted Na⁺\nconditions. In contrast, the potency of ORG25543 remains unchanged, likely due to its inherently\nhigh potency that renders it insensitive to local Na⁺ occupancy at the Na1 site (Fig. 1g,h).\nWhile Na⁺ clearly plays a central role in the transport mechanism, as shown by this expremint, the\nbinding of ORG25543 and RPI-GLYT2-82 is not dependent on Na⁺ occupancy. We further do not\nobserve Na+ density in the more buried Na2 or Na3 sites even in our highest resolution (2.49 Å)\nORG25543-bound GlyT2. As the transporter is locked in the outward-open state by the inhibitors,\nthe Na1 site is naturally exposed to the solvent and observing the ion density at this electrostatically\nfavourable, conserved binding pocket, is very common and dose not necessarily mean that the\ninhibition is dependent on Na+. We would like to emphasise further that the conserved Na1 binding\nsite is distorted in both inhibitor-bound states in the same way, where we note:\n“one of the main Na+coordinating residues required for the stable octahedral ion coordination\ngeometry, N213 (TM1b), had rotated away from Na1 to interact with the respective inhibitor\n(Fig. 3d, e).”\n5. In MD simulation results, RPI-GLYT2-82 has higher possibility to slip in the central vestibule of\nGlyT2. Is there additional non-protein density appear in this site? Moreover, transport activity\nmeasurements of different mutations in the allosteric site and central vestibule are suggested to\nverify roles of the residues in RPI-GLYT2-82 inhibition.\nWe thank the reviewer for this remark and note that there is no additional non-protein density in the\nvestibule of GlyT2 in the RPI-GLYT2-82-bound structure. The ‘slip’ of RPI-GLYT2-82 into the\nvestibule, which occurs on a nanosecond timescale, simply shows that it is not as tightly coordinated\n16\nin the binding site as ORG25543, and this movement likely corresponds to a first transient\nintermediate during inhibitor binding/unbinding dynamics. We note that MD simulations are\nstochastic and sample a range of conformational states, in the uptake, binding and release\npathways, and interpreting these transient pathways is beyond the scope of this manuscript. Instead,\nthe goal of the MD simulations was to sample the interaction of GlyT2 and inhibitors over ensembles\nconsistent with density observed in the cryo-EM structures. Given the pseudo-irreversible character\nof ORG25543, we were not attempting to sample the entire pathway of inhibitor binding and\nunbinding.\nAs recommended by the reviewer we have performed the suggested mutagenesis studies of the\nallosteric binding site for RPI-GLYT2-82 which support our expectation that W215 and D633 are\ncritical residues in the binding of RPI-GLYT2-82, similar to ORG25543. The “maximum” inhibition\n(inhibition at 10 μM) of GlyT2 by RPI-GLYT2-82 is significantly reduced (p < 0.0001; One-Way\nANOVA - Tukey’s Multiple Comparisons) with the W215F and D633E mutations. Here, with IC >\n50\n10 μM, there is a > 18-fold shift in potency. As expected, glycine-induced transport currents fully\nrecover almost instantly to pre-inhibition levels.\nAs the central vestibule contains the substrate-binding site, mutations in this region are most likely\nto abolish transport activity entirely. Such an outcome, while predictable, would preclude obtaining\nmechanistically informative data from these variants.\nWe have updated the main figure 4 to present the mutagenesis results:\nFig. 4| Glycine binds to the central site of hGlyT2, whereas ORG25543 and RPI-GLYT2-82 bind to an\nallosteric pocket. a Cryo-EM density of glycine (map contour level = 0.042 in ChimeraX). b Glycine (cyan)\nbound to the central site of hGlyT2. Interacting residues shown in sticks, H-bonds and ionic interactions shown\nas dashed grey lines. Na+ modelled into Na2 shown as a green circle, modelled Cl– shown as a purple sphere,\nand modelled water molecule shown as a red circle. c Glycine binds in distinct poses in GlyT2 and GlyT1.\nGlycine-bound hGlyT2 (pink) overlayed with glycine-bound GlyT1 (PDB ID 8WFI, grey). The amine group\nof glycine (purple) forms a water-mediated interaction with GlyT1 Y62, the glycine (cyan) amino group forms\n17\na H-bond with GlyT2 S479. Interacting residues shown as sticks (hGlyT2 = pink, hGlyT1 = black). d Cryo-\nEM density of ORG25543 (map contour level = 0.055 in ChimeraX). e Cryo-EM density of RPI-GLYT2-82\n(map contour level = 0.04 in ChimeraX). f ORG25543 (orange) bound to hGlyT2Δ185 (green). Interacting\nresidues shown as sticks and H-bonds and ionic interactions shown as grey dashed lines. N213 is likely\ninvolved in a weak hydrogen bond interaction with the polarised C–H of the methyl group attached to\npositively charged ammonium nitrogen of R4 substituent. g RPI-GLYT2-82 (purple) bound to hGlyT2Δ185\n(blue). Interacting residues shown as sticks, and H-bonds and ionic interactions shown as grey dashed lines. h\nChemical structure of ORG25543 and key chemical substituents R1 (blue), R2 (orange), R3 (green), and R4\n(yellow) outlined. i Chemical structure of RPI-GLYT2-82 and key chemical substituents (R1–R4) outlined. j\nORG25543 concentration-dependant inhibition of glycine transport currents of WT hGlyT2, and mutants\nW215F (red) and D633E (blue). Data normalised to transport currents generated by the EC concentration of\n50\nglycine for each transporter. hGlyT2WT IC = 11.4 nM, hGlyT2W215F IC = 32.2 nM, and hGlyT2D633E IC =\n50 50 50\n164 nM. k Reversibility of ORG25543 inhibition of glycine transport currents mediated by hGlyT2WT,\nhGlyT2W215F, and hGlyT2D633E mutants. l RPI-GLYT2-82 dose-response curves of WT hGlyT2, and mutants\nW215F (red) and D633E (blue). Data normalised to glycine transport currents generated by the EC\n50\nconcentration of glycine for each transporter. hGlyT2WT RPI-GLYT2-82 IC = 554 nM. The IC values for\n50 50\nhGlyT2W215F and hGlyT2D633E are greater than the maximal tested dose (> 10 µM). m RPI-GLYT2-82 is a\nreversible inhibitor of glycine transport by hGlyT2WT, hGlyT2W215F, and hGlyT2D633E mutants. Experiments\nfor j - m performed with n = 5 cells from at least two different batches of oocytes with error bars representing\nSD.\n6. The transport process or mechanism of GlyT2 need to be investigated in detail so that the\ninhibitory effect upon RPI-GLYT2-82 and ORG25543 binding would be easier to understand.\nObviously, glycine binding cannot trigger the conformational change from inward to outward. What\nare the possible factors that trigger conformational changes physiologically?\nWe thank the reviewer for this interesting question. The conformational cycle of GlyT2, like other\nSLC6 family members, follows the alternate access mechanism and is driven primarily by the\ncoupled transport of sodium and chloride ions with the substrate glycine. In this model, only one side\nof the binding site is accessible at a time, preventing ion and substrate leakage.\nIn the outward-facing conformation, sodium ions (and chloride ions, as required by eukaryotic SLC6\nfamily members) bind to their respective sites within the transporter from the extracellular side. The\nbinding of these driving ions creates a high-affinity environment for substrate binding, enabling\nglycine to subsequently bind. Once sodium, chloride, and glycine are all bound, the transporter\nundergoes a conformational change to the occluded state, in which the binding sites are sealed off\nfrom both sides of the membrane.\nFrom this occluded state, the transporter transitions to the inward-facing conformation, exposing the\nbinding sites to the cytoplasmic side. Here, the co-transported Na+ ions, glycine, and Cl– ion are\nreleased sequentially into the cytoplasm. This release destabilizes the inward-facing state, allowing\nthe transporter to undergo another conformational change and ultimately return to the outward-facing\nstate. Re-binding of sodium and chloride ions from the extracellular space stabilizes the outward-\nopen state and resets the transporter for another round of glycine uptake.\nIn details:\nIn NSS transporters, “two ligand-dependent networks” cooperate to close the extracellular pathway\nand allow the cytoplasmic pathway to open. 1 – One of these networks centers on the interactions\nof the substrate carboxyl group with a tyrosine in TM3 (Y287 in GlyT2) and with Na+ bound at the\nNa1 site. Mutation of this tyrosine residue in the bacterial homologue LeuT blocks the ability of the\nbound substrate to initiate the open-out to open-in conformational change that allows substrate and\nNa+ to dissociate to the cytoplasm. 2– The other network involves formation of the salt bridge at\nextracellular gate of the pathway between TM1 and TM10 (R216–D633 in GlyT2). Formation of this\nnetwork is facilitated by Cl− binding and depends on a conserved glutamine residue in the Cl− binding\nsite (Q473 in GlyT2). The interaction between Cl− and Q473 in GlyT2, disengages R216 so that it\ncan interact with D633 to close the extracellular pathway (ref.1,2). Glycine transporters, including\nGlyT2, are characterised by tight stoichiometric coupling of Na+, substrate, and Cl− transport (ref.\n18\n3). Therefore, we would expect that only when Na+, Cl−, and substrate are bound would their\ncombined conformational effects close the extracellular pathway and allow the cytoplasmic pathway\nof GlyT2 to fully open. The effect of Cl− binding, together with the influence of substrate binding on\nconformation, further stabilizes the closed extracellular pathway and facilitates opening of the\ncytoplasmic pathway to release substrate and ions to the cytoplasm.\nThe conserved leucine residue on TM1 (L211 in GlyT2, L25 in LeuT) has been shown to play a key\nrole in the inward-to-outward transition. In the absence of ions (Na+ and Cl–) and substrate, this\nleucine occupies the binding pocket, acting as a gatekeeper that prevents ion leak during the return\nto the outward-facing state (ref. 4). When the leucine side chain relocates out of the binding pocket,\nit creates space for ions binding. Binding of Na⁺ at the Na1 and Na2 sites, together with chloride\nbinding, stabilizes the outward-open conformation and primes the transporter for glycine binding and\nforward transport.\nBoth allosteric inhibitors of GlyT2, ORG25543 and RPI-GLYT2-82, “lock” the transporter in the\noutward-open state through two complementary mechanisms: (1) disrupting the extracellular gate\nby breaking the salt bridge involving R216 and D633, thereby preventing opening of the cytoplasmic\npathway and transition toward the inward-facing, and (2) constraining the conserved leucine residue\n(L211) outside of the substrate pocket, which further locks GlyT2 in the outward-open\nconformation (Because this residue serves as a critical gatekeeper for sodium entry and substrate binding,\nits immobilization locks GlyT2 in the outward-open state and prevents the conformational cycling\nrequired for transport). Together, these mechanisms explain how small-molecule binding\nallosterically arrests GlyT2 in a non-transporting state, thereby inhibiting glycine uptake.\nInspired by this question, we have added a detailed discussion of the mechanism of transport by\nGlyT2 (and other NSS) and inhibition by the allosteric inhibitors in the revised manuscript:\nIn NSS transporters, two ligand-dependent networks coordinate closure of the extracellular\npathway and opening of the cytoplasmic one. The first involves the substrate carboxyl group\ninteracting with a tyrosine in TM3 (Y287 in GlyT2) and Na⁺ at the Na1 site; mutation of this\ntyrosine in the bacterial homologue LeuT prevents the substrate-induced outward-to-inward\ntransition53. The second network forms a salt bridge between TM1 and TM10 (R216–D633 in\nGlyT2), stabilized by Cl⁻ binding through a conserved glutamine residue in the Cl− binding site\n(Q473 in GlyT2). The interaction between Cl− and Q473 in GlyT2, releases R216 to engage D633,\nsealing the extracellular gate54,55. In GlyT2, transport of Na⁺, Cl⁻, and glycine is tightly coupled,\nand only their combined binding drives full closure of the extracellular pathway and opening of\nthe cytoplasmic side where the ions and substrate are released56. The conserved leucine residue\non TM1 (L211 in GlyT2, L25 in LeuT) plays a key role in the inward-to-outward transition. The\nresidue L211 acts as a gatekeeper, occupying the substrate pocket in the absence of ions to prevent\nleak during the return to the outward-facing state57 and vacating the pocket upon Na⁺ and Cl⁻\nbinding to prime the transporter for glycine binding and forward transport.\nThe allosteric inhibitors ORG25543 and RPI-GLYT2-82 lock GlyT2 in the outward-open state\nthrough two complementary mechanisms: (1) disrupting the extracellular gate by breaking the\nsalt bridge involving R216 and D633, thereby preventing opening of the cytoplasmic pathway\nand transition toward the inward-facing state, and (2) constraining the conserved leucine residue\n(L211) outside of the substrate pocket, which further locks GlyT2 in the outward-open\nconformation (Fig. 4f,g, and 6). Immobilization of the critical gatekeeper residues blocks\nconformational cycling and traps GlyT2 in a non-transporting state, thereby inhibiting glycine\ntransport.\n19\n1- A. Ben-Yona, A. Bendahan, B. I. Kanner, A glutamine residue conserved in the neurotransmitter:sodium:symporters is\nessential for the interaction of chloride with the GABA transporter GAT-1. J. Biol. Chem. 286, 2826–2833 (2011),\n2- Zhang YW, Uchendu S, Leone V, Bradshaw RT, Sangwa N, Forrest LR, Rudnick G. Chloride-dependent conformational\nchanges in the GlyT1 glycine transporter. PNAS. 2021 118(10):e2017431118.\n3- M. J. Roux, S. Supplisson, Neuronal and glial glycine transporters have different stoichiometries. Neuron 25, 373–383\n(2000)\n4- Malinauskaite, L., Said, S., Sahin, C. et al. A conserved leucine occupies the empty substrate site of LeuT in the Na+-\nfree return state. Nat Commun 7, 11673 (2016)\nSome minor problems:\n1. The rationale for Cl- binding is needed. Structural alignment with GlyT1 would make it more\npersuasive to build Cl-. What is the possible function of Cl- binding? Is it a substrate or regulatory\nfactor for GlyT2?\nWe thank the reviewer for this remark and would like to elaborate that similar to Na+, Cl⁻ gradients\ndrive substrate transport in eukaryotic SLC6 transporters as in GlyT2. Binding and occlusion of the\nextracellular substrate glycine, depends on the Na+ and Cl− gradient in GlyT2, which is a secondary\nactive transporter. Please see also our response to point 4, page 13.\nAs recommended, we have added Supplementary Fig. 14 as a structural alignment of the chloride\nbinding sites of GlyT2 and GlyT1 (comparison made using similar conformations). Comparing GlyT1\nand GlyT2 coordination, we see that chloride ion in both protein is coordinated by similar conserved\nresidues.\nSupplementary Fig. 14| Chloride coordination in GlyT2 and GlyT1. a Substrate‑free hGlyT2Δ185(purple)\nand iclepertin-bound GlyT1 (PDB ID 9J8D) (yellow) in the inward-open state, b Glycine-bound hGlyT2Δ185\n(pink) and glycine bound GlyT1 (PDB ID 8WFI) (red) in an inward-occluded state, and c ORG25543‑bound\nhGlyT2Δ185 (green), RPI-GLYT2-82-bound hGlyT2Δ185 (blue) and SSR504734-bound GlyT1 (PDB ID 8WFK)\nin outward‑open conformations are overlayed, showing similar coordination of chloride ion across the three\nconformations. GlyT1 chloride ions are shown in dark green and GlyT2 chloride ions are shown in lighter\ngreen. In c, the chloride ion of RPI-GLYT2-82-bound hGlyT2Δ185 is shown in the lightest green.\nTransmembrane helices are labelled.\nWe know from previous studies that a Cl– ion is co-transported with the substrate glycine (references\n1-3 below). Observing the ion density at the electrostatically favourable, conserved binding pocket,\nis very common, even in the inward-open states of SLC6 transporters. Previous examples are\nnoradrenaline transporter (NET) and GABA transporter 1 (GAT1), serotonin transporter (SERT),\nand recently published GABA transporter 3 (GAT3) structures in the inward-open substrate-free states\nwhere the authors have observed a clear Cl– density and modelled the ion (references 4–7, below)\n– similar to our GlyT2 inward-open substrate-free state.\nPublished Cl–-bound inward-open substrate-free states of GAT1, NET, SERT and GAT3 (references\n4–7) are:\nProtein SLC PDB ID Reference Resolution\nGAT1 SLC6A1 7Y7V https://doi.org/10.1038/s41594-023-00983-z 2.2 Å\nNET SLC6A2 8HFE https://doi.org/10.1038/s41586-024-07719-z 2.5 Å\nGAT3 SLC6A11 9CP5 https://doi.org/10.1038/s41467-025-59066-w 3.5 Å\n1- Roux MJ, Supplisson S. 2000. Neuronal and glial glycine transporters have different stoichiometries. Neuron. 25(2):373-383.\n2- Le Guellec B, Rousseau F, Bied M, Supplisson S. 2022. Flux coupling, not specificity, shapes the transport and phylogeny of SLC6\nglycine transporters. Proc Natl Acad Sci U S A.;119(41):e2205874119.\n3- Erdem FA, Ilic M, Koppensteiner P, Gołacki J, Lubec G, Freissmuth M, Sandtner W. 2019. A comparison of the transport kinetics of\nglycine transporter 1 and glycine transporter 2. J Gen Physiol. 151(8):1035-1050.\n4- Zhu, A., Huang, J., Kong, F. et al. 2023. Molecular basis for substrate recognition and transport of human GABA transporter GAT1. Nat\nStruct Mol Biol 30, 1012–1022\n5- Tan, J., Xiao, Y., Kong, F. et al. 2024. Molecular basis of human noradrenaline transporter reuptake and inhibition. Nature 632, 921–929\n6- Dongxue Yang, Eric Gouaux, 2021. Illumination of serotonin transporter mechanism and role of the allosteric site.Sci. Adv.7, eabl3857\n7. Yadav, R., Han, G.W. & Gati, C. Molecular basis of human GABA transporter 3 inhibition.Nat Commun 16, 3830 (2025).\nhttps://doi.org/10.1038/s41467-025-59066-w\n2. In glycine-bound structure, although the current glycine conformation fits well to the density,\nthe cryo-EM map is insufficient for determination of the amino group orientation. The related statements\nof “…whereas in GlyT1, it adopts a different orientation to interact with the backbone of TM6\nresidues.” should be deemphasized.\nWe have deemphasized the phrasing of this interaction in the text as recommended:\nIn hGlyT2, the amino group of glycine appears to form a hydrogen bond with the side-chain\nhydroxyl of S479 (unwound region of TM6) and is further coordinated….\nWe would like to draw attention to Exhibit 2, which shows the density at 5.5 r.m.s.d., clearly following\nthe orientation of the glycine amino group in GlyT2 (green). The overlay with the GlyT1-bound\nsubstrate (blue, PDB ID 8wfi) illustrates that the substrate adopts a distinct pose in GlyT1. However,\nwe cannot be certain whether the GlyT2 state presented here represents a transitional conformation.\nExhibit 2 – Overlay of glycine-bound GlyT2 (green) and\nglycine-bound GlyT1 (dark blue, PDB ID 8wfi). Map\ncounter level: 5.5 r.m.s.d.\n3. What could be the possible reasons for the current recovery larger than 100% of D633E in fig.\n4k?\nWe thank the reviewer for pointing this out. The molecular gate is an intrinsic and necessary part of\nthe transport cycle. It is highly conserved with SERT being a rare instance in SLC6 transporters that\nhas a Glu instead of an Asp in this position. We speculate that there is cell-attributed drift in currents\nover a prolonged period of the electrophysiology recording in cells expressing this mutant-\ntransporter. Of note, one cell was particularly affected at time points 25 and 30 mins, contributing to\nthe large variance. Nevertheless, the trend of full-transport recovery is consistent across all cells.\n21\n4. The glycine binding site is far from the disulfide bond in EL2. What is the possible relation of\ndisulfide bond reduction and glycine binding?\nWe thank the reviewer for this interesting question. We do not believe that reduction of this disulfide\nbridge influences glycine binding. However, this was an intriguing observation, as we previously\nnoted the same phenomenon in the GABA-bound GAT3 structure (unpublished; BioRxiv\ndoi.org/10.1101/2025.03.27.645797). We also examined several other substrate-bound SLC6\ntransporters, including GABA-bound hGAT1 (PDB 7Y7W, EMDB 33672), glycine-bound hGlyT1\n(PDB 8WFI, EMDB 37492), and dopamine-bound hDAT (PDB 8Y2D, EMDB 38851), reported by\nother groups, and found no continuous cryo-EM density indicative of a formed disulfide bridge at the\nrecommended contour levels. This suggests that the disulfide bridge may also be reduced in other\nsubstrate-bound SLC6 transporters. This is also an effort to encourage the community to pay closer\nattention to the density during model building.\n5. The selectivity of RPI-GLYT2-82 to GlyT2 need more validation. What could be the possible\nreasons for absence of other SLC6s in the Off-target selectivity screening? Comparison with other\nSLC6 family proteins is suggested.\nWe agree with the reviewer and have now included a GlyT1 selectivity test. We found that the\ninhibitor does not act on GlyT1, showing only ~ 6% inhibition at a 10 µM concentration. We have\nrevised Fig. 1 to include this result. Please see our response to Major point #4 where we present the\nrevised figure 1 in full. Here the respective panel:\nFig. 1f RPI-GLYT2-82 inhibits GlyT2 but has minimal effect on GlyT1\nshowing only 6% inhibition at 10 µM (p < 0.0001). Mean inhibition ± SEM\nfrom 10 µM RPI-GLYT2-82 is plotted.\nWe performed the experiments on SLC6 members NET, SERT and DAT – For convenience, we have\ncut the full tables to only show the relevant data here, but the full tables and experimental procedure\nare presented in Supplementary data 1, Exhibits 10 and 11. In brief the selectivity screen shows:\nNET – 1.3% inhibition at 10 µM, DAT – 22% inhibition at 10 µM (both n=2), and SERT – IC for\n50\ninhibition of 4.7 µM.\nExhibit 10. RPI-GLYT2-82 selectivity screening. RPI-GLYT2-82 was tested at 1 × 10-5 M.\nCompound binding was calculated as a % inhibition of the binding of a ligand specific for each\ntarget (upper graph). Compound enzyme inhibition effect was calculated as a % inhibition of\ncontrol enzyme activity (bottom graph).\nReviewer #5 (Remarks to the Author):\nIn their work, Cantwell Chater and colleagues develop a reversible inhibitor of the neuronal glycine\ntransporter GlyT2 that provides analgesia in mouse models of neuropathic pain. To elucidate its\nmechanism of action, the authors resolved the structure of human GlyT2 for the first time alone and\nin complex with glycine and two different inhibitors, including the newly synthetized one.\nElectrophysiology and structural insights, aided by MD simulations, showed that the newly\nsynthetized compound acts as a reversible allosteric inhibitor that traps the transporter in an outward\nfacing conformation. Because of its relatively low affinity, the inhibitor shows no on-target side\neffects, making it a very promising therapeutical target to treat chronic neuropathic pain.\nI believe this study to be highly relevant for the field of neurotransmitter transporters. First, by\ncapturing GlyT2 at different conformations, including the one bound to its endogenous ligand, the\n23\nauthors set the ground for further mechanistic understanding of its transport cycle. More importantly,\nthe rational design of a new promising inhibitor of GlyT2 may lay the foundation for further\ndevelopment of non-opioid pain therapeutics.\nThe conclusions of the study are sound and supported by a vast array of different methods, including\nstructural and computational techniques, electrophysiology and animal models.\nThe manuscript is clear and well written; I found it quite easy to follow through the results although\nmost techniques used are outside my field of expertise.\nComments/suggestions:\n- Modulation of GlyT2 by cholesterol seems to be an important aspect of its biological function.\nAlthough the authors briefly describe the sterol densities found in their structures and even assess\ntheir stability and interactions with MD simulations (Supplementary Fig. 11), this is, in my opinion,\nnot emphasized enough in the main text. I would suggest to include a couple of sentences in the\nsecond section of the results summarizing the behavior of sterols at the proposed sites in MD\nsimulations (Supplementary Fig. 11d-i). I would even suggest changing the title of the second section\nof the results to “hGlyT2 adopts a LeuT fold stabilized by lipids”.\nWe thank the reviewer for this remark and agree that cholesterol plays an important role in regulation\nof GlyT2 and other SLC6 transporters. However, the sterols that were able to unambigously model\nin the cryo-EM maps, are present in all the three different conformational states of GlyT2, bound to\nsubstarte, inhibitors, and in substrate-free state. Therefore, we belive that these cholesterol/CHS\nmolecules play a structural part, rather than a functional role.\nRegarding the MD simulations, we note that cholesterol molecules are not restrained during\nsimulations and are free to diffuse within the membrane, including into and out of their binding sites.\nWe find that all four sterols identified in the ORG25543-bound GlyT2 structure remain in their binding\nsites throughout the combined 3000 ns of MD simulation. Regarding the sterols identified from the\nRPI-GLYT2-82-bound cryo-EM structure, CHOL2 (cholesterol) and CHOL5 (CHS) remain bound at\ntheir respective sites throughout the combined replicate simulations. The cholesterol in the CHOL3’\nsite remains in contact with the site, but does not retain its original cryo-EM binding pose, instead\nsamples a range of conformations within the CHOL3’ site. We speculate that cholesterol within the\nCHOL3’ site may be able to adopt multiple orientations, able to adapt to the local lipid\nenvironment. As recommended, we have expanded the main text to discuss role of cholesterol\ndynamics in more detail:\nWe further examined the stability of sterols in the inhibitor-bound GlyT2 structures and found\nthat most remained stably bound throughout the simulations (Supplementary Fig. 12d-l).\nHowever, the cholesterol molecule at the CHOL3’ site in the RPI-GLYT2-82-bound structure\nsampled multiple conformations while maintaining contact with the site, suggesting local\nflexibility in this region. Because this feature, along with other sterol molecules we modelled, is\nconsistently present in all hGlyT2Δ185 structures in three different conformational states, we\nsuggest that these sterols act primarily as important structural stabilisers of hGlyT2 rather than as\nregulatory elements. We did not observe any clear cholesterol-like densities at CHOL1 or CHOL4\nsites17.”\n- In Supplementary Fig. 15, RMSD values show that the backbone of the protein takes some time to\nequilibrate. RMSD of heavy atoms of the inhibitors also show fluctuations throughout the simulations.\nHowever, the authors perform MM-PBSA only on the first 50 ns of the trajectories, where the protein\nhas clearly not fully equilibrated. To me, it is not surprising that the MM-PBSA results agree with the\nstructural data if the initial structure did not have the time to relax. I strongly recommend to perform\nthe same MM-PBSA analysis using the full 500 ns trajectories. In case this drastically changes the\n24\nresults, more analysis might be needed, as this could suggest the existence of several different\nbinding modes of the ligands in the pocket.\nWe thank the reviwer for this remark and would like to point out that the purpose of the MM-PBSA\ncalculations was to “calculate the enthalpic contribution of each protein residue within the cryo-EM\nbinding site” to the protein/ligand interactions, to determine which interactions stabilised or\ndestabilised ligand binding. For this reason, the MM-PBSA was calculated on the first 50 ns of\nsimulation, during which each ligand was fully contained within its cryo-EM density envelope, when\nthere were minimal transient changes in the binding mode of RPI-GLYT2-82 and ORG25543\n(Supporting Figure 19). These transient changes in ligand RMSD are also noted by the Reviewer.\nTo further clarify, we would like to point out that the protein backbone structures, in fact, did have\nsufficient time to relax in the MD equilibration protocol described in the Methods section. Protein\nbackbone RMSD plateaued after the first 20 ns of simulation, then fluctuated around the mean of\n2.83+/0.44 Å with ORG25543 bound and 2.23 +/- 0.39 Å with RPI-GLYT2-82 bound, for the\nremaining 480 ns of simulation. This is well within thermal noise of the protein at 300 K\n(doi:10.1002/prot.340220308).\nWhile MM-PBSA remains a powerful “but theoretical tool”, our cryo-EM maps unambiguously depict\nthe ligands pose, making that the most reliable anchor for our mechanistic interpretation. Therefore,\nwhile we respect the suggestion, we strongly consider our cryo-EM data to provide a definitive\nexperimental foundation for how these inhibitors bind, which appropriately underpins the\ncomputational analysis.\nMinor comments:\n- At the beginning of page 17 in the discussion section, when they mention the efficacy of pregabalin,\nthe authors refer to Figure 4, but I believe they mean Figure 5.\nWe thank the reviewer for noticing this error. We have corrected the figure reference to (Fig. 5).\n- In the MD Methods section (page 26), change “ionizable residue” to “ionizable residues”.\nWe thank the reviewer for noticing this error. We have modified “ionizable residue” to “ionizable\nresidues” in the revised manuscript.\n- In the MD Methods section (page 26), it is not clear to me what the authors mean by “ORGH”,\nas this abbreviation has not been explained in the main text. I suggest changing “GlyT2-ORGH” by\neither “Glyt2-inhibitor” or by “Glyt2- ORG25543 and Glyt2-RPI-GlyT2-82” for clarity.\nWe thank the reviewer for pointing this error out. We have corrected “GlyT2-ORGH” to “GlyT2–\ninhibitors”\n- The text in Supp. Fig 17 a-b g-h is not very well readable. I would suggest to increase the font size\nin these plots.\nWe thank the reviewer for pointing this out. We have increased the font size of the figure\n(Supplementary Fig. 19 in the revised manuscript) to improve clarity.\nPublishing suggestion:\n- Publish with revisions\nReviewer #6 (Remarks to the Author):\n“I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part\nof the Nature Communications initiative to facilitate training in peer review and to provide appropriate\nrecognition for Early Career Researchers who co-review manuscripts.”\nTo the authors\nThe manuscript focuses on GlyT2 and a new allosteric inhibitor RPI-GLYT2-82 with peculiar\ncharacteristics; the structural information collected by CryEM is consistent, and the results agree\nwith previous data about other members of the SLC6 family, with a few novelties about the\ndeterminants involved in the interaction with the old and new inhibitor and the cholesterol binding\nsite. However, the pharmacological investigation is insufficient and limited if the aim, apparently\ndeclared, is to present a new potential therapy for pain. The conclusion of the abstract and the end\nof the introduction agree with a “structural goal” of the manuscript, but in other points, the focus\nseems the importance of RPI-GLYT2-82 as possible new drugs.\nTo better explain the point, it is important to ask what is the significance of the manuscript for the\nresearch community?\nIf the goal of the paper is to show the structure of the transporters in three different conformations,\nidentifying the Na, Cl, Gly binding site and the possible site of interaction of cholesterol and the of\ntwo allosteric inhibitors, and a possible new “inhibithors” that can represent the basis of the future\nwork, some new experiments are required but the manuscript can be considered for publication after\nimportant revision. Indeed, if it is the case, this manuscript reports interesting data as in other\nrecently published research about transporters of the same family inquiring and defining important\nbinding sites, interactions and poses.\nInstead, if the goal of the authors is to show that the molecule RPI-GLYT2-82 is a potential new pain\ntherapy, the manuscript needs to be completely reorganized. The study in vivo and in vitro must be\npart of the main manuscript. Details of the experimental plan and the investigation methods must be\nbetter explained, and the studies have to focus not only on GLYT2 but also on the other potential\ntarget of the PRI-GLYT2-82.\nFrom here, my comments will consider the “structural” aim to show the structure of the transporter\nand the binding site of a molecule that can serve as a future blueprint for potential new inhibitors.\nWe thank the reviewer for their insights and carefully going through our manuscript. As the reviewer\ncorrectly points out, the goal of the paper is in fact is to present structures of human GlyT2 in clinically\nrelevant conformational states and to provide a proof-of-concept for the rational design of GlyT2\ninhibitors.\nIn this case, first of all, to frame the results in the context of identifying a molecule suitable to act on\nGlyT2 specifically and without side effects in a way that may support future development of a\ntherapeutic, the manuscript should include some new experiments to demonstrate the specificity and\nthe action of RPI-GLYT2_82.\nFirst, the data collected about GlyT2 in Figure 1 must also be collected for GLYT1. The absence of\neffect, SERT, DAT and NET must be shown. These last two (DAT and NET) are to be tested because\nusually any compound interacting with SERT has an action also on the other monoamine\ntransporters (even if the data about the binding seems to exclude this possibility). This is important\nas the data reported in the table of the Off-Target Selectivity Screening of RPI-GLYT2-82 showed\nthat serotonin receptor and transport seem to be targets of RPI-GLYT2-82. Similarly, a fast screening\nof the effect on the 5-HTA receptors could better highlight the selectivity.\nWe agree with the reviewer that it will strengthen our study to show the selectivity of RPI-GLYT2-82\nat GlyT1 as well. We have performed this experiment and found that the inhibitor does not act on\nGlyT1, showing only ~ 6% inhibition at a 10 µM concentration. We have presented this result in panel\nf of the main figure 1:\ncurrents in the presence of different concentrations of RPI-GLYT2-82 showing non-competitive inhibition.\nThere was no statistically significant difference in the glycine EC values across all four conditions (p = 0.49)\n50\nbut there was a statistically significant reduction in I between 0 nM RPI-GLYT2-82 (mean I ± SEM=\nmax max\n1.07 ± 0.01) and the I at 600 nM RPI-GLYT2-82 (mean I ± SEM= 0.76 ± 0.04) (p < 0.0001 ) and also\nmax max\nthe I at 1.8 µM RPI-GLYT2-82 (mean I ± SEM= 0.39 ± 0.02) (p < 0.0001). Data analysed using nonlinear\nmax max\nregression (see Methods). b RPI-GLYT2-82 concentration-dependent inhibition of glycine transport currents.\nCurrent-responses were normalised to currents generated by 30 µM glycine. The difference between the\nhGlyT2WT IC = 554 nM (95% CI: 412 to 747 nM) and the hGlyT2Δ185 IC = 683 nM (95% CI: 370 to 996\n50 50\nnM) is not statistically significant (p = 0.45) c ORG25543 concentration-dependent inhibition of glycine\ntransport normalised to currents generated by 30 µM glycine. The difference between the hGlyT2WT IC =\n50\n11.4 nM (95% CI: 8.0 to 16.4 nM) and the hGlyT2Δ185 IC = 13.1 nM (95% CI: 9.4 to 16.8 nM) is not\n50\nstatistically significantly (p = 0.60). d RPI-GLYT2-82 is reversible and washes out within 5-minutes. Data\nfitted to a one-phase decay curve (WT not fitted due to full reversal before the first time point). e ORG25543\nhas prolonged inhibitory effects with minimal restoration of glycine-induced currents 30-minutes post-\nORG25543. Results normalised to currents generated by 30 µM glycine. f RPI-GLYT2-82 inhibits GlyT2 with\nminimal effect on GlyT1 showing only 6% inhibition at 10 µM (p < 0.0001). Mean inhibition ± SEM from 10\nµM RPI-GLYT2-82 is plotted. g RPI-GLYT2-82 is more potent at lower Na+ concentrations. The IC at 20\n50\nmM Na+ (IC = 159 nM (95% CI: 81 to 238 nM)) is significantly lower than that at 100 mM Na+ (IC = 279\n50 50\nnM (95% CI: 182 to 376 nM)) (p = 0.04). h ORG25543 potency does not change with decreasing Na+\nconcentration. The IC at 20 mM Na+ (IC = 18 nM (95% CI: 6.8 to 29 nM)) is not significantly different\n50 50\nthan that at 100 mM Na+ (IC = 17.6 nM (95% CI: 5.5 to 30 nM)) (p > 0.9999). i Normalised glycine-\n50\ndependent transport currents in hGlyT2WT- and hGlyT2Δ185-expressing Xenopus laevis oocytes showing\ncomparable transport activity in the two constructs (p = 0.78). EC , IC , washout and competition\n50 50\nexperiments performed with n = 5 cells, from at least two different batches of oocytes. Error bars represent\nSD.\nWe present the selectivity screening experiments on the close SLC6 members and respective data\nin the Supplementary data 1 – Exhibits 10–14 (please also see below) – For convenience, we have\ncut the full tables to only show the relevant data here, but the full tables and experimental procedures\n27\nare presented in Supplementary data 1. In brief the selectivity screen of the compound shows: NET\n– only 1.3% inhibition at 10 µM, DAT: 22% inhibition at 10 µM (both n=2). The IC value for inhibition\n50\nof SERT is 4.7 µM and IC value for ligand displacement at 5HT2A is 1.9 µM.\nGLYT2-82 clearer in the revised manuscript.\nExhibit 10. RPI-GLYT2-82 selectivity screening. RPI-GLYT2-82 was tested at 1 × 10-5 M.\nCompound binding was calculated as a % inhibition of the binding of a ligand specific for each target.\nrespective data in the Supplementary data 1 – Exhibits 10–14 (please see below).\nExhibit 10. RPI-GLYT2-82 selectivity screening. RPI-GLYT2-82 was tested at 1 × 10-5 M.\nCompound binding was calculated as a % inhibition of the binding of a ligand specific for each target.\n50\nwhen arguing that the compound’s analgesic effect is driven solely by high selectivity for GlyT2.\nHowever, as we noted in the manuscript, activity at these off-target sites is likely only a minor\ncontributor and becomes relevant only at higher concentrations (approximately five times the\nmaximal analgesic dose). This is consistent with the transient sedation observed in the first 30–60\nminutes, which we discussed as a potential side effect.\nRPI-GLYT2-82 action was tested in vitro in Xenopus laevis oocytes, defining it as a non-competitive\ninhibitor. How was the maximal current recorded (Imax), does it change by the presence of RPI-\nGLYT2-82? These data in a table will be useful.\nAbout Figure 1, most importantly, the data are not supported by statistical analysis. The effect of the\ninhibitor and the similarity between the two GLYT2 (wt and truncated) need to be statistically verified.\nWe agree with the reviewer and have modified Fig. 1a such that the decrease in Imax is more clearly\nshown. We have supported this with statistical analysis of both Imax and EC showing a statistically\n50\nsignificant decrease in Imax with increasing concentrations of RPI-GLYT2-82 but no change in EC .\nconcentrations of RPI-GLYT2-82 showing non-competitive behaviour. There\nwas no statistically significant difference in the glycine EC values across all\n50\nfour conditions but there was a statistically significant reduction in I between\nmax\nno RPI-GLYT2-82 (mean I ± SEM= 1.07 ± 0.01) and the I at 600 nM RPI-\nmax max\nGLYT2-82 (mean I ± SEM= 0.76 ± 0.04) (p < 0.0001 ) and also the I at\nmax max\n1.8 µM RPI-GLYT2-82 (mean I ± SEM= 0.39 ± 0.02) (p < 0.0001).\nmax\nSupplementary Table 1| RPI-GLYT2-82 displays non-competitive behavior.\n[RPI-GLYT2-82] (nM) Glycine EC ± SEM (µM) I/I ± SEM\n50 300\n0 18.0 ± 1.2 1.07 ± 0.01\n200 27.9 ± 3.3ns 0.98 ± 0.026ns\n600 26.7 ± 6.1ns 0.76 ± 0.039****\n1800 24.3 ± 6.3ns 0.39 ± 0.022****\nEC Statistics: Brown-Forsythe ANOVA with Dunnett’s T3 multiple comparisons (ns p > 0.05).\nIf the n=5 refers to the number of oocytes, from how many batches are they? In the figure capture,\nit is important to report both the number of oocytes and the number of batches.\nA dot plot showing the experimental points will clarify better how the experiment was planned and\nconducted.\nWe thank the reviewer for this comment and have included the details in the figure legend.\nEC , IC , washout and competition experiments performed with n = 5 cells, from at least two\n50 50\ndifferent batches of oocytes.\nSimilarly, to better understand the action of the old and new molecules, representative traces of the\nrecording in the presence and in the absence of the two inhibitors (old ORG and new RPI) alone and\nin the presence of glycine must be shown at least in the supplementary material.\nWe thank the reviewer for this remark and have added the representative traces of the recordings in\nthe supplementary figure 1 in the revised manuscript.\n82. Transport activity was characterized by two-electrode voltage clamp electrophysiology with\nXenopus laevis oocytes expressing hGlyT2WT, hGlyT2Δ185 or wild-type hGlyT1. Representative\nhGlyT2 expressing cells electrophysiology traces of a ORG25543 dose-response b RPI-GLYT2-82\ndose response, c RPI-GLYT2-82 activity in the absence glycine, d reversibility characterization of\nORG25543 and e reversibility characterization of RPI-GLYT2-82. f Representative trace of hGlyT1\nexpressing oocyte perfused with RPI-GLYT2-82 and glycine.\nOther points\nThe methods are very long and difficult to follow, and lack of statistical information about the\nstatistical analysis\nWe thank the reviewer for this comment. We have endeavoured to be as detailed as possible to\nensure reproducibility and clarity for readers. We have now amended our reporting of statistical\nsnalysis by adding a sub-section in the methods (Statistical analyses). We have also modified the\nsubsection “Expression and functional analysis of GlyT2WT, GlyT2Δ185, and mutant GlyT2 in\nXenopus laevis oocytes” to be more succinct and clearer. We would be willing to clarify further\nsections in the methods if they remain difficult to follow.\nIn the docking with glycine, looking at the Cysteine density. Chater et al. says that “However,\nalthough we observed clear densities for C311 and C320 in glycine-bound hGlyT2d185, we did not\ndetect a distinct density for the disulfide bridge, suggesting that the cysteine residues may be\nreduced in the substrate-bound state”. Does this mean that the C311 and C320 are involved in\nglycine binding or are essential in conformational changes induced by the binding of Gly?\nWe thank the reviewer for this interesting question. It is generally not well established how\nspecifically this disulfide bridge can affect the conformational state of GlyT2 or any other NSS\nmember, apart from the general stabilization of the extracellular side of the transporter and especially\nthe extracellular loop 2. However, the cysteines/disulfide bridge is crucial for protein folding, as seen\nfor example by mutations in GAT1 (p.Cys164Tyr) (Palmer S. et al. Pediatr Neurol, 2016). We can\nonly speculate as to what this observation means and given the inherent flexibility of the extracellular\nloop 2, we can’t conclude that any differences observed are due to the missing disulfide bridge.\nHowever, it is worth noting that in other substrate-bound SLC6 transporters such as GABA-bound\nhGAT1 (PDB 7Y7W, EMDB 33672), glycine-bound hGlyT1 (PDB 8WFI, EMDB 37492), and\ndopamine-bound hDAT (PDB 8Y2D, EMDB 38851) we do not observe a continuous cryo-EM density\nrepresentative of a formed disulfide bridge at the recommended contour levels, suggesting the\ndisulfide bridge might be reduced in other substrate-bound SLC6 transporters. The disulfide bridge\nmay also be reduced in other substrate-bound SLC6 transporters. This is also an effort to encourage\nthe community to pay closer attention to the density during model building.\nA docking of the RPI-GLYT2-82 and ORG25543 together with the glycine, both binding GLY2 is\nmissing.\nWe thank the reviewer for this remark and have accordingly performed a docking simulation and\nhave included the following in Results:\nTo assess whether glycine can bind to inhibitor-bound hGlyT2 conformations obtained by cryo-EM,\nwe performed 50 ns docking simulations. The observed exchange of solvent and ions between the\nextracellular space and the substrate-binding site, together with glycine’s mobility within the substrate\nbinding cavity, suggests that glycine can access the ORG25543- or RPI-GLYT2-82–bound\nhGlyT2Δ185 from the extracellular solvent. These findings support a mechanism in which allosteric\ninhibitors such as ORG25543 and RPI-GLYT2-82 inhibit glycine transport by preventing\nconformational cycling, a process independent of glycine binding (Supplementary Table 5,6 and\nSupplementary Fig. 20).\nduring glycine docking simulations.\nGlyT2 + ORG25543 + glycine GlyT2 + RPI-GLYT2-82 + glycine\nGlycine contact frequency (%)\nValues report the percentage of simulation frames in which any atom of the GlyT2 residue was within\n4 Å of any atom of the docked glycine molecule. Values are reported only for those residues in\ncontact with glycine for more than 40% of the combined replicate simulations for at least one\nsimulation system.\nGlyT2 complex. a Glycine (cyan sticks) binds to the vacant Na2 sodium binding site, located\nbetween TM1 (blue), TM6 (green), and TM8 (pink). ORG25543 (purple sticks) remains in the cryo-\nEM binding site, below EL4 (yellow). Sodium (cyan) and chloride (green) remain in their respective\nbinding sites. b Bound glycine is coordinated by the backbone amine groups of L211 and G212, the\nbackbone carbonyl groups of Y207 and L574, and the sidechains of Y287, D577, and T578 (not\nshown). c A schematic shows coordination of glycine by GlyT2 coordination residues, highlighting\ndirect interactions and frequent contacts. Coordination by T578 was frequent, with glycine/T578\ncontact present in 98% of frames, but occurred through a variety of modes, with T578 coordinating\neither the ammonium or carboxylate groups of glycine. Both possible coordination modes are shown,\nmarked with an asterisk.\nSince finding a specific inhibitor for GlyTs is important, it will be essential to verify if RPI-GLYT2-82\ncould interact with GlyT1 even by docking.\nWe agree with the reviewer that it will strengthen the study to show the selectivity of RPI-GLYT2-82\nat GlyT1 as well. As also stated in point 1, we have performed this experiment and found that the\ninhibitor does not act on GlyT1, showing only ~ 6% inhibition at a 10 µM concentration. Please see\nour response to major point 1 where we present the results in panel f of Figure 1 (page 27).\nMinor:\nGlyT2Δ185 was expressed in HE400AZ expression medium. something is missing in the sentence\nWe thank the reviewer for noticing the error. We have revised this to say:\n“The hGlyT2Δ185 protein was expressed in Expi293F cells cultured in HE400AZ expression\nmedium (GMEP Cell Technologies, Japan) in …. ”\nThe definition of Imax is not correct and not clear, as in general the reported description of\nelectrophysiological data analysis\nWe thank the reviewer for this comment, we have amended the definition in the equation:\nI is the calculated maximal current\nmax\nExpression and functional analysis of GlyT2WT, GlyT2Δ185, and mutant GlyT2 in Xenopus laevis\noocytes\nStock solutions of 50 mM ORG25543, and 20 mM RPI-GLYT2-82 were dissolved in dimethyl\nsulfoxide (DMSO) and diluted in frog Ringer’s solution (96 mM NaCl, 2 mM KCl, 1 mM MgCl ,\n2\n1.8 mM CaCl , 5 mM HEPES, pH 7.5) to desired concentrations. Final solutions contained less than\n2\n0.001% DMSO, a concentration that had no effect on transporter function.\nHuman GlyT2a wild type (herein referred to as GlyT2) and codon optimised hGlyT2Δ185\ncomplementary DNA were subcloned into the plasmid oocyte transcription vector (pOTV). Site-\ndirected mutagenesis was performed using traditional PCR techniques and sequenced by the\nAustralian Genome Research Facility (Sydney, Australia).\nPurified plasmid DNAs were linearised with SpeI (New England Biolabs, Cat#R3133S).\nComplementary RNA was transcribed by the T7 RNA polymerase using the mMESAGE\nmMACHINE T7 kit (Ambion, Cat#AM1344).\nStage V Xenopus laevis oocytes were detached from the lobe by digestion with 2 mg/mL collagenase\nA (Boehringer, Mannheim, Germany). Defolliculated stage V oocytes were injected with 20 ng of\ncRNA encoding hGlyT2WT, hGlyT2Δ185, or mutant GlyT2 (Drummond Nanoinject, Drummond\nScientific Co., Broomall, PA, USA). The oocytes were stored at 18 °C in frog Ringer’s solution,\nwhich was supplemented with 2.5 mM sodium pyruvate, 0.5 mM theophylline, 50 μg/mL gentamicin\nand 100 μg/mL tetracycline for 3-6 days, until transporter expression was adequate for measurement\nusing the two-electrode voltage clamp technique. Experiments were performed with at least n = 5,\nfrom cells precured from at least two individual Xenopus laevis.\nWhole-cell glycine transport currents were recorded at -60 mV using a Geneclamp 500 amplifier\n(Axon Instruments, Foster City, CA, USA) with a Powerlab 2/25 chart recorder (ADInstruments,\nSydney, Australia). LabChart version 8 software (ADInstruments, Sydney, Australia) was used to\nprocess and visualise current traces.\nGlycine concentration dependent transport currents were measured for GlyT2WT and hGlyT2Δ185 and\nthe mutant GlyT2s (n = 5) and the mutant GlyT2s (n = 5) and fit to the modified Michaelis Menton\nequation using GraphPad Prism (version 4.0, GraphPad Software, San Diego, USA):\nI/I = [Gly]/(EC + [Gly])\nmax 50\nwhere, I is the transport current generated by each glycine concentration (1-300 µM), I is the\nmax\ncalculated maximal current and EC is the concentration of glycine that generates a half maximal\n50\ncurrent.\nThe potency of inhibition of hGlyT2WT, hGlyT2Δ185, or mutant GlyT2 was measured by applying the\nEC glycine concentration for each transporter and then in the presence of the EC for glycine with\n50 50\nincreasing concentrations of inhibitors ORG25543 (0.3–300 nM) and RPI-GLYT2-82 (0.01–10 μM).\nCurrents were fit to the equation:\nI/I = Bottom + (Top- Bottom)/(1+10(x-logIC50))\nEC50\nwhere I is current (nA) generated by EC for glycine in the presence of the indicated concentration\n50\nof the inhibitor, and I is the current generated by the EC (μM) glycine concentration in the\nEC50 50\nabsence of inhibitor. Top and Bottom are the maximal and minimal (≥ 0) plateau responses\nrespectively, x is the concentration of inhibitor (nM), and IC is the concentration that causes 50%\n50\nmaximal inhibition.\nabove but with 300 μM glycine to ensure measurable transport currents were obtained at low sodium\nconcentrations. Choline chloride was used as a substitute salt when altering sodium concentrations.\nThe mechanism of inhibition of transport was determined by measuring glycine concentration-\ndependent transport currents with the co-application of RPI-GLYT2-82 at concentrations < 3-fold\nIC , IC , and > 3-fold IC .\n50 50 50\nReversibility from inhibition was determined by using the previously determined IC of ORG25543\n50\nor RPI-GLYT2-82 (or a maximal dose of 10 μM in cases where the IC is greater than 10 μM). The\n50\nglycine EC for hGlyT2WT, hGlyT2Δ185, or mutant hGlyT2 was applied and then the IC of the\n50 50\ninhibitor was co-applied until the current plateaued. The cell was then continuously perfused with\nfrog Ringer's solution to wash out the inhibitor from the bath solution. Every 5 min, glycine\n(EC ) was re-applied for 1 min, and repeated for a total wash period of 30 min or until complete\n50\ntransporter recovery. Washout kinetics were fitted using the equation below:\nRecovery = (Y – Plateau) × e-Kt + Plateau\nlevels (%), Y is the point which crosses the Y axis (≥ 0) and increases to the Plateau (≤ 100) in one\n0\nphase, K is the rate constant (1/time), and t is time (mins).\nWhat does it “biological samples” mean? In the case of oocytes, is it the number of oocytes or\nbatches?\nWe have modified the legends to indicate number of oocytes and number of batches:\nEC , IC , washout and competition experiments performed with n = 5 cells, from at least two\n50 50\ndifferent batches of oocytes\nFigure 1d: the fitting line for the wt is missing\nWe thank the reviewer for pointing this out. WT is not fitted due to rapid reversibility. At the 5 min\nmark, there is already complete restoration in current to pre-inhibitor levels and therefore a\ndissociation curve cannot be accurately fitted. We have included the following statement in the\nlegend of the figure in the revised manuscript:\nData fitted to a one-phase decay curve (WT not fitted due to full reversal before the first time\npoint).\nFigure 4b, the circle described in the capture representing Na+ and Cl- are not visible.\nWe thank the reviewer for noticing this error. We have now revised the figure to show Na+ and Cl-\nions.\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part\nof the Nature Communications initiative to facilitate training in peer review and to provide appropriate\nrecognition for Early Career Researchers who co-review manuscripts.\nWe thank the reviewer for their efforts.\n37\n- Reviewers comments\n- Our response\n- From the manuscript – Revised version (from the manuscript)\nReviewer #2 (Remarks to the Author):\nI am satisfied with most of the responses by the authors with one exception.\nThe conditioned place preference (CPP) paradigm should be performed. As outlined in my previous\ncomment, it will provide important additional data, as it allows assessing the aversive component of\npain. There are actually compounds (adenosine) that reduced withdrawal responses but have no\neffects in the CPP, potentially indicating that they lack analgesic efficacy despite being active in\nwithdrawal response assays.\nThe authors argue that the test «works best» with reenforcing drugs such as morphine. This is not\ncorrect. On the contrary, reinforcing properties are an important confounding factor. The authors cite\nSufka, Pain 1994 who writes “… the negatively reinforcing properties of analgesic drugs can be\nassessed via the CPP paradigm. In addition, this paradigm offers greater clinical relevance as\nanimals determine drug efficacy without the involvement of high-intensity, phasic nociceptive\nstimulation.” Already the first publication (on the use of CPP in analgesia research (King et al., Nat\nNeurosci, 2009; PMID 19783992) showed that CPP works well with drugs without reenforcing\nproperties (clonidine, conotoxin, lidocaine). Another more recent example is Neumann et al., PAIN\n2021 (PMID 32773599), who have successsfully studied a drug enhancing inhibitory\nneurotransmission in the spinal cord.\nWe thank the reviewer for this thoughtful comment. We agree that CPP can provide supportive\nevidence when a positive signal is observed; however, it is equally important to emphasize that\nabsence of an analgesic effect in CPP does not equate to a lack of analgesia. Across multiple drug\nclasses, clinically effective analgesics often produce inconsistent CPP outcomes. For example,\nNSAIDs, despite being robust and clinically reliable analgesics, often fail to produce CPP, even when\ntheir antinociceptive effects are clearly demonstrated in standard behavioural assays. Thus, CPP\ncan be informative when an analgesic effect is observed, but a lack of effect cannot be taken as\nconclusive evidence that a compound does not relieve pain. This makes CPP unsuitable as a\nuniversal confirmatory assay in early-stage analgesic studies. We consider that performing CPP in\nthe current manuscript would be unlikely to produce an unambiguous, mechanistically informative\nresult that changes the interpretation of our anti-allodynia findings. The CPP readout risks adding\ninterpretational ambiguity (reward vs analgesia) rather than clarity, especially at this early,\nmechanistic stage of drug development.\nWe have also directly addressed the reviewer’s concern that enhanced glycinergic inhibition might\nreduce withdrawal responses via motor weakness rather than genuine analgesia. To resolve this,\nwe incorporated targeted motor-control experiments (grip strength). These data demonstrate that at\nanalgesic doses there are no motor deficits coincident with the anti-allodynic window, and that mild,\ntransient impairment only appears at a supramaximal dose with a distinct time course from the\nanalgesic effect. These findings directly address the confound raised by the reviewer and support\nthe conclusion that reduced withdrawal responses arise from analgesia rather than motor weakness.\nIn addition, we would like to emphasise that the concern raised by the reviewer – that reduced\nwithdrawal responses may not reflect true analgesia, as seen with compounds like adenosine that\nreduce withdrawal endpoints but have no effects in the CPP, has now been directly addressed by\nthe inclusion of “cold allodynia” testing. Cold allodynia is a distinct and clinically relevant modality of\nneuropathic pain, and efficacy in this assay provides modality-independent evidence for analgesic\naction. RPI-GLYT2-82 produced robust and dose-dependent reversal of cold allodynia in the CCI\nmodel, with significant reductions in acetone-evoked responses at both 50 and 100 mg/kg and peak\nefficacy comparable to gabapentin (Fig. 5b). These effects occurred in the absence of motor deficits\nat analgesic doses, as demonstrated by rotarod and grip-strength assays, and therefore cannot be\nattributed to drug-induced weakness or impaired responsiveness. Together, the demonstration of\nefficacy across multiple sensory modalities (mechanical and cold allodynia), combined with specific\nmotor-control data, provides strong convergent evidence of true analgesic activity, independent of\nany motor confound or artefactual suppression of withdrawal behaviours.\nThe authors have addressed my questions. It is a pity that efforts to determine the binding affinity\nbetween ORG25543, RPI-GLYT2-82 and GlyT2 are failed. But the results in the author rebuttal is\nrather convincing. I understand that the micelle environment could interfere the ligand binding in the\npurified GlyT2. Other problems about sodium, chloride and cholesterols in GlyT2 transport are all\naddressed properly. The proposed inhibitory mechanism of ORG25543 and RPI-GLYT2-82 is\nreasonable.\nPersonally I think the manuscript is a good candidate to publish on Nature Communications.\nWe thank the reviewer for their contributions.\nReviewer #5 (Remarks to the Author):\nI thank the authors for addressing all my points. I just have two comments:\n1. Regarding the MM-PBSA, they wrote: \"We thank the reviewer for this remark and would like to\npoint out that the purpose of the MM-PBSA calculations was to “calculate the enthalpic contribution\nof each protein residue within the cryo-EM binding site” to the protein/ligand interactions, to\ndetermine which interactions stabilised or destabilised ligand binding.\" I thank the authors for the\nclarification. If the purpose of the MM-PBSA is solely to calculate the enthalpic contribution within\nthe cryo-EM structure, then it is indeed fine to just use the first 50 ns of the simulation. In my opinion,\nhowever, this point is not clearly made in the text. I did not find the quote the authors refer to\nanywhere in the current version, perhaps it got lost during the revision process. In any case, I strongly\nrecommend the authors to add the quoted sentence in the results section of the main text.\nWe thank the reviewer for this recommendation. We have now incorporated a clear note in the Main\ntext as:\nTo analyse MD simulation trajectories of inhibitor-bound hGlyT2 complexes, we calculated the\nbinding enthalpy over the first 50 ns of the replicate simulations, during which both inhibitors\nmaintained their cryo-EM binding conformations in hGlyT2. This revealed the significant\ncontribution of L211, W215, and D633 to ORG25543 binding and the 0.83–0.92-fold reduction in\nbinding enthalpy for RPI-GLYT2-82 compared to ORG25543 (95% C.I.) (Supplementary Fig. 19).\nA further source of misunderstanding is that, in Supplementary Figure 19, the MM-PBSA energy\ndecomposition plots are displayed in the same panel as the density heat maps; in the captions, the\nauthors write that the density heat maps were obtained from the total simulation time of 1500 ns, so\none gets the impression the MM-PBSA energy decomposition has been calculated for the same time\nrange. The authors only specify that they used only 50 ns of the simulation for the plot of the relative\nbinding enthalpy of ORG25543 and RPI-GLYT2-82, but I would strongly suggest to specify in the\nfigure caption that this is the case for all three MM-PBSA plots displayed.\nWe thank the reviewer for pointing this out. We have made the following changes to the\nSupplementary Fig. 19 legend:\ng Heat map of the relative position of ORG25543 across the 1500 ns of unbiased replicate MD\nsimulations, where each point on the plot corresponds to the probability of being in that position\nrelative to the most probable state (computed as a Boltzmann factor) and MM-PBSA per residue\nbinding enthalpy decomposition of key residues from the ORG25543 binding site. The binding\nenergies of the ORG25543 cryo-EM binding pose were calculated from the first 50 ns of each MD\nreplicate. The enthalpic contribution of the interaction between D633 and ORG25543 was less\nfavorable than that calculated for the D633/PBSA water interaction in simulations at 310 K. h Heat\nmap of the relative position of RPI-GLYT2-82 across the 1500 ns of unbiased replicate MD\nsimulations, where each point on the plot corresponds to the probability of being in that position\nrelative to the most probable state (computed as a Boltzmann factor). MM-PBSA per residue binding\nenthalpy decomposition of key residues in the RPI-GLYT2-82 binding site. The binding energies of\n2\nthe RPI-GLYT2-82 cryo-EM binding pose were calculated from the first 50 ns of each MD replicate.\nThe enthalpic contribution of the interaction between D633 and RPI-GLYT2-82 was less favorable\nthan that calculated for the D633/PBSA water interaction in simulations at 310 K. MM-PBSA\nestimated relative binding enthalpy of ORG25543 and RPI-GLYT2-82 over the first 50 ns of each\nreplicate simulation, with estimated fold difference in binding enthalpy labelled (95% C.I.).\n2. I noticed that the authors have added new docking results. These are in my opinion consistent\nwith the rest of the results and further strengthen the model of allosteric inhibition. I would just make\na small clarification in the nomenclature: I don’t like the term “docking simulation”, because it can be\nmisleading. Technically speaking, the authors first did a docking calculation of the binding pose, and\nthen used MD simulations to assess the stability of this binding pose. I recommend rephrasing the\nsentence “to assess whether glycine can bind to inhibitor-bound hGlyT2 conformations obtained by\ncryo-EM, we performed 50 ns docking simulations“ to something like “to assess whether glycine can\nbind to inhibitor-bound hGlyT2 conformations obtained by cryo-EM, we first docked this molecule\nonto the inhibitor-bound structure and assessed the stability of the binding poses using MD\nsimulations.”\nWe thank the reviewer for this suggestion and have incoroporated the changes:\nTo determine whether glycine can bind to inhibitor-bound hGlyT2 conformations obtained by cryo-\nEM, we first docked glycine into the inhibitor-bound structures and assessed the stability of the\nbinding poses using MD simulations. The observed exchange of solvent and ions between the\nextracellular space and the substrate-binding site, together with glycine’s mobility within the substrate\nbinding cavity, suggests that glycine can access the ORG25543- or RPI-GLYT2-82–bound\nhGlyT2Δ185 from the extracellular solvent. The findings support a mechanism in which allosteric\ninhibitors such as ORG25543 and RPI-GLYT2-82 inhibit glycine transport by preventing\nconformational cycling, a process independent of glycine binding (Supplementary Table 5,6 and\nSupplementary Fig. 20).\nAfter the authors have addressed these two points, I’m happy to suggest acceptance of this article.\nWe thank the reviewer for their contributions.\nReviewer #6 (Remarks to the Author):\nUpon re-examining the revised manuscript, I acknowledge that the authors have improved the quality\nof the functional data increasing the value of the data reported with new information that was\npreviously missing. Nevertheless, several substantial issues remain to be addressed.\nFirstly, after reviewing the authors’ responses to my earlier comments, I still do not find an answer.\nMy (and of another reviewer) original request concerned experiments involving the MATs\ntransporter, whereas the response provided refers instead to GlyT1(see the end of pag 26 beginning\nof 27 of the responses to the reviewers). Consequently, I must reiterate my initial question: \"First,\nthe data collected about GlyT2 in Figure 1 must also be collected for GLYT1. The absence of\neffect, SERT, DAT and NET must be shown. These last two (DAT and NET) are to be tested because\nusually any compound interacting with SERT has an action also on the other monoamine\ntransporters (even if the data about the binding seems to exclude this possibility). This is important\nas the data reported in the table of the Off-Target Selectivity Screening of RPI-GLYT2-82 showed\nthat serotonin receptor and transport seem to be targets of RPI-GLYT2-82. Similarly, a fast screening\nof the effect on the 5-HTA receptors could better highlight the selectivity\"\nMy request was to have these data obtained in the same model (Xenopus laevis oocytes) expressing\nMATs in the presence of the compound. This would be a easy and informative experiment, allowing\nthe authors to assess whether the compound exerts similar or distinct effects within the same\nexperimental system.\nWe thank the reviewer for the clarification. We would like to note that one of the requests in the first\nset of reviewer comments specifically concerned GlyT1, which is why the response on page 26–27\n3\nof our previous rebuttal addressed GlyT1 selectivity. Regarding the comment: “First, the data\ncollected about GlyT2 in Figure 1 must also be collected for GLYT1”, as shown in Fig. 1h, RPI-\nGLYT2-82 produces only ~6% inhibition at 10 μM on GlyT1. This absence of measurable activity is\nwhy the additional experiments (IC₅₀ curves, Na⁺-dependence, washout behaviour) cannot be\nperformed for GlyT1, as these assays require a compound that actually inhibits the transporter.\nWe agree with the reviewer that “compounds interacting with SERT usually also have an action on\nthe other monoamine transporters.” For this reason, we conducted a full MAT selectivity profile for\nRPI-GLYT2-82 early in the study. We also appreciate the comments from Reviewer #6 for proposing\nthat we repeat our experiments conducted in Figure 1 with monoamine transporters (MATs): SERT,\nDAT and NET. Whilst this is an admirable goal, it is unlikely to provide reliable data that would\nenhance the current study beyond what we have already provided as the effect of RPI-GLYT2-82\non the relevant monoamine transporters could not “technically” be studied in X. laevis oocytes used\nfor assessing the effect on GlyT1 and GlyT2. We would like to elaborate on this:\nOur in vitro characterization of RPI-GLYT2-82 was conducted in Xenopus laevis oocytes which is a\nrobust expression system for the glycine transporters (hGlyT1 and hGlyT2). However, “hNET does\nnot express in this expression system” and therefore neither two-electrode voltage clamp\nelectrophysiology nor radiolabeled uptake is possible in oocytes. Please see the following reference:\n(https://link.springer.com/chapter/10.1007/3-540-29784-7_2). As expected, we could not find any\nstudies that has successfully expressed hNET in oocytes or reported any current measurements.\nThe vast majority of studies on hSERT and hDAT utilise 3H-substrate uptake assays using\nmammalian cells expressing the recombinant transporters, or inhibition of 3H-antagonists binding,\nwhich are the gold standard methodologies that allow comparison with other studies. Oocytes have\nbeen used in ”only ~7 papers1-7 in the last 30 years” (out of many 1000’s of papers) and in most\ncases application of serotonin to oocytes expressing hSERT generate only ~20 nA currents. This is\nat about the limit for reliable measurements and in our experience (as we also tried hSERT in\noocytes) it has not provided a reliable signal to noise level to provide an accurate assessment of the\nactivity of inhibitors. Experiments with hDAT are even less reliable than hSERT and often involve\nthe ”murine version of DAT” to provide sufficient currents which are more robust compared to human\nDAT4,6.\nIn contrast, oocytes are an excellent system for measuring glycine transport currents because they\ngenerate currents in the range of 50-300 nA.\nIn lieu of this, we have provided data showing that 10 μM RPI-GLYT2-82 displaces ~85% of 3H-\nimipramine from hSERT expressed in CHO cells. To further explore this, we have provided data of\nradiolabbelled uptake in CHO cells and determined a RPI-GLYT2-82 IC of 4.7 μM at SERT. We\n50\nalso show that 10 μM RPI-GlyT2-82 displaces ~20% of 3H-BTCP from hDAT and <5% of 3H-\nnisoxetine from hNET expressed in CHO cells (exceprts from the manuscript). Regarding the\nreviewer’s concern “even if the data about the binding seems to exclude this possibility” , we would\nlike to emphasyse that we are not claiming that RPI-GlyT2-82 is a highly selective inhibitor, and\nrepeating these experiments in Xenopus laevis oocytes, where hSERT and hDAT are unlikely to\ngenerate measurable signal, and hNET does not express at all, would not provide\nany additional insight into the activity of RPI-GLYT2-82 beyond what is already presented in\nour manuscript and will not change our conclusions.\nTo make it clearer for the reader, we have further discussed the selectivity data on SERT, DAT and\nNET currently included in Supplementary Data 1 more thoroughly, and have provided direct\nreference to relevant graphs and figures as:\nThis additional activity may contribute to its suppression of pain signals. Indeed, antagonism of 5-\nHT2A has previously been reported for other ORG25543-based GlyT2 inhibitors, including\nopiranserin37,38. RPI-GLYT2-82 also exhibits appreciable inhibitory activity at SERT (IC = 4.7 µM,\nnorepinephrine (hNET) transporters (10 μM RPI-GLYT2-82 displaces ~20% of 3H-1-[1-(2-\nbenzo[b]thiopheneyl)cyclohexyl]piperidine hydrochloride (3H-BTCP) from hDAT and <5% of 3H-\n4\nnisoxetine from hNET, Supplementary Data 1, Supplementary Fig. 22). Additionally, it is inactive at\nother off-target sites covered by our Cerep panel screen that are known to promote sedation (γ-\naminobutyric acid type A (GABA ), histamine (H1), adenosine, adrenergic, and muscarinic\nA\nreceptors) (Supplementary Fig. 22, Supplementary Table 16-17).\nWe have further provided the source data behind all the main and supplementary graphs for further\nclarity.\nReferences\n1. Solis et al., 2017 (PMID: 28530234)\n2. De Felice et al., 2012 (PMID: 22291010)\n3. Rodriguez-Manchaca et al., 2011 (PMID: 22014068)\n4. Romanazzi et al., 2021 (PMID: 34957043)\n5. Sandtner et al., 2013 (PMID: 24251585)\n6. Borre et al., 2014 (PMID: 25063810)\n7. Saha et al., 2015 (PMID: 25502630)\nFor the remaining points raised by this reviewer, the responses and the data provided are\nsatisfactory. I would, however, like to point out one typo error: the equation referenced is the\nMichaelis–Menten equation, not “Monten.”\nWe thank the reviewer for pointing this out and have corrected the typo.\nIn conclusion, the manuscript has gratly improved, but addressing the experimental point above is\nnecessary to fully substantiate the authors’ claims.\nWe thank the reviewer for their contributions.\nReviewer #7 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part\nof the Nature Communications initiative to facilitate training in peer review and to provide appropriate\nrecognition for Early Career Researchers who co-review manuscripts.\nWe thank the reviewer for their contributions.\nReviewer #8 (Remarks to the Author):\nIn this manuscript “A reversible allosteric inhibitor of GlyT2 alleviates neuropathic pain without on-\ntarget side effects” Cantwell Chater et al. synthesized and developed a reversible inhibitor of GlyT2\nnamed RPI-GLYT2-82, and analogue of ORG25543 and characterized both inhibitors mode of action\nutilizing a combination of Cryo-EM and in vitro and in vivo pharmacology. The work has several\nhighlights as the authors captured the GlyT2 transporter in the absence of substrate, in the presence\nof glycine or in the presence of either RPI-GLYT2-82 or ORG25543 which allow to see three distinct\nconformations of the transporter: inward-open, inward-occluded and outward-open. Additionally,\nmolecular simulations and mutagenesis allowed to compare and dissect key structural features of\nthe reversibility profile of RPI-GLYT2-82. Finally, the authors conducted in vivo studies to assess the\nanalgesic potential and reward properties of RPI-GLYT2-82. Overall, the manuscript is easy to\nfollow, very well written and organized and represents a move forward in the field of both\ntransmembrane transporters and neuropathic pain research.\nHere are suggestions and comments that if addressed and, in my opinion, will enhance the\npresentation of the manuscript:\nMain text\n-The results section (page 3 line 62) starts with the introduction of ORG25543, the downside of this\ncompound and the structural modification that culminated in the development of RPI-GLYT2-82,\nyet there is no structure of these compounds until figure 4. For both, chemist and not chemist readers,\nthe manuscript will benefit from moving the structures from figure 4 to figure 1 or to make a rational\ndesign structure figure and incorporate that into figure 1.\nWe thank the reviewer for this comment and have added the structures of both compounds as panels\na and b in figure 1.\nFig. 1| RPI-GLYT2-82 is a non-competitive inhibitor of hGlyT2. Chemical structure of a ORG25543 and\nb RPI-GLYT2-82. c Glycine concentration-dependent currents in the presence of different concentrations of\nRPI-GLYT2-82 showing non-competitive inhibition. There was no statistically significant difference in the\nglycine EC values across all four conditions (p = 0.49) but there was a statistically significant reduction in\nmax max max\n(mean I ± SEM= 0.76 ± 0.04) (p < 0.0001 ) and also the I at 1.8 µM RPI-GLYT2-82 (mean I ± SEM\nmax max max\n= 0.39 ± 0.02) (p < 0.0001). Data analysed using nonlinear regression (see Methods). d RPI-GLYT2-82\nconcentration-dependent inhibition of glycine transport currents. Current-responses were normalised to\ncurrents generated by 30 µM glycine. The difference between the hGlyT2WT IC = 554 nM (95% CI: 412 to\n50\n747 nM) and the hGlyT2Δ185 IC = 683 nM (95% CI: 370 to 996 nM) is not statistically significant (p = 0.45)\n50\ne ORG25543 concentration-dependent inhibition of glycine transport normalised to currents generated by 30\nµM glycine. The difference between the hGlyT2WT IC = 11.4 nM (95% CI: 8.0 to 16.4 nM) and the\n50\nhGlyT2Δ185 IC = 13.1 nM (95% CI: 9.4 to 16.8 nM) is not statistically significantly (p = 0.60). f RPI-GLYT2-\n50\n82 is reversible and washes out within 5-minutes. Data fitted to a one-phase decay curve (WT not fitted due to\nfull reversal before the first time point). g ORG25543 has prolonged inhibitory effects with minimal restoration\nof glycine-induced currents 30-minutes post-ORG25543. Results normalised to currents generated by 30 µM\nglycine. h RPI-GLYT2-82 inhibits GlyT2 with minimal effect on GlyT1 showing only 6% inhibition at 10 µM\n(p < 0.0001). Mean inhibition ± SD from 10 µM RPI-GLYT2-82 is plotted. i RPI-GLYT2-82 is more potent\nat lower Na+ concentrations. The IC at 20 mM Na+ (IC = 159 nM (95% CI: 81 to 238 nM)) is significantly\n50 50\nlower than that at 100 mM Na+ (IC = 279 nM (95% CI: 182 to 376 nM)) (p = 0.04). j ORG25543 potency\n50\n6\ndoes not change with decreasing Na+ concentration. The IC at 20 mM Na+ (IC = 18 nM (95% CI: 6.8 to 29\n50 50\nnM)) is not significantly different than that at 100 mM Na+ (IC = 17.6 nM (95% CI: 5.5 to 30 nM)) (p >\n50\n0.9999). k Normalised glycine-dependent transport currents in hGlyT2WT- and hGlyT2Δ185-expressing Xenopus\nlaevis oocytes showing comparable transport activity in the two constructs (p = 0.78). EC , IC , washout and\n50 50\ncompetition experiments performed with n = 5 cells, from at least two different batches of oocytes. Error bars\nrepresent SD.\n-One of the main characters of this manuscript is RPI-GLYT2-82, although the manuscript fails to\nprovide more details about the rational design of this compound. In fact, besides knowing it is an\nanalogue of ORG25543 the authors did not provide any information about how they rationalized that\nan incorporation of ring-constrained systems will result in an analogue with tuned potency, was this\nserendipitous or there was a reason for doing this? The manuscripts will benefit to add a couple of\nsentences addressing this information.\nWe thank the reviewer for this comment. We have addressed how we designed RPI-GLYT2-82 we\nhave added the following text:\nThe narrow therapeutic index of ORG25543 has been attributed to its slow dissociation rate,\nsuggesting that ORG25543-based compounds with improved reversibility may decrease on-target\nside effects and toxicity29,39,40. To overcome the liabilities associated with ORG25543 while\nsimultaneously introducing chemical novelty into the GlyT2 inhibitor scaffold, we pursued the design\nof rigidified benzamide derivatives with reduced conformational flexibility, a strategy anticipated to\nenhance passive permeability and improve overall drug-like properties. Among the structural variants\nexamined, ring-constrained heterocycles, most notably indolines, emerged as effective and\nstructurally innovative isosteric replacements for the tri-alkoxy benzamide motif of ORG25543,\nmaintaining both GlyT2 inhibitory potency and reversible binding kinetics. A subsequent integrated\ndrug discovery effort focused on these conformationally restricted indoline scaffolds ultimately\nyielded the allosteric GlyT2 inhibitor RPI-GLYT2-82 (Fig. 1a,b, Supplementary methods).\n- For the in vivo characterization, 50 mg/Kg of RPI-GLYT2-82 seems like a high amount, why this\nconcentration was utilized from the start? Did the authors try any lower concentration?\nWe thank the reviewer for this question and have now clarified this choice within the text:\nTo evaluate the in vivo utility of our reversible GlyT2 inhibitor RPI-GLYT2-82, we performed plasma\nand central nervous system pharmacokinetic (PK) profiling in mice and calculated unbound drug\nconcentrations in plasma (C ) and brain (C ) at 30- and 120-minutes post-administration\nᵤ,p ᵤ,b\n(Supplementary Data 1, Supplementary Fig. 21, Supplementary Table 7, 9-13,). We initially selected\na 50 mg/kg i.p. dose to obtain a preliminary PK readout and quantify unbound exposures in both\ncompartments. Notably, this dose produced unbound brain concentrations that approximated the in\nvitro IC of RPI-GLYT2-82, indicating that it would likely afford sufficient target engagement and\n50\nthus serve as a rational starting point for assessing analgesic efficacy (Supplementary Table 14-15).\nIndeed, RPI-GLYT2-82 achieved a C of 597.89 nM at 30 minutes after dosing (Fig. 1d,\nᵤ,b\nSupplementary Table 7), approximately equal to its hGlyT2 IC , supporting the expectation of\n50\nachieving pharmacologically meaningful GlyT2 occupancy in vivo (Fig. 1b, Supplementary Table 7).\nSupplementary information:\n- I am unsure if it was a format mistake or if the SI I received missed the first page but usually SI\nhave a front page associated with the manuscript (title, author, affiliations… etc). I suggest adding\nthis in case it is indeed missing.\ncover page which also contains an overview of Supplementary figure and Tables for clarity.\n- Additionally, the SI is quite big so it will benefit from a table of content.\nWe agree with the reviewer and have now included a table of contents.\n- The format of the SI needs to be homogenized, it seems like from page 1 to 34 was done in one\nway and from page 35 to the end differently. The initial portion uses “supplemental fig X or\nsupplemental table. X” whereas from page 35 uses “exhibit”, is there a particular reason?\nWe thank the reviewer for this comment and have now amended the SI to have a more consistent\nformatting. We have maintained the terminology Exhibits only for the “Supplementary methods –\nsynthesis of RPI-GLYT2-82” and Supplementary Fig. and Supplementary Table for the SI.\n- There are two “exhibit 1” one in page 37, the other one in page 59.\nWe thank the reviewer for noticing this mistake and have now amended this.\n- I will suggest changing exhibit 1 from page 59 to “scheme 1”\nWe thank the reviewer for this comment and have amended it to Scheme 1.\n- Although this might be more of a style suggestion, I founded very hard to follow the chemistry in\nthe current presentation. For instance, the synthesis starts with the chemical name of RPI-GLYT2-\n82 but in fact the firs step (Step A) is the synthesis of intermediate 2. So, this makes an initial\nconfusion, I respectfully suggest using the chemical name of intermediate 2 and then the procedure,\nsomething similar to this:\n4-(dimethylamino)tetrahydro-2H-pyran-4-carbonitrile (2): To a solution of tetrahydro-4H-pyran-4-one\n(1, 10.0 g,100 mmol) in H2O (500 mL) was added N,N-dimethylamine hydrochloride (8.15 g, 100\nmmol) followed by KCN (6.50 g, 100 mmol). The reaction mixture was stirred at rt for 16 h with\ncompletion of the reaction determined by TLC. The reaction mixture was extracted with Et2O (3 Å~\n500 mL), and the combined organic layers were washed sequentially with H2O (2 Å~ 500 mL) and\nsaturated brine solution (200 mL). The organic phase was dried over anhydrous Na2SO4, filtered,\nand the solvent was removed under reduced pressure to afford 4 (dimethylamino)tetrahydro-2H-\npyran-4-carbonitrile (2) as a colorless oil (10.0 g, 65% yield). Amino nitrile 2 was used in the next\nstep without further purification. .H NMR (600 MHz, CD3OD) d 3.9 4.0 (m, 2 H), 3.58-3.62 (m, 2 H),\n2.35 (s, 6 H), 2.11-2.15 (m, 2 H), 1.68–1.73 (m,2 H).\nWe thank the reviewer for this suggestion and have changed it to match this format for clarity.\n- Then continue with chemical name of intermediate 3 and so on. I will also suggest removing the\n“Step A, step B”, unless is highlighted on the actual synthetic scheme, this can be confusing. Or at\nleast, make “step a” in bold. I do think this suggestion will make the chemistry section much easy to\nfollow.\nWe thank the reviewer for this suggestion and have made the suggested changes.\n- The H-NMR of compound 7 has two 3.64 ppm signals both are t, J= 11.3 Hz, 2H, this seems a bit\nodd, perhaps a typo?\nIt was indeed a typo which has now been fixed.\n- The LCMS method is missing the temperature used.\nWe have now included the temperature used:\nA Shimadzu Nexcol C18 5μM, 50 × 3.0 mm was used at room temperature.\n- The HRMS method is also missing, you only mentioned the instrument used.\nWe thank the reviewer for pointing out this oversight and have now included the method:\nAn X Bridge C18 5μm 4.6 × 150 mm was used. High-resolution mass spectrometry (HRMS) data\nwere acquired on a Thermo Scientific Orbitrap Eclipse Tribrid mass spectrometer equipped with an\nESI source. Samples were dissolved in CH OH and infused directly into the ion source at a flow rate\n3\n8\nof 3–10 μL/min. Spectra were collected in positive ion mode using the Orbitrap mass analyzer at a\nresolving power of 120,000 (at m/z 200). Source parameters were set as follows: spray voltage 3.5\nkV, sheath gas 10–20 arbitrary units, auxiliary gas 3–5 arbitrary units, and capillary temperature 300–\n320 °C. Mass calibration was performed immediately prior to sample analysis using the\nmanufacturer’s recommended calibration solutions. The [M+H]⁺ ion was reported with a mass\naccuracy typically within ±3 ppm.\n- You report the use of a melting point apparatus then you do not report any melting points.\n- On the same line, can you please provide the melting point of the final compound?\nWe thank the reviewer for pointing these out. With regards to both points, the melting point apparatus\ninformation was removed (a carry-over typo). We did not obtain a mp as the compound was\namorphous due to lyophilization.\n- In some intermediates the authors report the 13C whereas in other intermediates they don’t, is\nthere any particular reason? I suggest keeping the characterization consistent across the\nintermediates and of course the final compound should definitely have 13C.\nWe thank the reviewer for this comment. 13C NMR data for intermediates have been removed to\nremain consistent. We have maintained 13C data for the final compound.\n- On the same line, when you report 13C (intermediate 3 and 6, and in RPI-GLYT2-82) the frequency\nreported [13C NMR (600 MHz, DMSO-d6)] of the carbons seems odd, are you sure is 600MHz?\nusually 600 MHz instruments provide a 150 MHz Carbon. I suggest to double check this information.\nWe thank the reviewer for suggesting a correction. This was a typo and the 150 MHz was\nadded/corrected for the 13C NMR data.\n- In the first step of synthesis of intermediate 7 (oxalyl chloride…) the scheme is missing the solvent\n(CH2Cl2), please add.\nWe have now added CH2Cl2 into the Scheme 1| Synthesis of compound RPI-GLYT2-82.\n9"}, {"round": 2, "reviewer_comments": {"reviewer_2": "I am satisfied with most of the responses by the authors with one exception.\nThe conditioned place preference (CPP) paradigm should be performed. As outlined in my previous comment, it will provide important additional data, as it allows assessing the aversive component of pain. There are actually compounds (adenosine) that reduced withdrawal responses but have no effects in the CPP, potentially indicating that they lack analgesic efficacy despite being active in withdrawal response assays.\nThe authors argue that the test «works best» with reenforcing drugs such as morphine. This is not correct. On the contrary, reinforcing properties are an important confounding factor. The authors cite Sufka, Pain 1994 who writes “… the negatively reinforcing properties of analgesic drugs can be assessed via the CPP paradigm. In addition, this paradigm offers greater clinical relevance as animals determine drug efficacy without the involvement of high-intensity, phasic nociceptive stimulation.” Already the first publication (on the use of CPP in analgesia research (King et al., Nat Neurosci, 2009; PMID 19783992) showed that CPP works well with drugs without reenforcing properties (clonidine, conotoxin, lidocaine). Another more recent example is Neumann et al., PAIN 2021 (PMID 32773599), who have successsfully studied a drug enhancing inhibitory neurotransmission in the spinal cord.", "reviewer_3": "The authors have addressed my questions. It is a pity that efforts to determine the binding affinity between ORG25543, RPI-GLYT2-82 and GlyT2 are failed. But the results in the author rebuttal is rather convincing. I understand that the micelle environment could interfere the ligand binding in the purified GlyT2. Other problems about sodium, chloride and cholesterols in GlyT2 transport are all addressed properly. The proposed inhibitory mechanism of ORG25543 and RPI-GLYT2-82 is reasonable.\nPersonally I think the manuscript is a good candidate to publish on Nature Communications.", "reviewer_5": "I thank the authors for addressing all my points. I just have two comments:\n1. Regarding the MM-PBSA, they wrote: \"We thank the reviewer for this remark and would like to point out that the purpose of the MM-PBSA calculations was to “calculate the enthalpic contribution of each protein residue within the cryo-EM binding site” to the protein/ligand interactions, to determine which interactions stabilised or destabilised ligand binding.\" I thank the authors for the clarification. If the purpose of the MM-PBSA is solely to calculate the enthalpic contribution within the cryo-EM structure, then it is indeed fine to just use the first 50 ns of the simulation. In my opinion, however, this point is not clearly made in the text. I did not find the quote the authors refer to anywhere in the current version, perhaps it got lost during the revision process. In any case, I strongly recommend the authors to add the quoted sentence in the results section of the main text.\nA further source of misunderstanding is that, in Supplementary Figure 19, the MM-PBSA energy decomposition plots are displayed in the same panel as the density heat maps; in the captions, the authors write that the density heat maps were obtained from the total simulation time of 1500 ns, so one gets the impression the MM-PBSA energy decomposition has been calculated for the same time range. The authors only specify that they used only 50 ns of the simulation for the plot of the relative binding enthalpy of ORG25543 and RPI-GLYT2-82, but I would strongly suggest to specify in the figure caption that this is the case for all three MM-PBSA plots displayed.\n2. I noticed that the authors have added new docking results. These are in my opinion consistent with the rest of the results and further strengthen the model of allosteric inhibition. I would just make a small clarification in the nomenclature: I don’t like the term “docking simulation”, because it can be misleading. Technically speaking, the authors first did a docking calculation of the binding pose, and then used MD simulations to assess the stability of this binding pose. I recommend rephrasing the sentence “to assess whether glycine can bind to inhibitor-bound hGlyT2 conformations obtained by cryo-EM, we performed 50 ns docking simulations“ to something like “to assess whether glycine can bind to inhibitor-bound hGlyT2 conformations obtained by cryo-EM, we first docked this molecule onto the inhibitor-bound structure and assessed the stability of the binding poses using MD simulations.”\nAfter the authors have addressed these two points, I’m happy to suggest acceptance of this article.", "reviewer_6": "Upon re-examining the revised manuscript, I acknowledge that the authors have improved the quality of the functional data increasing the value of the data reported with new information that was previously missing. Nevertheless, several substantial issues remain to be addressed.\nFirstly, after reviewing the authors’ responses to my earlier comments, I still do not find an answer. My (and of another reviewer) original request concerned experiments involving the MATs transporter, whereas the response provided refers instead to GlyT1(see the end of pag 26 beginning of 27 of the responses to the reviewers). Consequently, I must reiterate my initial question: \"First, the data collected about GlyT2 in Figure 1 must also be collected for GLYT1. The absence of effect, SERT, DAT and NET must be shown. These last two (DAT and NET) are to be tested because usually any compound interacting with SERT has an action also on the other monoamine transporters (even if the data about the binding seems to exclude this possibility). This is important as the data reported in the table of the Off-Target Selectivity Screening of RPI-GLYT2-82 showed that serotonin receptor and transport seem to be targets of RPI-GLYT2-82. Similarly, a fast screening of the effect on the 5-HTA receptors could better highlight the selectivity\"\nMy request was to have these data obtained in the same model (Xenopus laevis oocytes) expressing MATs in the presence of the compound. This would be a easy and informative experiment, allowing the authors to assess whether the compound exerts similar or distinct effects within the same experimental system.\nFor the remaining points raised by this reviewer, the responses and the data provided are satisfactory. I would, however, like to point out one typo error: the equation referenced is the Michaelis–Menten equation, not “Monten.”\nIn conclusion, the manuscript has gratly improved, but addressing the experimental point above is necessary to fully substantiate the authors’ claims.", "reviewer_7": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "- Reviewers comments\n- Our response\n- From the manuscript – Revised version (from the manuscript)\nReviewer #2 (Remarks to the Author):\nI am satisfied with most of the responses by the authors with one exception.\nThe conditioned place preference (CPP) paradigm should be performed. As outlined in my previous comment, it will provide important additional data, as it allows assessing the aversive component of pain. There are actually compounds (adenosine) that reduced withdrawal responses but have no effects in the CPP, potentially indicating that they lack analgesic efficacy despite being active in withdrawal response assays.\nThe authors argue that the test «works best» with reenforcing drugs such as morphine. This is not correct. On the contrary, reinforcing properties are an important confounding factor. The authors cite Sufka, Pain 1994 who writes “… the negatively reinforcing properties of analgesic drugs can be assessed via the CPP paradigm. In addition, this paradigm offers greater clinical relevance as animals determine drug efficacy without the involvement of high-intensity, phasic nociceptive stimulation.” Already the first publication (on the use of CPP in analgesia research (King et al., Nat Neurosci, 2009; PMID 19783992) showed that CPP works well with drugs without reenforcing properties (clonidine, conotoxin, lidocaine). Another more recent example is Neumann et al., PAIN 2021 (PMID 32773599), who have successsfully studied a drug enhancing inhibitory neurotransmission in the spinal cord.\nWe thank the reviewer for this thoughtful comment. We agree that CPP can provide supportive evidence when a positive signal is observed; however, it is equally important to emphasize that absence of an analgesic effect in CPP does not equate to a lack of analgesia. Across multiple drug classes, clinically effective analgesics often produce inconsistent CPP outcomes. For example, NSAIDs, despite being robust and clinically reliable analgesics, often fail to produce CPP, even when their antinociceptive effects are clearly demonstrated in standard behavioural assays. Thus, CPP can be informative when an analgesic effect is observed, but a lack of effect cannot be taken as conclusive evidence that a compound does not relieve pain. This makes CPP unsuitable as a universal confirmatory assay in early-stage analgesic studies. We consider that performing CPP in the current manuscript would be unlikely to produce an unambiguous, mechanistically informative result that changes the interpretation of our anti-allodynia findings. The CPP readout risks adding interpretational ambiguity (reward vs analgesia) rather than clarity, especially at this early, mechanistic stage of drug development.\nWe have also directly addressed the reviewer’s concern that enhanced glycinergic inhibition might reduce withdrawal responses via motor weakness rather than genuine analgesia. To resolve this, we incorporated targeted motor-control experiments (grip strength). These data demonstrate that at analgesic doses there are no motor deficits coincident with the anti-allodynic window, and that mild, transient impairment only appears at a supramaximal dose with a distinct time course from the analgesic effect. These findings directly address the confound raised by the reviewer and support the conclusion that reduced withdrawal responses arise from analgesia rather than motor weakness.\nIn addition, we would like to emphasise that the concern raised by the reviewer – that reduced withdrawal responses may not reflect true analgesia, as seen with compounds like adenosine that reduce withdrawal endpoints but have no effects in the CPP, has now been directly addressed by the inclusion of “cold allodynia” testing. Cold allodynia is a distinct and clinically relevant modality of neuropathic pain, and efficacy in this assay provides modality-independent evidence for analgesic action. RPI-GLYT2-82 produced robust and dose-dependent reversal of cold allodynia in the CCI model, with significant reductions in acetone-evoked responses at both 50 and 100 mg/kg and peak efficacy comparable to gabapentin (Fig. 5b). These effects occurred in the absence of motor deficits at analgesic doses, as demonstrated by rotarod and grip-strength assays, and therefore cannot be attributed to drug-induced weakness or impaired responsiveness. Together, the demonstration of efficacy across multiple sensory modalities (mechanical and cold allodynia), combined with specific motor-control data, provides strong convergent evidence of true analgesic activity, independent of any motor confound or artefactual suppression of withdrawal behaviours.\nThe authors have addressed my questions. It is a pity that efforts to determine the binding affinity between ORG25543, RPI-GLYT2-82 and GlyT2 are failed. But the results in the author rebuttal is rather convincing. I understand that the micelle environment could interfere the ligand binding in the purified GlyT2. Other problems about sodium, chloride and cholesterols in GlyT2 transport are all addressed properly. The proposed inhibitory mechanism of ORG25543 and RPI-GLYT2-82 is reasonable.\nPersonally I think the manuscript is a good candidate to publish on Nature Communications.\nWe thank the reviewer for their contributions.\nReviewer #5 (Remarks to the Author):\nI thank the authors for addressing all my points. I just have two comments:\n1. Regarding the MM-PBSA, they wrote: \"We thank the reviewer for this remark and would like to point out that the purpose of the MM-PBSA calculations was to “calculate the enthalpic contribution of each protein residue within the cryo-EM binding site” to the protein/ligand interactions, to determine which interactions stabilised or destabilised ligand binding.\" I thank the authors for the clarification. If the purpose of the MM-PBSA is solely to calculate the enthalpic contribution within the cryo-EM structure, then it is indeed fine to just use the first 50 ns of the simulation. In my opinion, however, this point is not clearly made in the text. I did not find the quote the authors refer to anywhere in the current version, perhaps it got lost during the revision process. In any case, I strongly recommend the authors to add the quoted sentence in the results section of the main text.\nWe thank the reviewer for this recommendation. We have now incorporated a clear note in the Main text as:\nTo analyse MD simulation trajectories of inhibitor-bound hGlyT2 complexes, we calculated the binding enthalpy over the first 50 ns of the replicate simulations, during which both inhibitors maintained their cryo-EM binding conformations in hGlyT2. This revealed the significant contribution of L211, W215, and D633 to ORG25543 binding and the 0.83–0.92-fold reduction in binding enthalpy for RPI-GLYT2-82 compared to ORG25543 (95% C.I.) (Supplementary Fig. 19).\nA further source of misunderstanding is that, in Supplementary Figure 19, the MM-PBSA energy decomposition plots are displayed in the same panel as the density heat maps; in the captions, the authors write that the density heat maps were obtained from the total simulation time of 1500 ns, so one gets the impression the MM-PBSA energy decomposition has been calculated for the same time range. The authors only specify that they used only 50 ns of the simulation for the plot of the relative binding enthalpy of ORG25543 and RPI-GLYT2-82, but I would strongly suggest to specify in the figure caption that this is the case for all three MM-PBSA plots displayed.\nWe thank the reviewer for pointing this out. We have made the following changes to the Supplementary Fig. 19 legend:\ng Heat map of the relative position of ORG25543 across the 1500 ns of unbiased replicate MD simulations, where each point on the plot corresponds to the probability of being in that position relative to the most probable state (computed as a Boltzmann factor) and MM-PBSA per residue binding enthalpy decomposition of key residues from the ORG25543 binding site. The binding energies of the ORG25543 cryo-EM binding pose were calculated from the first 50 ns of each MD replicate. The enthalpic contribution of the interaction between D633 and ORG25543 was less favorable than that calculated for the D633/PBSA water interaction in simulations at 310 K. h Heat map of the relative position of RPI-GLYT2-82 across the 1500 ns of unbiased replicate MD simulations, where each point on the plot corresponds to the probability of being in that position relative to the most probable state (computed as a Boltzmann factor). MM-PBSA per residue binding enthalpy decomposition of key residues in the RPI-GLYT2-82 binding site. The binding energies of 2 the RPI-GLYT2-82 cryo-EM binding pose were calculated from the first 50 ns of each MD replicate. The enthalpic contribution of the interaction between D633 and RPI-GLYT2-82 was less favorable than that calculated for the D633/PBSA water interaction in simulations at 310 K. MM-PBSA estimated relative binding enthalpy of ORG25543 and RPI-GLYT2-82 over the first 50 ns of each replicate simulation, with estimated fold difference in binding enthalpy labelled (95% C.I.).\n2. I noticed that the authors have added new docking results. These are in my opinion consistent with the rest of the results and further strengthen the model of allosteric inhibition. I would just make a small clarification in the nomenclature: I don’t like the term “docking simulation”, because it can be misleading. Technically speaking, the authors first did a docking calculation of the binding pose, and then used MD simulations to assess the stability of this binding pose. I recommend rephrasing the sentence “to assess whether glycine can bind to inhibitor-bound hGlyT2 conformations obtained by cryo-EM, we performed 50 ns docking simulations“ to something like “to assess whether glycine can bind to inhibitor-bound hGlyT2 conformations obtained by cryo-EM, we first docked this molecule onto the inhibitor-bound structure and assessed the stability of the binding poses using MD simulations.”\nWe thank the reviewer for this suggestion and have incoroporated the changes:\nTo determine whether glycine can bind to inhibitor-bound hGlyT2 conformations obtained by cryo-EM, we first docked glycine into the inhibitor-bound structures and assessed the stability of the binding poses using MD simulations. The observed exchange of solvent and ions between the extracellular space and the substrate-binding site, together with glycine’s mobility within the substrate binding cavity, suggests that glycine can access the ORG25543- or RPI-GLYT2-82–bound hGlyT2Δ185 from the extracellular solvent. The findings support a mechanism in which allosteric inhibitors such as ORG25543 and RPI-GLYT2-82 inhibit glycine transport by preventing conformational cycling, a process independent of glycine binding (Supplementary Table 5,6 and Supplementary Fig. 20).\nAfter the authors have addressed these two points, I’m happy to suggest acceptance of this article.\nWe thank the reviewer for their contributions.\nReviewer #6 (Remarks to the Author):\nUpon re-examining the revised manuscript, I acknowledge that the authors have improved the quality of the functional data increasing the value of the data reported with new information that was previously missing. Nevertheless, several substantial issues remain to be addressed.\nFirstly, after reviewing the authors’ responses to my earlier comments, I still do not find an answer. My (and of another reviewer) original request concerned experiments involving the MATs transporter, whereas the response provided refers instead to GlyT1(see the end of pag 26 beginning of 27 of the responses to the reviewers). Consequently, I must reiterate my initial question: \"First, the data collected about GlyT2 in Figure 1 must also be collected for GLYT1. The absence of effect, SERT, DAT and NET must be shown. These last two (DAT and NET) are to be tested because usually any compound interacting with SERT has an action also on the other monoamine transporters (even if the data about the binding seems to exclude this possibility). This is important as the data reported in the table of the Off-Target Selectivity Screening of RPI-GLYT2-82 showed that serotonin receptor and transport seem to be targets of RPI-GLYT2-82. Similarly, a fast screening of the effect on the 5-HTA receptors could better highlight the selectivity\"\nMy request was to have these data obtained in the same model (Xenopus laevis oocytes) expressing MATs in the presence of the compound. This would be a easy and informative experiment, allowing the authors to assess whether the compound exerts similar or distinct effects within the same experimental system.\nWe thank the reviewer for the clarification. We would like to note that one of the requests in the first set of reviewer comments specifically concerned GlyT1, which is why the response on page 26–27 3 of our previous rebuttal addressed GlyT1 selectivity. Regarding the comment: “First, the data collected about GlyT2 in Figure 1 must also be collected for GLYT1”, as shown in Fig. 1h, RPI-GLYT2-82 produces only ~6% inhibition at 10 μM on GlyT1. This absence of measurable activity is why the additional experiments (IC₅₀ curves, Na⁺-dependence, washout behaviour) cannot be performed for GlyT1, as these assays require a compound that actually inhibits the transporter.\nWe agree with the reviewer that “compounds interacting with SERT usually also have an action on the other monoamine transporters.” For this reason, we conducted a full MAT selectivity profile for RPI-GLYT2-82 early in the study. We also appreciate the comments from Reviewer #6 for proposing that we repeat our experiments conducted in Figure 1 with monoamine transporters (MATs): SERT, DAT and NET. Whilst this is an admirable goal, it is unlikely to provide reliable data that would enhance the current study beyond what we have already provided as the effect of RPI-GLYT2-82 on the relevant monoamine transporters could not “technically” be studied in X. laevis oocytes used for assessing the effect on GlyT1 and GlyT2. We would like to elaborate on this:\nOur in vitro characterization of RPI-GLYT2-82 was conducted in Xenopus laevis oocytes which is a robust expression system for the glycine transporters (hGlyT1 and hGlyT2). However, “hNET does not express in this expression system” and therefore neither two-electrode voltage clamp electrophysiology nor radiolabeled uptake is possible in oocytes. Please see the following reference: (https://link.springer.com/chapter/10.1007/3-540-29784-7_2). As expected, we could not find any studies that has successfully expressed hNET in oocytes or reported any current measurements.\nThe vast majority of studies on hSERT and hDAT utilise 3H-substrate uptake assays using mammalian cells expressing the recombinant transporters, or inhibition of 3H-antagonists binding, which are the gold standard methodologies that allow comparison with other studies. Oocytes have been used in ”only ~7 papers1-7 in the last 30 years” (out of many 1000’s of papers) and in most cases application of serotonin to oocytes expressing hSERT generate only ~20 nA currents. This is at about the limit for reliable measurements and in our experience (as we also tried hSERT in oocytes) it has not provided a reliable signal to noise level to provide an accurate assessment of the activity of inhibitors. Experiments with hDAT are even less reliable than hSERT and often involve the ”murine version of DAT” to provide sufficient currents which are more robust compared to human DAT4,6.\nIn contrast, oocytes are an excellent system for measuring glycine transport currents because they generate currents in the range of 50-300 nA.\nIn lieu of this, we have provided data showing that 10 μM RPI-GLYT2-82 displaces ~85% of 3H-imipramine from hSERT expressed in CHO cells. To further explore this, we have provided data of radiolabbelled uptake in CHO cells and determined a RPI-GLYT2-82 IC of 4.7 μM at SERT. We 50 also show that 10 μM RPI-GlyT2-82 displaces ~20% of 3H-BTCP from hDAT and <5% of 3H-nisoxetine from hNET expressed in CHO cells (exceprts from the manuscript). Regarding the reviewer’s concern “even if the data about the binding seems to exclude this possibility” , we would like to emphasyse that we are not claiming that RPI-GlyT2-82 is a highly selective inhibitor, and repeating these experiments in Xenopus laevis oocytes, where hSERT and hDAT are unlikely to generate measurable signal, and hNET does not express at all, would not provide any additional insight into the activity of RPI-GLYT2-82 beyond what is already presented in our manuscript and will not change our conclusions.\nTo make it clearer for the reader, we have further discussed the selectivity data on SERT, DAT and NET currently included in Supplementary Data 1 more thoroughly, and have provided direct reference to relevant graphs and figures as:\nThis additional activity may contribute to its suppression of pain signals. Indeed, antagonism of 5-HT2A has previously been reported for other ORG25543-based GlyT2 inhibitors, including opiranserin37,38. RPI-GLYT2-82 also exhibits appreciable inhibitory activity at SERT (IC = 4.7 µM, 50 Supplementary Fig. 23), but has no significant binding activity at the dopamine (hDAT) or norepinephrine (hNET) transporters (10 μM RPI-GLYT2-82 displaces ~20% of 3H-1-[1-(2- benzo[b]thiopheneyl)cyclohexyl]piperidine hydrochloride (3H-BTCP) from hDAT and <5% of 3H- 4 nisoxetine from hNET, Supplementary Data 1, Supplementary Fig. 22). Additionally, it is inactive at other off-target sites covered by our Cerep panel screen that are known to promote sedation (γ- aminobutyric acid type A (GABA ), histamine (H1), adenosine, adrenergic, and muscarinic A receptors) (Supplementary Fig. 22, Supplementary Table 16-17).\nWe have further provided the source data behind all the main and supplementary graphs for further clarity.\nReferences\n1. Solis et al., 2017 (PMID: 28530234)\n2. De Felice et al., 2012 (PMID: 22291010)\n3. Rodriguez-Manchaca et al., 2011 (PMID: 22014068)\n4. Romanazzi et al., 2021 (PMID: 34957043)\n5. Sandtner et al., 2013 (PMID: 24251585)\n6. Borre et al., 2014 (PMID: 25063810)\n7. Saha et al., 2015 (PMID: 25502630)\nFor the remaining points raised by this reviewer, the responses and the data provided are satisfactory. I would, however, like to point out one typo error: the equation referenced is the Michaelis–Menten equation, not “Monten.”\nWe thank the reviewer for pointing this out and have corrected the typo.\nIn conclusion, the manuscript has gratly improved, but addressing the experimental point above is necessary to fully substantiate the authors’ claims.\nWe thank the reviewer for their contributions.\nReviewer #7 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nWe thank the reviewer for their contributions.\nReviewer #8 (Remarks to the Author):\nIn this manuscript “A reversible allosteric inhibitor of GlyT2 alleviates neuropathic pain without on-target side effects” Cantwell Chater et al. synthesized and developed a reversible inhibitor of GlyT2 named RPI-GLYT2-82, and analogue of ORG25543 and characterized both inhibitors mode of action utilizing a combination of Cryo-EM and in vitro and in vivo pharmacology. The work has several highlights as the authors captured the GlyT2 transporter in the absence of substrate, in the presence of glycine or in the presence of either RPI-GLYT2-82 or ORG25543 which allow to see three distinct conformations of the transporter: inward-open, inward-occluded and outward-open. Additionally, molecular simulations and mutagenesis allowed to compare and dissect key structural features of the reversibility profile of RPI-GLYT2-82. Finally, the authors conducted in vivo studies to assess the analgesic potential and reward properties of RPI-GLYT2-82. Overall, the manuscript is easy to follow, very well written and organized and represents a move forward in the field of both transmembrane transporters and neuropathic pain research.\nHere are suggestions and comments that if addressed and, in my opinion, will enhance the presentation of the manuscript:\nMain text\n-The results section (page 3 line 62) starts with the introduction of ORG25543, the downside of this compound and the structural modification that culminated in the development of RPI-GLYT2-82, yet there is no structure of these compounds until figure 4. For both, chemist and not chemist readers, the manuscript will benefit from moving the structures from figure 4 to figure 1 or to make a rational design structure figure and incorporate that into figure 1.\nWe thank the reviewer for this comment and have added the structures of both compounds as panels a and b in figure 1.\nFig. 1| RPI-GLYT2-82 is a non-competitive inhibitor of hGlyT2. Chemical structure of a ORG25543 and b RPI-GLYT2-82. c Glycine concentration-dependent currents in the presence of different concentrations of RPI-GLYT2-82 showing non-competitive inhibition. There was no statistically significant difference in the glycine EC values across all four conditions (p = 0.49) but there was a statistically significant reduction in 50 I between 0 nM RPI-GLYT2-82 (mean I ± SEM= 1.07 ± 0.01) and the I at 600 nM RPI-GLYT2-82 max max max (mean I ± SEM= 0.76 ± 0.04) (p < 0.0001 ) and also the I at 1.8 µM RPI-GLYT2-82 (mean I ± SEM max max max = 0.39 ± 0.02) (p < 0.0001). Data analysed using nonlinear regression (see Methods). d RPI-GLYT2-82 concentration-dependent inhibition of glycine transport currents. Current-responses were normalised to currents generated by 30 µM glycine. The difference between the hGlyT2WT IC = 554 nM (95% CI: 412 to 50 747 nM) and the hGlyT2Δ185 IC = 683 nM (95% CI: 370 to 996 nM) is not statistically significant (p = 0.45) 50 e ORG25543 concentration-dependent inhibition of glycine transport normalised to currents generated by 30 µM glycine. The difference between the hGlyT2WT IC = 11.4 nM (95% CI: 8.0 to 16.4 nM) and the 50 hGlyT2Δ185 IC = 13.1 nM (95% CI: 9.4 to 16.8 nM) is not statistically significantly (p = 0.60). f RPI-GLYT2- 50 82 is reversible and washes out within 5-minutes. Data fitted to a one-phase decay curve (WT not fitted due to full reversal before the first time point). g ORG25543 has prolonged inhibitory effects with minimal restoration of glycine-induced currents 30-minutes post-ORG25543. Results normalised to currents generated by 30 µM glycine. h RPI-GLYT2-82 inhibits GlyT2 with minimal effect on GlyT1 showing only 6% inhibition at 10 µM (p < 0.0001). Mean inhibition ± SD from 10 µM RPI-GLYT2-82 is plotted. i RPI-GLYT2-82 is more potent at lower Na+ concentrations. The IC at 20 mM Na+ (IC = 159 nM (95% CI: 81 to 238 nM)) is significantly 50 50 lower than that at 100 mM Na+ (IC = 279 nM (95% CI: 182 to 376 nM)) (p = 0.04). j ORG25543 potency 50 does not change with decreasing Na+ concentration. The IC at 20 mM Na+ (IC = 18 nM (95% CI: 6.8 to 29 50 50 nM)) is not significantly different than that at 100 mM Na+ (IC = 17.6 nM (95% CI: 5.5 to 30 nM)) (p > 50 0.9999). k Normalised glycine-dependent transport currents in hGlyT2WT- and hGlyT2Δ185-expressing Xenopus laevis oocytes showing comparable transport activity in the two constructs (p = 0.78). EC , IC , washout and 50 50 competition experiments performed with n = 5 cells, from at least two different batches of oocytes. Error bars represent SD.\n-One of the main characters of this manuscript is RPI-GLYT2-82, although the manuscript fails to provide more details about the rational design of this compound. In fact, besides knowing it is an analogue of ORG25543 the authors did not provide any information about how they rationalized that an incorporation of ring-constrained systems will result in an analogue with tuned potency, was this serendipitous or there was a reason for doing this? The manuscripts will benefit to add a couple of sentences addressing this information.\nWe thank the reviewer for this comment. We have addressed how we designed RPI-GLYT2-82 we have added the following text:\nThe narrow therapeutic index of ORG25543 has been attributed to its slow dissociation rate, suggesting that ORG25543-based compounds with improved reversibility may decrease on-target side effects and toxicity29,39,40. To overcome the liabilities associated with ORG25543 while simultaneously introducing chemical novelty into the GlyT2 inhibitor scaffold, we pursued the design of rigidified benzamide derivatives with reduced conformational flexibility, a strategy anticipated to enhance passive permeability and improve overall drug-like properties. Among the structural variants examined, ring-constrained heterocycles, most notably indolines, emerged as effective and structurally innovative isosteric replacements for the tri-alkoxy benzamide motif of ORG25543, maintaining both GlyT2 inhibitory potency and reversible binding kinetics. A subsequent integrated drug discovery effort focused on these conformationally restricted indoline scaffolds ultimately yielded the allosteric GlyT2 inhibitor RPI-GLYT2-82 (Fig. 1a,b, Supplementary methods).\n- For the in vivo characterization, 50 mg/Kg of RPI-GLYT2-82 seems like a high amount, why this concentration was utilized from the start? Did the authors try any lower concentration?\nWe thank the reviewer for this question and have now clarified this choice within the text:\nTo evaluate the in vivo utility of our reversible GlyT2 inhibitor RPI-GLYT2-82, we performed plasma and central nervous system pharmacokinetic (PK) profiling in mice and calculated unbound drug concentrations in plasma (C ) and brain (C ) at 30- and 120-minutes post-administration ᵤ,p ᵤ,b (Supplementary Data 1, Supplementary Fig. 21, Supplementary Table 7, 9-13,). We initially selected a 50 mg/kg i.p. dose to obtain a preliminary PK readout and quantify unbound exposures in both compartments. Notably, this dose produced unbound brain concentrations that approximated the in vitro IC of RPI-GLYT2-82, indicating that it would likely afford sufficient target engagement and 50 thus serve as a rational starting point for assessing analgesic efficacy (Supplementary Table 14-15). Indeed, RPI-GLYT2-82 achieved a C of 597.89 nM at 30 minutes after dosing (Fig. 1d, ᵤ,b Supplementary Table 7), approximately equal to its hGlyT2 IC , supporting the expectation of 50 achieving pharmacologically meaningful GlyT2 occupancy in vivo (Fig. 1b, Supplementary Table 7).\nSupplementary information:\n- I am unsure if it was a format mistake or if the SI I received missed the first page but usually SI have a front page associated with the manuscript (title, author, affiliations… etc). I suggest adding this in case it is indeed missing.\n- Additionally, the SI is quite big so it will benefit from a table of content.\nWe agree with the reviewer and have now included a table of contents.\n- The format of the SI needs to be homogenized, it seems like from page 1 to 34 was done in one way and from page 35 to the end differently. The initial portion uses “supplemental fig X or supplemental table. X” whereas from page 35 uses “exhibit”, is there a particular reason?\nWe thank the reviewer for this comment and have now amended the SI to have a more consistent formatting. We have maintained the terminology Exhibits only for the “Supplementary methods – synthesis of RPI-GLYT2-82” and Supplementary Fig. and Supplementary Table for the SI.\n- There are two “exhibit 1” one in page 37, the other one in page 59.\nWe thank the reviewer for noticing this mistake and have now amended this.\n- I will suggest changing exhibit 1 from page 59 to “scheme 1”\nWe thank the reviewer for this comment and have amended it to Scheme 1.\n- Although this might be more of a style suggestion, I founded very hard to follow the chemistry in the current presentation. For instance, the synthesis starts with the chemical name of RPI-GLYT2- 82 but in fact the firs step (Step A) is the synthesis of intermediate 2. So, this makes an initial confusion, I respectfully suggest using the chemical name of intermediate 2 and then the procedure, something similar to this:\n4-(dimethylamino)tetrahydro-2H-pyran-4-carbonitrile (2): To a solution of tetrahydro-4H-pyran-4-one (1, 10.0 g,100 mmol) in H2O (500 mL) was added N,N-dimethylamine hydrochloride (8.15 g, 100 mmol) followed by KCN (6.50 g, 100 mmol). The reaction mixture was stirred at rt for 16 h with completion of the reaction determined by TLC. The reaction mixture was extracted with Et2O (3 Å~ 500 mL), and the combined organic layers were washed sequentially with H2O (2 Å~ 500 mL) and saturated brine solution (200 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and the solvent was removed under reduced pressure to afford 4 (dimethylamino)tetrahydro-2H- pyran-4-carbonitrile (2) as a colorless oil (10.0 g, 65% yield). Amino nitrile 2 was used in the next step without further purification. .H NMR (600 MHz, CD3OD) d 3.9 4.0 (m, 2 H), 3.58-3.62 (m, 2 H), 2.35 (s, 6 H), 2.11-2.15 (m, 2 H), 1.68–1.73 (m,2 H).\nWe thank the reviewer for this suggestion and have changed it to match this format for clarity.\n- Then continue with chemical name of intermediate 3 and so on. I will also suggest removing the “Step A, step B”, unless is highlighted on the actual synthetic scheme, this can be confusing. Or at least, make “step a” in bold. I do think this suggestion will make the chemistry section much easy to follow.\nWe thank the reviewer for this suggestion and have made the suggested changes.\n- The H-NMR of compound 7 has two 3.64 ppm signals both are t, J= 11.3 Hz, 2H, this seems a bit odd, perhaps a typo?\nIt was indeed a typo which has now been fixed.\n- The LCMS method is missing the temperature used.\nWe have now included the temperature used:\nA Shimadzu Nexcol C18 5μM, 50 × 3.0 mm was used at room temperature.\n- The HRMS method is also missing, you only mentioned the instrument used.\nWe thank the reviewer for pointing out this oversight and have now included the method:\nAn X Bridge C18 5μm 4.6 × 150 mm was used. High-resolution mass spectrometry (HRMS) data were acquired on a Thermo Scientific Orbitrap Eclipse Tribrid mass spectrometer equipped with an ESI source. Samples were dissolved in CH OH and infused directly into the ion source at a flow rate 3 8 of 3–10 μL/min. Spectra were collected in positive ion mode using the Orbitrap mass analyzer at a resolving power of 120,000 (at m/z 200). Source parameters were set as follows: spray voltage 3.5 kV, sheath gas 10–20 arbitrary units, auxiliary gas 3–5 arbitrary units, and capillary temperature 300– 320 °C. Mass calibration was performed immediately prior to sample analysis using the manufacturer’s recommended calibration solutions. The [M+H]⁺ ion was reported with a mass accuracy typically within ±3 ppm.\n- You report the use of a melting point apparatus then you do not report any melting points.\n- On the same line, can you please provide the melting point of the final compound?\nWe thank the reviewer for pointing these out. With regards to both points, the melting point apparatus information was removed (a carry-over typo). We did not obtain a mp as the compound was amorphous due to lyophilization.\n- In some intermediates the authors report the 13C whereas in other intermediates they don’t, is there any particular reason? I suggest keeping the characterization consistent across the intermediates and of course the final compound should definitely have 13C.\nWe thank the reviewer for this comment. 13C NMR data for intermediates have been removed to remain consistent. We have maintained 13C data for the final compound.\n- On the same line, when you report 13C (intermediate 3 and 6, and in RPI-GLYT2-82) the frequency reported [13C NMR (600 MHz, DMSO-d6)] of the carbons seems odd, are you sure is 600MHz? usually 600 MHz instruments provide a 150 MHz Carbon. I suggest to double check this information.\nWe thank the reviewer for suggesting a correction. This was a typo and the 150 MHz was added/corrected for the 13C NMR data.\n- In the first step of synthesis of intermediate 7 (oxalyl chloride…) the scheme is missing the solvent (CH2Cl2), please add.\nWe have now added CH2Cl2 into the Scheme 1| Synthesis of compound RPI-GLYT2-82.\n9"}, {"round": 3, "reviewer_comments": {"reviewer_8": "Thank you to the authors for addressing all the comments and suggestions. I suggest acceptance of the manuscript in its current version."}, "author_response": ""}]

Decision letter