Exploiting human fucosyltransferase 8 allostery with a covalent inhibitor for core fucosylation suppression

Domain: neuroscience
Published: 2026-02-10
Editorial outcome: major_revision

Abstract
Core fucosylation, catalyzed by fucosyltransferase 8 (FUT8), plays critical roles in cancer progression, immune evasion, and drug resistance, making it a compelling therapeutic target. However, development of selective FUT8 inhibitors has been hindered by shared substrate specificity of fucosyltransferases. Here, we report the discovery of a previously unrecognized allosteric site on FUT8 and the development of a low-toxicity covalent inhibitor, CAIF (stearic acid-N-hydroxysuccinimide ester-dimethylimidazolium bromide), through structure-based drug design. High-throughput screening and crystallographic studies reveal that small molecules such as NH125 bind to a channel-like allosteric pocket, inducing conformational changes that disrupt FUT8 activity. Leveraging these insights, we design CAIF to covalently target lysine K216 within the allosteric site. CAIF exhibits minimal cytotoxicity and significantly inhibits core fucosylation and cancer cell invasion in cellular assays. This work establishes CAIF as a lead compound for further optimization and development, offering a framework for targeting glycosyltransferases through allosteric and covalent inhibition strategies.

Introduction
Glycosylation, a common post-translational modification of proteins, plays pivotal roles in regulating diverse physiological functions in living organisms1. Core fucosylation, specifically catalyzed by fucosyltransferase 8 (FUT8) in mammals2, involves the transfer of fucose from guanosine 5’-diphospho-β-L-fucose (GDP-Fuc) to the innermost N-acetylglucosamine residue of N-glycans attached to asparagine residues in proteins3–5. Dysregulation of FUT8 expression is linked to the progression of several diseases, notably including metastatic melanoma6, triple-negative breast cancer7, head and neck squamous cell carcinoma8, and non-small cell lung cancer9. In these conditions, elevated FUT8 levels result in increased core fucosylation of glycoproteins such as L1 cell adhesion molecule, B7 homolog 3, and semaphorin 7A, which promotes tumor invasion, metastasis, and immune evasion6–9. These findings underscore the clinical significance of targeting FUT8 activity.

Inhibition of FUT8, through genetic knockout or small-molecule inhibitors, enhances anti-tumor immunity by downregulating immune checkpoint molecules and reducing tumor growth and metastasis. For example, blocking core fucosylation of programmed cell death protein 1 (PD-1) by inhibiting FUT8 reduces cell-surface expression of PD-1 and enhances T cell activation, leading to more efficient tumor eradication10. FUT8 is thus considered a promising therapeutic target for cancer immunotherapy11. Additionally, FUT8-mediated core fucosylation plays a critical role in promoting targeted drug resistance and chemoresistance12,13, as its expression is broadly correlated with resistance to multiple targeted therapies, especially epidermal growth factor receptor (EGFR) and insulin-like growth factor receptor inhibitors12. Suppression of FUT8 through RNA interference enhances drug-induced cell killing and sensitizes resistant clones, while its overexpression rescues resistance, highlighting its pivotal role in modulating drug sensitivity12. Beyond its role in immune regulation and drug resistance, core fucosylation also plays a critical role in regulating tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL)-induced apoptosis. FUT8 knockdown enhances sensitivity to TRAIL in SW480 cells and potentiates mitochondrial-dependent apoptosis via caspase-9 activation, highlighting its role in modulating cell death pathways in colorectal cancer13. Furthermore, FUT8-mediated core fucosylation enhances RNA viral replication by suppressing retinoic acid-inducible gene I (RIG-I)-mediated antiviral immunity and activating EGFR signaling14, highlighting its significance as a potential target in antiviral therapy. Core fucosylation can reduce the efficacy of antibody-dependent cellular cytotoxicity15–17, necessitating the suppression or inhibition of FUT8 in the production of therapeutic antibodies and cellular immunotherapies16,18.

To rationally develop effective inhibitors targeting FUT8, a deep understanding of its structure and catalytic mechanism is essential. FUT8 is a type II transmembrane protein composed of an N-terminal cytoplasmic region, a transmembrane domain, a stem region (V30–L108), a coiled-coil domain (G109–T173), a GT-B catalytic domain (D174–N503 and E563–K575), and an Src homology 3 (SH3) domain (N503-E563)19–21. FUT8 forms a dimer in solution, with the dimerization interface comprising an extended four-helix bundle contributed by two helices from each monomer, further stabilized by interactions between the bundle and the SH3 domain22,23. The catalysis by FUT8 resembles an SN2-type displacement mechanism, involving a series of loops and α-helices that form the binding sites22. A pocket formed by one of these loops and the SH3 domain is responsible for recognizing the branched glycan and establishing specific contacts with the α-1,3-GlcNAc branch, which is essential for catalysis23. The interaction between GDP-Fuc and the donor-binding site primarily involves hydrogen bonds between the nucleotide base and the donor-binding pocket. Upon GDP-Fuc binding, the highly flexible loop region of FUT8 becomes ordered, and an R365–D368–R441 motif encloses the sugar nucleotide, locking the donor in place23,24. Binding of the glycan acceptor via the O6 hydroxyl group of the GlcNAc residue, which acts as the nucleophile, leads to deprotonation of E373. A subsequent SN2-type attack on the C1 carbon of the donor sugar results in fucose transfer, with proton shuttling from E373 via K369 to the β-phosphate leaving group, thereby completing the glycosylation reaction23,24. These structural insights reveal distinct binding sites for both the donor and acceptor substrates, providing a critical framework for designing specific inhibitors.

Over the past three decades, considerable effort has been directed towards the development of FUT8 inhibitors25–27, encompassing both substrate analogs and non-sugar-related compounds. As the fucosyltransferase family uses GDP-Fuc as a shared donor substrate25, fucose and GDP-Fuc analogs, such as 2-fluorofucose (2FF)28, β-carbafucose18, and fluorinated rhamnosides29, act as pan-inhibitors of fucosylation. However, these pan-inhibitors may cause significant side effects, as seen with the discontinuation of 2FF after thromboembolic events were reported during phase I clinical trials for solid tumors28. Previous studies have also made significant strides in identifying non-sugar FUT8 inhibitors, such as FDW028, through virtual screening of FUT8’s active site, demonstrating low micromolar binding affinity30. In a recent study, high-throughput screening and structural optimization led to the discovery of a FUT8-selective inhibitor that exhibits strong binding to FUT8 in the presence of GDP31. Despite these promising developments, the exact binding sites of these inhibitors on FUT8 remain unknown, and their mechanisms of action (MoAs) have not been clearly defined. Together, these studies underscore the progress made in FUT8 inhibitor discovery while highlighting the critical need for further mechanistic insights to fully realize their therapeutic potential.

Given the important value of FUT8 inhibitors and the current lack of inhibitors with well-defined MoAs, our study aims to address this critical gap by integrating advanced screening techniques with structural biology approaches. Through high-throughput screening of FUT8 enzymatic activity, X-ray crystallography, and chemical synthesis, we have discovered a series of FUT8 inhibitors. Notably, we have identified an allosteric covalent regulatory site on FUT8, which, to our knowledge, has not been previously reported in human fucosyltransferases. This breakthrough provides valuable insights into the design of highly selective inhibitors within the fucosyltransferase family, offering a strategic framework for future therapeutic development.

Results
Identification and characterization of FUT8 inhibitors
To discover core fucosylation inhibitors, we expressed and purified the human FUT8 (68-575) (Supplementary Fig. 1a–c) and established a robust luminescence-based activity assay for high-throughput screening (HTS) (Supplementary Fig. 1d, e). Using this assay, we performed an HTS of approximately 6248 compounds from Selleck Chemicals (Fig. 1a and Supplementary Fig. 1f). The screening demonstrated excellent quality, with average Z´-factors of 0.85 and 0.90 for the two independent screening sets (Fig. 1a, b and Supplementary Fig. 1f). From the initial hits, we identified three compounds—mitoxantrone, NH125, and chlorhexidine (CHX)—that exhibited consistent inhibitory activity in both the primary luminescence assay and a secondary validation assay (Transcreener GDP FI assay) (Supplementary Table 1 and Fig. 1c). Dose-response experiments revealed that NH125 and CHX inhibited FUT8 with IC50 values of about 15.3 and 14.4 μM, respectively, while mitoxantrone was less potent (Fig. 1d, Supplementary Fig. 2, and Supplementary Table 2). As mitoxantrone is a frequent hit in screenings against diverse targets, suggesting potential non-specificity, we focused subsequent studies on NH125 and CHX.

We next evaluated the cytotoxicity and the ability to inhibit cellular core fucosylation of NH125 and CHX on A375 cells. NH125 and CHX showed some toxicity to inhibit cell proliferation in A375 cells, with IC50 values of about 13.2 and 6.9 μM, respectively (Supplementary Fig. 3a). Using fluorescein isothiocyanate (FITC) labeled lentil lectin (LCA), which specifically recognizes core fucosylated N-glycans, we found that NH125 and CHX both significantly alleviated the core fucosylation on A375 cell surface in confocal microscopy and flow cytometry experiments (Fig. 1e and Supplementary Fig. 3b, c). Consistent with previous discovery that inhibiting or knocking out FUT8 suppresses the metastatic and invasive capabilities of melanoma6, both CHX and NH125 demonstrated robust inhibitory effects on the in vitro invasion of A375 cells (Fig. 1f and Supplementary Fig. 3d, e). These phenotypes, potentially arising from the dual effects of suppressed glycosylation and inhibited cell growth, suggest that NH125 and CHX represent promising lead compounds for the development of FUT8-targeting therapeutics. Further optimization of these compounds to minimize their cell growth inhibitory effects while retaining FUT8-targeting activity will help more specifically elucidate the functional roles of core fucosylation.

NH125 and CHX bind to an allosteric inhibitory site of FUT8
To investigate the inhibitory mechanisms of NH125 and CHX on FUT8, we determined the crystal structures of the complexes formed by these two small molecules with FUT8. The FUT8-CHX complex was solved to a resolution of 3.0 Å, and the FUT8-NH125 complex was solved to a resolution of 2.5 Å (Supplementary Table 3). They both belong to the C121 space group with similar cell parameters. The crystal structures revealed a homodimer organization facilitated by the first two N-terminal α-helices (namely, the stem region) of FUT8 (Fig. 2a, b). These helices arrange to form a 4-helix bundle, which aligns with previously reported structures of FUT821–23. The active center of FUT8 is situated at the intersection of Rossmann 1, 2, and SH3 domains22,23. Specifically, the binding site of GDP-Fuc is mainly formed by the Rossmann domain 2, while the receptor sugar binds to the furrow between the three domains of Rossmann 1, 2 and SH3 (Fig. 2c, d).

Surprisingly, neither NH125 nor CHX binds to the substrate binding site of FUT8. Instead, they induce a channel-like binding pocket between the Rossmann domains 1 and 2 of FUT8, located in proximity to the GDP-Fuc binding site (Fig. 2c, d). Both NH125 and CHX possess a chain-like structure and are bound by FUT8 almost exclusively through hydrophobic interactions. The residues directly involved in binding include I214, K216, Y220, Q223, L224, W255, F259, I336, R337, P338, E343, I346, L447, I451, I454, H455, V474, I478, etc. (Fig. 2e–h). Notably, the only polar interaction is a hydrogen bond between the I336 main chain oxygen atom and an imino nitrogen atom in CHX (Fig. 2e, f).

Importantly, the binding of CHX or NH125 would not result in direct steric rejection of the FUT8 substrate GDP-Fuc or the receptor sugar from the structure. Therefore, they are identified as allosteric inhibitors of FUT8.

Allosteric site binders change the conformation and dynamics of FUT8
In order to further understand the MoAs of CHX and NH125 as allosteric inhibitors, we compared the FUT8-CHX and FUT8-NH125 complexes with the FUT8-GDP-sugar complex (PDB code: 6TKV, referred to as holo) reported previously23.

In the holo structure, the NWRYATG loop plays a role in binding the sugar donor substrate. Specifically, the phenol hydroxyl group on the Y250 side chain and the two oxygen atoms on GDP ribose can form hydrogen bond interactions in the holo structure. However, in both the FUT8-CHX and FUT8-NH125 structures, the inhibitors induce significant conformational changes in the Y220 residue. If the NWRYATG loop was structured as in the holo state in the inhibitor-bound structures, the side chain rotamer of Y220 would clash with Y250 (Fig. 3a, and Supplementary Fig. 4a, b). Furthermore, CHX or NH125 would directly clash with W248 under the same condition (Fig. 3a, and Supplementary Fig. 4a, b). Consequently, in the inhibitor-bound structures, the NWRYATG loop becomes disordered, thereby losing its interactions with the sugar donor substrate.

It is worth mentioning that in the FUT8-CHX structure, only one molecule of the FUT8 dimer binds to a CHX, while the other molecule remains in its apo state (Supplementary Fig. 5a, b). In the FUT8-NH125 structure, both molecules (chains A and B) of the dimer bind to NH125 (Supplementary Fig. 5c, d). Overall, the architecture of the FUT8 protein remains largely unchanged, with no major domain-scale conformational differences observed between the FUT8-NH125 and FUT8-holo complex structures (Fig. 3b). Consistent with the location of the inhibitor-binding pocket distal to the dimer interface, ligand binding also induces no notable structural perturbations at the FUT8 dimer interface (Fig. 3b). Despite this overall structural stability, a detailed comparison of the FUT8_apo, FUT8-CHX, FUT8-NH125_A, FUT8-NH125_B, and FUT8-holo structures reveals intrinsic flexibility in the NKG loop (Fig. 3c). Specifically, in the FUT8-CHX and FUT8-NH125_B structures, K216 undergoes a conformational change, flipping from forming hydrogen bonds with D295 and the N-acetylglucosamine moiety of the sugar receptor, to having its side chain directed towards the allosteric pocket (Fig. 3c). This change can potentially be utilized in future drug design, as the primary amino group in the side chain of lysine exhibits nucleophilic properties and can be specifically targeted by FUT8 allosteric site binders that carry corresponding covalent warheads.

It has been demonstrated that protein kinetics influences the catalytic efficiency and drug resistance of enzymes32,33. Using the FUT8-NH125 structure as the initial conformation, and molecular dynamics (MD) simulations were performed both in the presence and absence of bound NH125 (Supplementary Fig. 6a, b). The per-residue Root Mean Square Fluctuation (RMSF) profile from our MD simulations and the B-factor profile from our crystal structure show a remarkably high degree of concordance (Supplementary Fig. 6c). This strong correlation validates the reliability of our MD simulations and confirms that the dynamic regions captured in the crystal lattice are consistent with the solution dynamics we observed in silico. The RMSF analysis revealed that NH125 binding induces asymmetric changes in flexibility across the structure. Although the overall RMSF increased in the NH125-bound state—likely due to the intrinsic stochasticity of molecular dynamics simulations—this increase did not exceed the standard deviation observed across three fully independent replicates, indicating that the changes are not statistically significant. Notably, two loops critical for natural substrate binding, RTDKVGTEA and SISWSAGLHNRYTENS, exhibited the most pronounced increases in flexibility (Supplementary Fig. 6d). In contrast, a slight decrease in RMSF was observed in some regions, such as parts of the SH3 domain that are involved in receptor glycan binding (Supplementary Fig. 6d). Furthermore, the NKG loop exhibited higher flexibility than adjacent residues, but its RMSF showed no significant change upon NH125 binding (Supplementary Fig. 6d). These results indicate that the dynamic nature of the NKG loop is an innate property of the FUT8 structure and is not induced or significantly altered by allosteric inhibitor binding. This suggests that the functional role of this loop may be related to its intrinsic conformational plasticity, which appears to be preserved rather than perturbed upon NH125 binding. These dynamic changes provide a potential mechanistic explanation for the allosteric inhibition of substrate binding. Importantly, residues at the extensive dimer interface showed minimal changes in flexibility upon NH125 binding (Supplementary Fig. 6d), suggesting that the allosteric inhibition mechanism operates independently of quaternary structural perturbations.

In addition, comparison of the free energy landscape (FEL) projected along the first two principal components (PC1 and PC2) revealed that NH125 binding remodels the conformational ensemble of FUT8 (Fig. 3d and Supplementary Fig. 7). In the Apo state, the FEL exhibits four metastable basins with their relative free energies of 0 kJ/mol (upper-right), 0.63 kJ/mol (upper-middle), 0.42 kJ/mol (central), and 9.15 kJ/mol (lower-middle). These basins reflect a dynamic conformational distribution, with several low-energy states likely representing functionally relevant conformations accessible during catalytic cycles. The basin with higher free energy (∼9.15 kJ/mol) may correspond to a less populated, transient state. Upon NH125 binding, the FEL is significantly reconfigured, also featuring four distinct basins located with relative minima at 0.95 kJ/mol (upper-left), 0 kJ/mol (upper-middle), 9.78 kJ/mol (central), and 1.42 kJ/mol (lower-middle) (Fig. 3d). Notably, NH125 stabilizes a different global minimum in the upper-middle region (0 kJ/mol)—a state not prominently sampled in the Apo form—while destabilizing several native basins, as reflected by increased energies in other regions. Although conformational diversity is retained, the reshaped energy landscape features altered barrier heights and basin distributions. This remodeling suggests that NH125 binding perturbs the thermodynamic preferences of FUT8, potentially trapping the enzyme in non-productive states and disrupting the conformational cycling required for catalysis. Therefore, our simulations indicate that NH125 may act as an allosteric inhibitor not only through binding but also by reprogramming the free energy landscape of FUT8, thereby impairing its functional dynamics.

The allosteric site of FUT8 can be covalently targeted
The identification of allosteric sites offers a distinct strategy to selectively inhibit FUT8, potentially addressing the challenges posed by the shared substrate specificity among fucosyltransferases in the development of selective inhibitors. Since the allosteric regulatory site possesses a long, channel-like shape, we selected and tested four additional compounds with chain-like structures: olanexidine, sulfosuccinimidyl oleate sodium (SSO), crocetin, and crocin II (Fig. 4a). Among these four compounds, they demonstrated varying levels of inhibition against FUT8.

Notably, SSO completely suppressed the relative activity of FUT8, reducing it to baseline levels with an IC50 value of 36.8 μM (Fig. 4b, Supplementary Fig. 2d, and Supplementary Table 2). SSO is distinguished by its unique structural composition, which includes an N-hydroxysuccinimidyl (NHS) ester moiety—a covalent warhead commonly used in molecular probes. This observation led us to hypothesize that SSO may also act as a covalent inhibitor of FUT8.

To elucidate the MoA of SSO, we conducted co-crystallization experiments involving FUT8 and SSO. The FUT8-SSO complex structure was solved to a resolution of 2.7 Å (Supplementary Table 4). In this structure, SSO occupies the allosteric sites, similar to CHX and NH125. And indeed, SSO functions as a covalent inhibitor of FUT8, covalently binding to the K216 residue of FUT8 (Fig. 4c, d and Supplementary Fig. 8), thereby leading to an irreversible inhibition of FUT8’s activity. This finding is consistent with the prior functional observation that mutation of this residue (K216A) completely abrogates FUT8 catalysis, underscoring its essential role in the enzyme’s function23,24.

Development of a low-toxic allosteric covalent inhibitor of FUT8
Given that both NH125 and SSO share a remarkably similar long straight-chain aliphatic structure, we subsequently employed a fragment merging strategy to develop an integrated compound that combines the advantageous features of both molecules for enhanced FUT8 inhibition (Fig. 5a). Structural analysis of FUT8-NH125 complexes (FUT8-NH125_A and FUT8-NH125_B) revealed conformational flexibility in the benzene moiety of NH125, suggesting its limited contribution to stable receptor interactions (Supplementary Fig. 8b). Consequently, we excluded this structural element in our compound design. Furthermore, to reduce potential nonspecific protein modifications, we removed the sulfonic acid group from the maleimide ring of SSO (Fig. 5a).

The resulting compound, 1-(18-((2,5-dioxopyrrolidin-1-yl)oxy)-18-oxooctadecyl)-2,3-dimethyl-1H-imidazol-3-ium bromide (Supplementary Note 1), demonstrated significantly improved FUT8 inhibitory activity, exhibiting an IC50 value of 5.7 μM (Fig. 5b, Supplementary Fig. 2e, f, and Supplementary Table 2). This represents a 2.5-fold enhancement compared to NH125 (IC50 about 15 μM) and a 6-fold improvement over SSO (IC50 about 37 μM). To confirm that the observed inhibition was specifically due to targeting FUT8 and not the detection system, we performed additional control experiments. Incubation of varying concentrations of CAIF with the coupling enzymes and GDP substrate (in the absence of FUT8) revealed no inhibitory effect up to 1 mM (Supplementary Fig. 9a). We next evaluated the selectivity of CAIF against other members of the same protein family, FUT3 and FUT4, which represent the most likely potential sources of off-target activity. Despite this close phylogenetic relationship, the results are highly encouraging: CAIF exhibited no activity against FUT4 and only weak inhibition against FUT3 (Supplementary Fig. 9b, c). This translates to a remarkable selectivity index of over 114-fold for FUT8 over FUT3. To further evaluate the selectivity of CAIF and assess its potential off-target effects, we computationally screened the human proteome for structural homologs of the GT-B domain of FUT8 using FoldSeek34. Only two proteins (Prob. > 0.23), POFUT135 and POFUT236, were identified as close structural homologs of FUT8. Despite low sequence identity (11% and 12%, respectively), their structural similarity suggested potential shared binding features. We subsequently expressed and purified both POFUT1 and POFUT2 and evaluated the inhibitory activity of CAIF. Remarkably, CAIF showed no significant inhibition against either POFUT1 or POFUT2, even at the highest tested concentration of 1 mM (Supplementary Fig. 9d, e). This finding robustly underscores the advantage of targeting the unique allosteric pocket of FUT8 for achieving selective inhibition.

To elucidate the MoAs of this putative covalent allosteric inhibitor of FUT8 (CAIF), we also determined the co-crystal structure of the FUT8-CAIF complex at 2.0 Å resolution (Supplementary Table 4). The structural data confirmed our design rationale, revealing CAIF binding at the allosteric site. The inhibitor occupies a pocket formed by Y220, L224, W248, W255, V258, F259, I336, P338, E343, I346, L447, I451, I454, H455, and I478, and forms a covalent amide bond with the ε-amino group of K216 (Fig. 5c, d and Supplementary Fig. 8c, d). To functionally validate the importance of Y220 suggested by the structure, we tested the inhibitory activity of CAIF against the Y220A mutant. Interestingly, its inhibitory activity was drastically reduced, with an IC50 value exceeding 503 μM (Supplementary Fig. 9f). This represents a greater than 83-fold loss of potency compared to the wild-type protein (IC50 about 6 μM). This is consistent with our proposed mechanism in which the binding of CAIF triggers a conformational change that positions Y220 to sterically clash with Y250, thereby destabilizing the active state of FUT8.

We subsequently evaluated the cytotoxicity profile of CAIF across four distinct cell lines: HeLa, A375, GAK, and HEK293T. The cytotoxicity assays revealed that CAIF exhibited minimal cellular toxicity with all tested cell lines. Notably, even at the maximum tested concentration of 500 μM, CAIF showed no detectable cytotoxicity against HeLa and GAK cells (Supplementary Fig. 10a). This favorable toxicity profile enabled us to investigate CAIF’s effects on core fucosylation without interference from cell viability concerns. We employed high-content cellular imaging with fucose-specific lectins LCA and Aleuria aurantia lectin (AAL). CAIF treatment showed significant effect in inhibiting HeLa and A375 cells core fucosylation (Fig. 5e and Supplementary Fig. 10b). Quantitative analysis in GAK cells yielded an EC50 of about 40 μM (Fig. 5f and Supplementary Fig. 10c). In addition, CAIF treatment outperformed 2FF in suppressing HeLa cell migration and invasion in the in vitro invasion assay. (Fig. 5g and Supplementary Fig. 10d).

In summary, the development of CAIF represents a significant advancement in FUT8 inhibition, combining structural insights from NH125 and SSO to achieve enhanced potency, minimal cytotoxicity, and superior efficacy in modulating core fucosylation across multiple cancer cell lines. This inhibitor demonstrates robust activity in suppressing cancer cell migration and invasion, highlighting the broad therapeutic potential of FUT8 inhibition in diverse tumor models.

Methods
Plasmid construction
The DNA sequences encoding the catalytic domains of human FUT8 (residues 68–575), FUT3 (residues 35–361), and FUT4 (residues 181–530), POFUT1 (residues 24–384), and POFUT2 (residues 22–429) were codon-optimized for expression in human cells and synthesized (Tsingke Biotechnology). Each sequence was cloned into an expression vector featuring an N-terminal 3×Flag tag; FUT8, FUT4, POFUT1, and POFUT2 were cloned into a modified pSectag2A vector, while FUT3 was cloned into the pcDNA3.4 vector. The FUT8 Y220A mutant and the FUT8 (105–575) truncation variant were subsequently generated using the FUT8 (68–575) construct as a template. The DNA sequences encoding human FA9 (residues 93–129) and human thrombospondin-1 TSP1 (residues 379–429) were codon-optimized for expression in E. coli and synthesized and cloned into a pET28a vector with an N-terminal His-MBP-TEV tag (Tsingke Biotechnology). The synthesized cDNA sequences and the corresponding deduced mature protein sequences for all constructs are provided in Supplementary Note 2.

Protein expression and purification
Recombinant proteins were expressed in Expi293F cells (Thermo Fisher Scientific) cultured in suspension in 293F Hi-exp Medium supplemented with L-glutamine (OPM, Cat# AC601501). Cells were maintained at 37°C, 5% CO2, 50% humidity, and agitated at 110 rpm. Transfection was performed using linear polyethylenimine (PEI-40000, YEASEN) at a concentration of 1 mg/mL. Specific transfection parameters varied by construct: For FUT8 and FUT4, POFUT1, and POFUT2, transfections were performed at a density of 3.0 × 106 cells/mL using 3 mg of plasmid DNA and 9 mg of PEI per liter of culture. For FUT3, transfections were performed at a density of 2.0 × 106 cells/mL using 3 mg of plasmid DNA and 4.5 mg of PEI per liter of culture. The FUT8 mutants were expressed using the same parameters as wild-type FUT8. Following transfection, cultures were incubated for 4–5 days before harvesting by centrifugation at 6000 × g for 15 min. The clarified supernatants were loaded onto anti-FLAG M2 affinity resin (GenScript). After extensive washing with a buffer containing 25 mM HEPES-NaOH (pH 7.5) and 150 mM NaCl for FUT3, FUT4, and FUT8, and 25 mM HEPES-NaOH (pH 8.0), 300 mM NaCl and 10 mM MgCl2 for POFUT1 and POFUT2, bound proteins were eluted with the same wash buffer supplemented with 0.2 mg/mL Flag peptide. The eluted proteins were further purified by size-exclusion chromatography (Superdex 200, Cytiva) equilibrated in the purification buffer. Protein concentrations were determined by measuring ultraviolet absorbance at 280 nm a NanoDrop spectrophotometer, with extinction coefficients calculated from the amino acid sequence. Typical yields from the purification process were approximately 1.5 mg/L for FUT8, 0.5 mg/L for FUT3 and the FUT8 mutants, and POFUT2, 0.8 mg/L for FUT4, and 1 mg/L for POFUT1.

Plasmids encoding Human FA9 (residues 93–129) and human TSP1 (residues 379–429) were transform into SHuffle T7 competent E. coli. A single colony was inoculated into 100 mL LB medium containing 50 µg/mL kanamycin and incubated at 37°C, 200 rpm for 6 hours. The culture was then transferred into 1 L fresh LB medium at a 1:200 dilution and incubated at 37°C until the OD600 reached 0.6–0.8. Protein expression was induced by adding 0.5 mM IPTG, followed by incubation at 16°C, 200 rpm for 20 hours. Cells were harvested by centrifugation at 6000 × g for 15 min at 4°C, resuspended in lysis buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 30 mM imidazole) and lysed using a high pressure cell disruptor. The lysate was clarified by centrifugation at 18,000 × g for 30 min at 4°C. The cleared lysate was loaded onto a pre-equilibrated Ni–NTA affinity chromatography (HisTrap FF column, 5 mL, Cytiva) using an AKTA FPLC system at 4°C. The resin was washed with 10 column volumes of wash buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 30 mM imidazole) and then eluted with a linear imidazole gradient (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 500 mM imidazole). The eluate was further purified by an anion exchange chromatography (HiTrap Q HP column, 5 mL, Cytiva) using an AKTA FPLC system at 4°C. The resin was washed with 10 column volumes of wash buffer (25 mM Tris-HCl, pH 7.5, 50 mM NaCl) and then eluted with a linear NaCl gradient (25 mM Tris-HCl, pH 7.5, 1 M NaCl). Pure FA9 and TSP1 were aliquoted and stored at −80°C in 25 mM Tris-HCl, pH 7.5, 150 mM NaCl.

High-throughput screening (HTS)
High-throughput screening experiments consisted of two steps: the FUT8 reaction and the GDP detection reaction (Supplementary Table 5). In the first step, small molecules (or DMSO) incubated with FUT8 enzyme assay system, which consisted of 10 nM FUT8, 10 μM GDP-Fuc, and 5 μM receptor glycans (a gift from Prof. Ping Wang) in 50 mM HEPES-NaOH (pH 7.5), 100 mM NaCl, 0.01% (v/v) Triton X-100, and 0.2% (w/v) BSA. Two compound libraries from Selleck Chemicals were screened: the U.S. Food and Drug Administration (FDA)-approved Drug Library (2024 compounds) and the Express-Pick Library (4224 compounds). The former was tested at a final concentration of 100 μM, and the latter at 10 μM. Due to the lack of a highly potent FUT8 inhibitor, the experimental system without FUT8 was used as a surrogate positive control, while the DMSO group served as the negative control. The reaction system was prepared in a 96-well PCR plate (19.8 μL/well), followed by the addition of 0.2 μL of either the test compounds or DMSO using a Gryphon automated liquid handler (ART Technologies), resulting in a final DMSO concentration of 1% (v/v). The plate was sealed and incubated at 37°C for 1 hour. After incubation, 10 μL of the reaction mixture from each well was transferred to a white 384-well plate (Corning) and mixed with an equal volume (10 μL) of GDP detection reagent. The plate was then protected from light and incubated at room temperature for 1 hour. GDP production was quantified by luminescence measurement using an EnVision® Multimode Plate Reader (PerkinElmer).

The quality of the HTS assay was assessed by calculating the Z’-factor, a measure of assay robustness in the absence of any test compounds, as described by Zhang et al.43. The formula used was: Z’ = 1 − [3 × (σp + σn) / |μp − μn|], where μp and μn are the means, and σp and σn are the population standard deviations (calculated using the STDEV.P function in Excel) of the positive (p) and negative (n) controls, respectively. A Z’-factor value of 0.5 is considered indicative of an excellent assay43. All screening data were normalized against the plate control values. Compounds that reduced FUT8 enzymatic activity by more than 40% (i.e., normalized signal < 0.6) were selected for further validation.

TRANSCREENER GDP FI assay
The experiment was conducted in a two-step procedure. In the initial step, both the experimental system setup and control group configuration were maintained identical to those established in the HTS protocol: 10 nM FUT8, 10 μM GDP-Fuc, and 5 μM receptor glycans in 50 mM HEPES-NaOH (pH 7.5), 100 mM NaCl, 0.01% (v/v) Triton X-100, and 0.2% (w/v) BSA. The reactions were carried out in eight-tube strips, each containing a total reaction volume of 25 μL. GDP production was quantified using the TRANSCREENER® GDP FI Assay (BellBrook Labs) according to the manufacturer’s protocol. Briefly, 20 μL aliquots of the reaction mixture were transferred to a black 96-well plate (Corning) and combined with 20 μL of detection reagent mixture containing GDP antibody-IrDye® Qc-1 and GDP Alexa 594 Tracer in Stop & Detect Buffer B (1:1 ratio). After thorough mixing, the plate was incubated at room temperature for 60 minutes. Fluorescence intensity was measured at λex 580 nm and λem 620 nm. All experiments were performed in quadruplicate.

Validation of CAIF selectivity in the GDP-detection system
To verify that CAIF does not interfere with the GDP-detection system, we performed control experiments in a total reaction volume of 20 μL (10 μL assay mixture + 10 μL detection reagent). The assay mixture contained 10 μM GDP and CAIF at concentrations ranging from 0 to 1 mM in assay buffer (50 mM HEPES-NaOH, pH 7.5, 100 mM NaCl, 0.01% (v/v) Triton X-100, 0.2% (w/v) BSA). After incubation, 10 μL of GDP-detection reagent was added (1:1 ratio) in white 384-well plates (Corning), followed by 1-hour incubation in the dark at room temperature. Luminescence signals were measured using either an EnVision® Multimode Plate Reader (PerkinElmer) or a GloMax Multi-detection system (TECAN). Data analysis was performed using linear regression in GraphPad Prism 7, with quadruplicate measurements for each condition. These experiments confirmed that CAIF at all tested concentrations showed no detectable effect on the GDP-detection system (Supplementary Fig. 9a), demonstrating its specific inhibition of FUT8.

Dose-response enzyme activity inhibition assays
The enzymatic activities of FUT8, FUT3, FUT4, POFUT1, and POFUT2 were assessed using the GDP-Glo™ Glycosyltransferase Assay (Promega), which quantifies the GDP released during the glycosyltransferase reaction. Compounds were solubilized in DMSO to generate 200× stock solutions, which were serially diluted to generate eleven-point concentration curves. Each dilution was added to the assay mixture at a 1:100 ratio (v/v). The enzyme-compound mixtures were pre-incubated on ice for 2 hours. This extended pre-incubation protocol was employed in accordance with established practices for characterizing covalent inhibitors, particularly when the determination of kinact/KI is experimentally challenging44,45. The reaction was initiated by combining the pre-incubated mixture with an equal volume of substrate solution, yielding final reaction conditions of 10 nM FUT8 or 25 nM FUT3 or 50 nM FUT4 or 20 nM POFUT1 or 20 nM POFUT2, 0.5% (v/v) DMSO, and the following components in their respective buffers: FUT8: 10 μM GDP-Fuc, 5 μM acceptor glycan in 50 mM HEPES-NaOH (pH 7.5), 100 mM NaCl, 0.01% (v/v) Triton X-100, and 0.2% (w/v) BSA. FUT3: 10 μM GDP-Fuc, 50 μM LacNAc in 50 mM HEPES-NaOH (pH 7.5), 100 mM NaCl, 0.01% (v/v) Triton X-100, 0.2% (w/v) BSA and 1 mM MnCl2. FUT4: 10 μM GDP-Fuc, 50 μM N-acetyllactosamine (Tokyo Chemical Industry) in 50 mM HEPES-NaOH (pH 7.5), 100 mM NaCl, 0.01% (v/v) Triton X-100, and 0.2% (w/v) BSA. POFUT1: 10 μM GDP-Fuc, 10 μM FA9 in 50 mM HEPES-NaOH (pH 7.5), 100 mM NaCl, 0.01% (v/v) Triton X-100, and 0.2% (w/v) BSA, 1 mM MgCl2. POFUT2: 10 μM GDP-Fuc, 10 μM TSP1 in 50 mM HEPES-NaOH (pH 7.5), 100 mM NaCl, 0.01% (v/v) Triton X-100, and 0.2% (w/v) BSA, 1 mM MgCl2. Reactions were incubated at 37°C for 1 hour (FUT8, FUT4, POFUT1, POFUT2) or 2 hours (FUT3) before being stopped by adding an equal volume of GDP-Glo detection reagent. After a further 1-hour incubation at room temperature, luminescence was measured using an EnVision® Multimode Plate Reader (PerkinElmer) or a GloMax® Discover Microplate Reader (Promega). Dose-response curves and half-maximal inhibitory concentration (IC50) values were calculated using nonlinear regression analysis in GraphPad Prism 7.

The cell lines
The human malignant melanoma cell lines A375 and GAK, human cervical adenocarcinoma cell line HeLa, and human embryonic kidney cell line HEK293T cell lines were purchased from the American Type Culture Collection and cultured in Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS; Gibco-Gibco) in 6 cm culture dishes. Their selection was based on their expression of FUT8 and their relevance to studying cancer biology and core fucosylation6,46. For specific experiments, cells were seeded at appropriated densities in various culture vessels, including 6 cm dishes, 12-well plates, or 96-well plates, depending on experimental requirements.

Cell viability assessment
Cell viability was evaluated using the Cell Counting Kit-8 (CCK-8) assay according to the manufacturer’s instructions. Briefly, cells were seeded into transparent 96-well plates at a density of 5 × 103 cells per well in 100 μL of complete medium and cultured overnight under standard culture conditions (37°C, 5% CO2). The next day, cells were treated with serially diluted compounds, while the negative control group received an equivalent volume of DMSO. Four replicate wells were used for each treatment condition. Blank wells containing culture medium with the corresponding concentrations of compounds (without cells) were included to correct for non-specific background absorbance. After 72 hours of compound exposure, 10 μL of CCK-8 reagent (APExBIO, Cat# K1018) was added to each well. Plates were subsequently incubated for an additional 4 hours at 37°C. Absorbance was measured at 450 nm using an EnVision® Multimode Plate Reader (PerkinElmer), and data were analyzed using GraphPad Prism 7 software. All experiments were performed in quadruplicate.

Lectin fluorescence-based cell imaging
GAK or A375 cells were seeded into black, clear-bottom 96-well plates (Greiner) at a density of 5 × 103 cells per well and cultured overnight under standard conditions (37°C, 5% CO2). After attachment, cells were treated with CAIF at concentrations ranging from 0 to 300 μM (from a 100× DMSO stock diluted in medium) for 72 hours. An equivalent volume of DMSO was added to the control wells. Following treatment, cells were washed three times with PBS and fixed with 4% paraformaldehyde (PFA) at room temperature for 20 min. After fixation, cells were blocked with 5% (w/v) BSA in PBS at room temperature for 2 h. For fucosylation detection, FITC-conjugated AAL or LCA lectins were diluted in PBS containing 2.5% (w/v) BSA to final concentrations of 2 μg/mL and 5 μg/mL, respectively, and incubated with the cells for 1 h at room temperature in the dark. After lectin staining, cells were washed and counterstained with Hoechst 33342 (1 μg/mL in PBS) for nuclear visualization. Fluorescence imaging was performed using the Operetta High-Content Imaging System (PerkinElmer). Image analysis was conducted using the Multiwavelength Cell Scoring module in MetaXpress software (Molecular Devices). The average integrated fluorescence intensity was quantified and normalized to cell number per field. Four fields per well were analyzed, with a minimum of four replicate wells per condition. Fucosylation levels were normalized to DMSO-treated controls and analyzed using GraphPad Prism 7 software.

Flow cytometry
A375 cells were seeded into 6-well plates at a density of 1 × 105 cells per well and maintained in DMEM supplemented with 10% FBS and 1% Penicillin-Streptomycin. Cells were treated for 72 hours with either DMSO control, CHX, or NH125. Following treatment, cells were harvested and stained with fluorescein-conjugated LCA. Fluorescence intensity was quantified using a Guava EasyCyte™ 8HT flow cytometer, and data were analyzed using FlowJo software.

In vitro invasion assay
Cell invasion was assessed using Corning® Transwell® 24-well plates (Sigma-Aldrich, Cat# CLS3421). A375 or HeLa cells were pretreated with varying concentrations of CHX, NH125, or CAIF for 48 hours prior to the assay. For migration analysis, 5 × 104 cells in serum-free medium were seeded into the upper chamber pre-coated with Matrigel matrix (Corning, Cat# 356234), while the lower chamber contained complete medium with 20% FBS as a chemoattractant. Following 24-hour incubation at 37°C, migrated and invaded cells were stained with 5 μg/mL Calcein AM (Sangon Biotech, China) for 30 minutes at 37°C. Cell invading was quantified by counting five random fields per insert using fluorescence microscopy.

Structural determination of FUT8-inhibitor complexes
To elucidate the molecular mechanisms of FUT8 inhibition, we expressed and purified the human FUT8 (105-575) for co-crystallization studies with candidate inhibitors (NH125, CHX, SSO, and CAIF). High-quality co-crystals were obtained for all FUT8-inhibitor complexes. X-ray diffraction data were collected at the BL19U1 beamline at the National Facility for Protein Science in Shanghai, and beamlines BL02U1 and BL10U1 of the Shanghai Synchrotron Radiation Facility47. The data were processed using beamline-integrated software packages (autoPROC, Propoise, and xia2) for unit cell determination, space group assignment, data integration, and scaling48,49. Molecular replacement was performed using the Phaser program in the CCP4 suite with the apo-FUT8 structure (PDB: 6X5S) as the search model22,50. Iterative model building and refinement were conducted using Coot and Phenix51,52. The data collection and model statistics are given in Supplementary Tables 3 and 4.

Molecular dynamics (MD) simulation and analysis
MD simulations were performed using GROMACS 2024.2 to study the dynamics of both apo-FUT8 and the FUT8–NH125 complex53. The CHARMM36-July 2022 force field was employed for its accurate parametrization of proteins and small molecule ligands54, in conjunction with the TIP3P water model55. Each system contained a FUT8 dimer solvated in a dodecahedral periodic box with a minimum distance of 1.0 nm between the protein and the box boundary. The system was neutralized with Na+ ions (Supplementary Table 6). The protonation states of all ionizable residues were set to their default values at pH 7.0. Energy minimization was carried out using the steepest descent algorithm until the maximum force was below 10 kJ/mol. The minimized system was then used as the starting point for all subsequent independent replicates. The equilibration protocol consisted of two phases: For each independent replicate, a 100 ps NVT simulation was first performed at 300 K using the V-rescale thermostat (tau_t = 0.1 ps) with initial velocities generated from a Maxwell-Boltzmann distribution at 300 K using a random seed (gen_seed = −1). This was followed by a 100 ps NPT simulation at 300 K and 1.0 bar using the C-rescale barostat (tau_p = 2.0 ps). During equilibration, the protein and ligand heavy atoms were restrained to their initial positions using a harmonic force constant of 1000 kJ/mol/nm2.

For all simulations, the Verlet cutoff scheme was employed. Short-range nonbonded interactions were treated with a cutoff of 1.2 nm. Long-range electrostatics were handled using the Particle Mesh Ewald (PME) method with a Fourier spacing of 0.16 nm and an interpolation order of 4. The van der Waals interactions were modified with a force-switch function, smoothly shifting the potential to zero between 1.0 and 1.2 nm. The neighbor list was updated every 20 steps. Bonds involving hydrogen atoms were constrained using the LINCS algorithm (lincs_order = 4, lincs_iter = 1), allowing for a 2-fs integration timestep. Production simulations were then conducted for 100 ns under NPT conditions using the Parrinello–Rahman barostat. All restraints were removed for the production phase. Thus, for each system (apo and NH125-bound), three truly independent replicates were generated, differing only in their initial random velocity assignments from the start of the NVT equilibration phase. All analyses were performed on the production-phase trajectories from each independent replicate after confirming stability via root-mean-square deviation (RMSD) analysis. For each replicate, a stable trajectory segment was selected for subsequent calculations to characterize the dynamic behavior of FUT8 in both the inhibitor-bound and apo states.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Overall assessment\nThis is a well-written manuscript describing a wide-ranging study, encompassing protein expression and purification,\nenzyme assays including high-throughput screening, X-ray crystallography, computational studies and chemical synthesis.\nThe study described is a significant advance on those in the literature, showing that targeted inhibition of the enzyme can be\nachieved with selectivity against other fucosyl transferases. The authors have developed a covalent allosteric inhibitor. I am\nnot convinced that such compounds will be druggable but nevertheless this manuscript is an important proof-of-concept\nstudy.\nThe kinetic characterization of the synthesized compound is insufficient since potency has been measured using an IC50\nvalue. It has long been recognized that this is inappropriate for covalent inhibitors since the derived value is dependent on\nthe exposure time of the target to the inhibitor. Although there is a suggestion in the literature that IC50 values can be used\nwith long incubation times are used (Thorarensen, et al., Bioorg. Med. Chem. 29 (2021) 115865), the accepted way to do this\nis to measure the rate of inactivation at several inhibitor concentrations and derive the kinactivation/KI value for their\ncompounds. The authors are referred to the textbook by Robert Copeland, Evaluation of enzyme inhibitors in drug discovery:\nA guide for medicinal chemists and pharmacologists, 2nd ed. Wiley.\nAs the authors are using a coupled assay, they need to provide evidence that inhibition of the target is happening and that\nCAIF is not inhibiting the coupling enzymes (control experiments).\nThere are some additional details that need to be added to the methods and elsewhere as detailed here:\n• Buffers need to be properly defined including the counter-ion. HEPES is presumably HEPES-NaOH. % concentrations\nneed to be specified (presumably these are % v/v for Triton X-100 and % w/v for BSA).\n• For the HTS and other enzyme assay, what was the final DMSO concentration? Was this consistent throughout? How long\nwere compounds pre-incubated with enzyme before measuring activity? This is a key consideration for covalent inhibitors as\ninhibition will be dependent on pre-incubation time.\n• Please confirm that Z’ was calculated using the population standard deviations (the paper by Zhang et al., 1999 used\npopulation standard deviations although this has been subsequently criticised).\n• Figure 1D. The dose-response curve must use log10 molar drug concentrations. The values should be quoted using pIC50\n± SEM, since the distribution of IC50 estimates is skewed and therefore non-parametric. The dose-response curve for\nmitoxantrone needs to be repeated with higher drug concentrations because there is insufficient plateau on the right-hand\nside of the curve. The legend says “Experiments were performed in quadruplicate, with error bars representing the standard\ndeviation (n = 4, mean ± SD).” Presumably the displayed dose-response curve is a representative independent repeat, with\ndata collected at each drug concentration more than once. N numbers refer to independent repeats. Identical comments\napply to the dose-response curves for figures 4 and 5 and those in the supplementary information.\n• Figure S2d. What statistical correction was used for this data (Bonferroni?). Details of the statistical methods need to be\ngiven in the supplementary information.\n• Both synthesized compounds (compound 2 and CAIF) are novel. Novel compounds generally require 5 pieces of\ncharacterization data; why is there no 13C data and spectra for CAIF? The authors should report 1H and 13C spectra,\nHRMS and IR as a minimum.\n• There is one error in the supplementary crystallographic Table 2 - Structure FUT8-CAIF (9L65), the authors have entered\n'7824' solvent molecules were identified in the structure - which need to be corrected.", "reviewer_2": "Please see attached document", "reviewer_3": "The manuscript presents a novel allosteric site on FUT8 and the development of CAIF, a covalent inhibitor, through\nstructure-based drug design. The work integrates high-throughput screening, crystallography, and cellular assays to\ndemonstrate CAIF’s efficacy in suppressing core fucosylation and cancer cell invasion. While the structural and mechanistic\ninsights are commendable, critical gaps in validation, selectivity, and translational relevance significantly weaken the study’s\nimpact. Below are detailed concerns from this reviewer\nMajor Concerns\n1.CAIF’s low cytotoxicity and anti-invasive effects are demonstrated only in cellular assays. Without animal model data (e.g.,\ntumor growth/metastasis suppression, pharmacokinetics, or toxicity profiles), the therapeutic potential of CAIF remains\nspeculative. The absence of in vivo experiments undermines the clinical relevance of the findings.\n2.FUT8 belongs to a family of fucosyltransferases with shared structural motifs. While the authors claim CAIF’s selectivity via\nits allosteric site, no data are provided to confirm that CAIF does not inhibit other FUT enzymes. Selectivity assays against\nFUT1–FUT7/9 or homology modeling to compare binding sites are essential to validate specificity claims.\n3.The unresolved loops (e.g., residues 77–104) and conformational changes in the 215NKG217215​NKG217​ loop are not\nrigorously analyzed. A correlation between B-factors (crystallography) and RMSF values (MD simulations) would clarify\ndynamic contributions to allostery.\n4.The MD-derived free energy plots (Fig. 3c–f) lack quantitative descriptors (e.g., energy barriers, metastable states). Markov\nstate modeling (MSM) or residue-level hydrogen bonding/fluctuation analyses would strengthen mechanistic insights into\nhow NH125/CAIF binding alters FUT8 dynamics.\n5.The K216-CAIF covalent interaction lacks orthogonal validation (e.g., mass spectrometry, activity assays under reducing\nconditions). Mutagenesis (K216A) is also required to confirm its functional role in inhibitor binding.\n6.As a covalent inhibitor, CAIF’s potential for off-target modifications is not addressed. Proteomic profiling or kinetic assays\nwith unrelated lysine-dependent enzymes are needed to assess specificity.\n7.CAIF’s long aliphatic chain raises concerns about solubility and bioavailability. Pharmacokinetic studies (absorption,\ndistribution, metabolism, excretion) are absent and critical for translational evaluation.\n8.The proposed steric clash between Y220 and Y250 (Fig. 3a) lacks experimental support. Mutating Y220 to alanine and\ntesting inhibitor efficacy would clarify its role in GDP ribose binding and allosteric inhibition.\n9.The dimeric state of FUT8 under physiological conditions and its impact on inhibitor binding (e.g., cooperative effects) are\nnot discussed. Disruption of the dimer interface (via mutagenesis) could reveal its functional relevance.\nMinor\n1.Fig. 1a: Labels for positive/negative controls and hits are unclear; a legend is needed.\n2.Fig. 3c–f: Axes for free energy landscapes are unlabeled; quantitative energy values should be included.\n3.Tables: IC50 values for inhibitors are scattered; a consolidated table in supplementary materials would improve clarity.", "reviewer_5": "The manuscript reports the discovery of a novel allosteric site on FUT8 and the development of a low-toxicity covalent\ninhibitor, CAIF (stearic acid-N-hydroxysuccinimide ester-dimethylimidazolium bromide), through structure-based drug\ndesign. High-throughput screening and crystallographic studies revealed that small molecules, such as NH125, bind to a\nchannel-like allosteric pocket, inducing conformational changes that disrupt FUT8 activity. Leveraging these insights, they\ndesigned CAIF to covalently target lysine K216 within the allosteric site of FUT8. CAIF exhibits minimal cytotoxicity and\nsignificantly inhibits core fucosylation and cancer cell invasion in cellular assays. This work establishes CAIF as a lead\ncompound for further optimization and development, offering a framework for targeting\nglycosyltransferases through allosteric and covalent inhibition strategies.\nThe manuscript is well written and has significant results and therefore can be considered for publication in Nature\nCommunication after the responses to following comments are incorporated.\n1. The authors claimed CAIF as a specific FUT8 inhibitor based on its interaction with allosteric site. However, as all 12\nisoforms of FUTs share significant structural similarity and thus may possess similar allosteric sites as well. In this context,\nhow authors hypothesized that CAIF will specifically inhibit FUT8, and will not affect the other isoforms of FUTs.\n2. Authors mentioned that CAIF exhibits minimal cytotoxicity, while significantly inhibiting the core fucosylation, and cancer\ncell invasion in cellular assays. This statement appears to be self-contradictory, and thus needs further explanation.\n3. Various pro- and eukaryotic cell lines are mentioned in the manuscript. Authors needs to explain the selection criteria of\nthese cell lines with respect to FUT8 in the manuscript. The details of these cell lines also needs to be mentioned.\n4. Since, FUT8 is a bi-substrate enzyme and has two distinct donor and acceptor substrate binding sites. The 2nd and 3rd\nparagraph in the introduction section should be merged and reduced in a single paragraph. While a new paragraph needs to\nbe added highlighting the key structural details of both the binding sites of FUT8.\n5. Authors have not provided any details regarding the protein (FUT8) expression and purification in the Results Section.\nThis needs to be incorporated in the revised manuscript.\n6. They also need to mention the level of purity and yield of FUT8 protein.\n7. In in-vitro FUT8 inhibition assays, IC50 values are mentioned without Standard Error of Mean (S.E.M). Authors need to\nincorporate the S.E.M with all mentioned IC50 values.\n8. 1H and 13C -NMR data of compound 2 is provided in supplementary materials. However, it is not clear which compound\nis compound 2?\n9. 13C-NMR chemical shift data of the CAIF is missing.\n10. Citations of figures need to be checked by the authors.", "reviewer_6": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "Response to Reviewers' Comments\nManuscript ID: NCOMMS-25-06314-T\nManuscript Title: Exploiting human fucosyltransferase 8 allostery with a covalent inhibitor\nfor core fucosylation suppression\nReviewer #1:\nOverall assessment\nThis is a well-written manuscript describing a wide-ranging study, encompassing protein\nexpression and purification, enzyme assays including high-throughput screening, X-ray\ncrystallography, computational studies and chemical synthesis. The study described is a\nsignificant advance on those in the literature, showing that targeted inhibition of the enzyme\ncan be achieved with selectivity against other fucosyltransferases. The authors have\ndeveloped a covalent allosteric inhibitor. I am not convinced that such compounds will be\ndruggable but nevertheless this manuscript is an important proof-of-concept study.\nResponse: We sincerely thank the reviewer for the thoughtful evaluation of our manuscript\nand the recognition of its significance as \"an important proof-of-concept study\".\nThe kinetic characterization of the synthesized compound is insufficient since potency has\nbeen measured using an IC50 value. It has long been recognized that this is inappropriate for\ncovalent inhibitors since the derived value is dependent on the exposure time of the target to\nthe inhibitor. Although there is a suggestion in the literature that IC50 values can be used with\nlong incubation times are used (Thorarensen, et al., Bioorg. Med. Chem. 29 (2021) 115865),\nthe accepted way to do this is to measure the rate of inactivation at several inhibitor\nconcentrations and derive the kinactivation/KI value for their compounds. The authors are\nreferred to the textbook by Robert Copeland, Evaluation of enzyme inhibitors in drug\ndiscovery: A guide for medicinal chemists and pharmacologists, 2nd ed. Wiley.\nResponse: We fully agree with the reviewer that the derivation of k /K is the gold standard\ninact I\nfor covalent inhibitors, as it provides a time-independent measure of potency. However, our\ncompound's allosteric covalent mechanism introduces extra experimental challenges: unlike\n1\nactive-site covalent inhibitors, allosteric inhibitors cannot be competitively displaced by high\nconcentrations of substrate, making it hard to quench the covalent reaction using regular\nmethods. Therefore, we attempted to quench the reaction with excess lysine. However, due to\nthe complex nature of the FUT8 assay system and unknown quenching efficiency, and despite\nextensive optimization attempts, we were unable to establish a stable k /K assay. As a\ninact I\nreference, we have included one representative dataset below showing time-dependent\ninactivation at four inhibitor concentrations. From this dataset, we estimated approximate\nkinetic parameters: k = 3.7 min-1, K = 24.5 µM, yielding k /K = 0.15 µM-1·min-1. The\ninact I inact I\ndata indicate that covalent modification reaches near-plateau within 2 minutes.\nFigure for review. Determination of Covalent Inhibition Kinetics for CAIF. (A)\nTime-dependent inactivation of FUT8 by CAIF at varying concentrations. Residual\nenzyme activity (%) is plotted against incubation time (minutes) for four inhibitor\nconcentrations (1, 10, 30, and 100 µM). Error bars represent SD of four technical repeats.\n(B) Dependence of observed inactivation rates (k ) on CAIF concentration.\nobs\nIn our study, we determined the IC values for CAIF inhibitors using a 2-hour\n50\npre-incubation period to ensure complete covalent modification before measuring residual\nenzyme activity. This approach aligns with the reviewer's recommended literature, where\nprolonged incubation IC values are accepted for covalent inhibitors when kinetic assays are\n50\nimpractical.\nWe have now explicitly detailed this IC determination protocol in the Methods section\n50\nof our revised manuscript (Page 18, Lines 18–21), with appropriate references to the\nreviewer's recommended literature.\ntarget is happening and that CAIF is not inhibiting the coupling enzymes (control\nexperiments).\nResponse: We appreciate the reviewer's insightful suggestion regarding the need to\ndemonstrate that CAIF specifically inhibits the target enzyme FUT8 rather than the coupling\nenzymes in our assay system.\nTo address this important concern, we have now performed additional control\nexperiments where we directly incubated varying concentrations of CAIF with the detection\nsystem (containing the coupling enzymes and GDP substrate, but without FUT8). The results\n(newly added Supplementary Figure 8a) clearly demonstrate that CAIF shows no inhibitory\neffect on the coupling enzymes up to 1 mM, confirming that the observed inhibition in our\nmain assays is indeed due to specific targeting of FUT8. This control experiment strengthens\nour conclusion about CAIF's selectivity and addresses the potential confounding factor raised\nby the reviewer. We thank the reviewer for this valuable suggestion, which has helped us\nimprove the rigor of our mechanistic studies.\nSupplementary Figure 8 | Selectivity of CAIF and its dependence on the allosteric\nsite of FUT8. a) CAIF titration against a negative control assay, showing no inhibitory\nactivity.\nThere are some additional details that need to be added to the methods and elsewhere as\ndetailed here:\n• Buffers need to be properly defined including the counter-ion. HEPES is presumably\nHEPES-NaOH. % concentrations need to be specified (presumably these are % v/v for Triton\nX-100 and % w/v for BSA).\n(Page 16, Lines 7, 20; Page 17, Line 18; Page 18, Lines 3, 25, 26, 29) to ensure full\nreproducibility.\n• For the HTS and other enzyme assay, what was the final DMSO concentration? Was this\nconsistent throughout? How long were compounds pre-incubated with enzyme before\nmeasuring activity? This is a key consideration for covalent inhibitors as inhibition will be\ndependent on pre-incubation time.\nResponse: We have now clarified the following points in the revised manuscript: In the\nhigh-throughput screening (HTS), the final DMSO concentration was 1% (v/v) (Page 16,\nLine 29). For the concentration-dependent inhibition assays, a lower final DMSO\nconcentration of 0.5% (v/v) was used to minimize any potential nonspecific solvent effects on\nenzyme activity at higher compound concentrations (Page 18, Line 23). All control groups\nwere matched to the same DMSO concentration as the corresponding test compounds within\neach experiment.\nCompounds were pre-incubated with FUT8 on ice for 2 hours before activity\nmeasurement which is a sufficient time for covalent modification. These details have been\nadded to the Methods section (Page 18, Line 19). We appreciate the reviewer’s emphasis on\nthese parameters, which are indeed pivotal for interpreting covalent inhibition data.\n• Please confirm that Z’ was calculated using the population standard deviations (the paper by\nZhang et al., 1999 used population standard deviations although this has been subsequently\ncriticised).\nResponse: We appreciate the reviewer's careful attention to the statistical notation in our\nmanuscript. We sincerely regret the incorrect use of the population standard deviation symbol\n(σ) in our original Methods section, which may have caused confusion. We confirm that all\ncalculations were actually performed using sample standard deviations (s) with Bessel's\ncorrection (denominator = n-1), which is the appropriate approach for our experimental data.\nWe have now corrected the Methods section (Page 17, Lines 6–9) to explicitly state this\n4\ncorrection, now clearly specifying that standard deviations were calculated as sample\nstatistics using Excel's STDEV.S function.\n• Figure 1D. The dose-response curve must use log10 molar drug concentrations. The values\nshould be quoted using pIC50 ± SEM, since the distribution of IC50 estimates is skewed and\ntherefore non-parametric. The dose-response curve for mitoxantrone needs to be repeated\nwith higher drug concentrations because there is insufficient plateau on the right-hand side of\nthe curve. The legend says “Experiments were performed in quadruplicate, with error bars\nrepresenting the standard deviation (n = 4, mean ± SD).” Presumably the displayed\ndose-response curve is a representative independent repeat, with data collected at each drug\nconcentration more than once. N numbers refer to independent repeats. Identical comments\napply to the dose-response curves for figures 4 and 5 and those in the supplementary\ninformation.\nResponse: We thank the reviewer for these critical and constructive suggestions regarding the\nanalysis and presentation of our dose-response data. We have revised the figures and legends\naccordingly. In accordance with the recommendation, the x-axes of all dose-response curves\n(Figures 1d, 4b, 5b, and relevant Supplementary Figures) now represent log molar drug\n10\nconcentration. The inhibitory potency is now quantified and reported as pIC ± SE to account\n50\nfor the skewed distribution of IC estimates, as suggested.\nincreased to 3 mM. While the new data (newly added Supplementary Figure 2c) show a\nclearer trend towards a plateau, full saturation was not achieved even at this highest soluble\nconcentration. Therefore, we conservatively labeled the activity as pIC < 4.16 in original\n50\nmitoxantrone was less potent” without claiming a definitive value (Page 6, Line 11).\nWe have meticulously updated all relevant figure legends to unambiguously define the\nnature of the data and error bars. For example, the legend for Fig. 1d now reads: “Data points\nshow the mean ± SD of quadruplicate technical repeats from one representative independent\nexperiment. The dose-response curve from another independent replicate is provided in\n• Figure S2d. What statistical correction was used for this data (Bonferroni?). Details of the\nstatistical methods need to be given in the supplementary information.\nResponse: We appreciate the reviewer's inquiry about the statistical methods in Figure S2d.\nPlease note that during the revision, the figure numbering has been updated; the panel in\nquestion is now presented as Supplementary Figure 3d. We would like to clarify that:\nSupplementary Figure 3d (previously S2d) directly analyzes the data from Supplementary\nFigure 3c (which itself is an independent biological replicate of Fig. 1f). The statistical\ncomparison in Supplementary Figure 3d was performed between treatment groups within the\nSupplementary Figure 3c experiment only. Therefore, we used two-tailed unpaired Student's\nt-test without multiplicity correction. The analysis in Supplementary Figure 3d uses five\nimaging fields (technical replicates) from the Supplementary Figure 3c experiment. We have\nrevised the figure legend to read:\n“d) Quantitative analysis of invasion counts from panel c. Data represent mean ± SD of\nfive imaging fields from one biological replicate experiment, analyzed by two-tailed unpaired\nStudent's t-test (****P < 0.0001). Biological reproducibility is demonstrated in Fig. 1f and\nSupplementary Figure 3c.”\n• Both synthesized compounds (compound 2 and CAIF) are novel. Novel compounds\ngenerally require 5 pieces of characterization data; why is there no 13C data and spectra for\nCAIF? The authors should report 1H and 13C spectra, HRMS and IR as a minimum.\nResponse: We apologize for the initial omission and thank the reviewer for prompting this\nimportant clarification. We have now supplemented the 1H NMR, 13C NMR, HRMS, and IR\nspectra for both compounds in the Supporting Information (SI) Page 18.\n• There is one error in the supplementary crystallographic Table 2 - Structure FUT8-CAIF\n(9L65), the authors have entered '7824' solvent molecules were identified in the structure -\nwhich need to be corrected.\nfor solvent molecules has now been corrected from ‘7824’ to ‘824’ in the revised table on\nSupporting Information (SI) Page 5.\nSummary and overall comment\nThe manuscript describes the discovery of an allosteric covalent inhibitor of FUT8, which is\nimplicated in cancer metastasis. The authors initially discovered FUT8 inhibitors through a\nlarge-scale screening of small molecules. Subsequently, through careful structural\ninvestigation and rational design, the authors discovered an allosteric covalent inhibitor of\nFUT8, which significantly reduces the degree of fucosylation and cell invasion. The\nmanuscript is clearly described and figures well illustrated.\nResponse: We thank the reviewer for their positive evaluation of our FUT8 inhibitor\ndiscovery approach and results. Their recognition of the study’s structural insights, inhibitor\ndesign, and clear presentation is greatly encouraging as we advance this research program.\nThe main concern here is the author’s claim on specificity without direct supporting evidence.\nThere are no measurements of the off-target activities of CHX, NH125, SSO, or CAIF on\nother fucosyltransferases/other structurally similar proteins. Please add data showing the\neffects of these molecules on other fucosyltransferases. Titration experiments as shown in Fig\n1d, 4b/5b and computational structural analyses may be appropriate.\nResponse: We thank the reviewer for raising this critical point regarding the selectivity of our\ninhibitors. We fully agree that assessing activity against related enzymes is essential to\nvalidate specificity. In response, we have conducted additional selectivity profiling against\nother fucosyltransferases FUT3 and FUT4.\nThe results are highly encouraging: (1) CAIF exhibited no inhibitory activity against\nFUT4 at the tested concentrations. (2) CAIF showed only weak inhibition against FUT3\n(IC > 689 µM). This translates to a remarkable selectivity index of 114-fold for FUT8 over\nThese new data provide direct experimental evidence that CAIF is a selective FUT8\ninhibitor, even when tested against its close relatives. We have now included these selectivity\ndata in Supplementary Figure 8 and described them in the Results section (Page 12, Lines 8–\n12) of the revised manuscript.\nthank the reviewer again for this valuable suggestion.\nSupplementary Figure 8 | Selectivity of CAIF and its dependence on the\nallostericsite of FUT8. b) Dose-response inhibition curve of CAIF against the closely\nrelated fucosyltransferase FUT3. CAIF exhibited very weak activity against FUT3 (pIC50<\n3.2, IC50>689 µM). c) Dose-response inhibition curve of CAIF against the closely related\nfucosyltransferase FUT4. No inhibitory activity was observed.\nMain text -\nSome texts in “Identification and characterization of FUT8 inhibitors” of the Results section\nmay be moved to the Methods section.\nResponse: We thank the reviewer for this suggestion, which helps to improve the clarity and\nlogical flow of the manuscript.\nAs suggested, we have carefully moved the methodological details from the Results\nsection to the corresponding subsections in the Methods. Specifically: The details regarding\nthe composition of the compound libraries screened have been integrated into the\n“High-throughput screening (HTS)” subsection (Page 16, Lines 19–26). The revised Results\nsection now concisely presents the screening outcomes, hit validation, and functional\ncharacterization (Page 6, Lines 1–13). We believe this restructuring makes the manuscript\nmore streamlined and adherent to standard scientific writing conventions.\nIn ‘The allosteric site of FUT8 can be covalently targeted” part of the Results section, it is\nunclear to me whether authors have tested other compounds besides the four named in the\nparagraph. If so, please clarify.\nour manuscript. We confirm that we tested only these four additional compounds (olanexidine,\nSSO, crocetin, and crocin II) in this specific experiment. The wording in the original\nmanuscript was ambiguous. We have now revised the text to make this point explicitly clear\n(Page 10, Lines 25–27).\nFigures -\nFig 1a – There appear to be many other blue dots besides CHX, NH215, and mitoxantrone\nthat lie towards the bottom of the plot near the minus FUT8 controls. However, they are not\ndiscussed in the text. Please add clarification on what they are and why they were not\nincluded in further analyses.\nResponse: We thank the reviewer for this insightful observation. The reviewer is correct that\nFig. 1a shows additional blue dots near the minus FUT8 controls beyond CHX, NH125, and\nmitoxantrone.\nThese additional hits identified in the primary FUT8 inhibitor screening were\nsubsequently subjected to orthogonal validation using an immunofluorescence-based\nsecondary assay. This validation step was critical to distinguish true FUT8 inhibitors from\nfalse positives that may have arisen due to assay-specific artifacts or non-specific effects in\nthe primary screening.\nOur validation results demonstrated that only CHX, NH125, and mitoxantrone\nconsistently exhibited FUT8 inhibitory activity across both screening platforms. The other\ncompounds shown in Fig. 1a failed to confirm activity in the secondary assay and were\ntherefore not pursued for further characterization, including dose-response titration\nexperiments.\nTo provide full transparency of our screening workflow and results, we have now\nincluded Supplementary Table 1: HTS Screening and Transcreener GDP FI Assay\nValidation Results in the supplementary materials. This table documents all compounds\ntested. The rigorous two-tiered screening approach employed in our study ensures that only\ncompounds with reproducible activity across orthogonal assay platforms are advanced.\nside chains in all structure figures.\nResponse: We thank the reviewer for their valuable suggestion to enhance the clarity and\nrigor of our structural figures. We have implemented this feedback in the following ways:\nIn direct response to the suggestion, we have now updated Fig. 2e and Fig. 2g to include\nthe electron density maps (2Fo-Fc) for the bound ligands (CHX and NH125) and their key\ninteracting residues. These updated panels now provide a clear and direct visualization of the\nelectron density supporting our models.\nRegarding Fig. 3 and Supplementary Figure 3 (now Supplementary Figure 4), which\nare designed as structural superposition analyses to compare binding modes across different\ncomplexes, we were concerned that overlaying individual density maps from all superposed\nstructures might compromise the clarity of the key comparative message. However, the\nelectron density for every ligand and key residue featured in these analyses has been\nrigorously validated and is already prominently displayed in the foundational panels (Fig. 2e,\n2g, and new Supplementary Figure 5) that these comparisons are based upon.\nFor Supplementary Figure 6 (now Supplementary Figure 7), we are pleased to confirm\nthat panels Supplementary Figure 7a, 7c, and 7d already include the relevant electron density\nmaps. Panel 7b focuses on comparing the conformation of NH125 between two protein chains,\nand the unambiguous electron density for the ligand in each chain is already presented in Fig.\n2g and Supplementary Figure 5.\nWe hope that our updated figures and this clarification fully address the reviewer's\nconcern. We believe the manuscript now provides a complete and rigorous presentation of the\nstructural data, maintaining both analytical clarity and experimental validation.\nFig 3b – It is difficult for me to see K216 “flipping” in this figure. Please revise to clarify.\nResponse: We thank the reviewer for pointing out the lack of clarity in visualizing the\nconformational change of K216. In direct response to this comment, we have revised Fig. 3b\n(now Fig. 3c) to significantly improve its clarity: We have removed the background protein\ncartoon representation to eliminate visual clutter and allow the focus to rest entirely on the\n11\ncritical loop region containing K216. We have added a double-headed arrow to explicitly\nhighlight the direction of the side chain movement between the two conformational states.\nThese modifications now provide a clear and unambiguous visualization of the “flipping”\nmotion of the K216 side chain. We believe the revised figure effectively addresses the\nreviewer's concern and thank them for this suggestion, which has improved the clarity of our\nmanuscript.\nFig 3c/d/e/f – Please add to the main text the new insights gained from these energy landscape\nexperiments in further details, besides that they are different in the absence and presence of\nNH125.\nResponse: We thank the reviewer for this insightful suggestion. In response, we have\nperformed a more integrated and comparable analysis by collectively aligning the three\nindependent replicate simulations of both the Apo and NH125-bound states to a common\nreference structure before conducting the principal component analysis (PCA). This approach\nallows for a direct and unbiased comparison of the free energy landscapes (FELs) between the\ntwo states on the same conformational basis.\nThe revised FEL analysis, now presented in new Fig. 3d, provides a more detailed\nmechanistic interpretation of how NH125 binding remodels the conformational energy\nlandscape of FUT8. The expanded discussion explicitly describes the stabilization of a new\nglobal minimum representative of a non-productive state, and the destabilization of native\nfunctional basins. These changes collectively suggest that NH125 \"traps\" the enzyme in an\ninactive conformation, thereby providing a dynamic structural basis for its allosteric\ninhibitory mechanism.\nNow this section (Page 10, Lines 1–19) reads:\n“In addition, comparison of the free energy landscape (FEL) projected along the first\ntwo principal components (PC1 and PC2) revealed that NH125 binding remodels the\nconformational ensemble of FUT8 (Fig. 3d). In the Apo state, the FEL exhibits four\nmetastable basins with their relative free energies of 0 kJ/mol (upper-right), 0.63 kJ/mol\n(upper-middle), 0.42 kJ/mol (central), and 9.15 kJ/mol (lower-middle). These basins reflect a\n12\ndynamic conformational distribution, with several low-energy states likely representing\nfunctionally relevant conformations accessible during catalytic cycles. The basin with higher\nfree energy (∼9.15 kJ/mol) may correspond to a less populated, transient state. Upon NH125\nbinding, the FEL is significantly reconfigured, also featuring four distinct basins located with\nrelative minima at 0.95 kJ/mol (upper-left), 0 kJ/mol (upper-middle), 9.78 kJ/mol (central),\nand 1.42 kJ/mol (lower-middle) (Fig. 3d). Notably, NH125 stabilizes a new global minimum\nin the upper-middle region (0 kJ/mol)—a state not prominently sampled in the Apo\nform—while destabilizing several native basins, as reflected by increased energies in other\nregions. Although conformational diversity is retained, the reshaped energy landscape\nfeatures altered barrier heights and basin distributions. This remodeling suggests that NH125\nbinding perturbs the thermodynamic preferences of FUT8, potentially trapping the enzyme in\nnon-productive states and disrupting the conformational cycling required for catalysis.\nTherefore, our simulations indicate that NH125 may act as an allosteric inhibitor not only\nthrough binding but also by reprogramming the free energy landscape of FUT8, thereby\nimpairing its functional dynamics.”\nMethods -\nProtein expression and purification – please indicate protein yield and methods for calculating\nprotein concentration/amount.\nResponse: We have now added the requested details on protein yield and concentration\nquantification to the Methods section of the manuscript (Page 15, Lines 20–23).\nThe revised text now reads:\n“Protein concentrations were determined by measuring the ultraviolet absorbance at\n280 nm using a NanoDrop spectrophotometer, with extinction coefficients calculated from the\namino acid sequence. Typical yields from the purification process were approximately 1.5\nmg/L for FUT8, 0.5 mg/L for FUT3 and the FUT8 mutants, and 0.8 mg/L for FUT4.”\nFUT8 activity assay – please add the concentrations of enzyme used in each experiment.\nResponse: We thank the reviewer for pointing out this omission. We have now explicitly\n13\nstated the concentration of FUT8 enzyme (10 nM) used in the activity assays in the Methods\nsection. (Page 16, Line 19; Page 17, Line 17; Page 18 Line 23).\nCell viability assessment – Please provide in detail the methods of cell treatment referred to in\nthe first sentence. Please indicate controls and standard curves used in the experiments.\nResponse: We have now added the requested details on cell viability assessment (Page 19,\nLines 20–27).\nThe revised text now reads:\n“Cell viability was evaluated using the Cell Counting Kit-8 (CCK-8) assay according to\nthe manufacturer's instructions. Briefly, cells were seeded into transparent 96-well plates at a\ndensity of 5×103 cells per well in 100 µL of complete medium and cultured overnight under\nstandard culture conditions (37 °C, 5% CO ). The next day, cells were treated with serially\n2\ndiluted compounds, while the negative control group received an equivalent volume of DMSO.\nFour replicate wells were used for each treatment condition. Blank wells containing culture\nmedium with the corresponding concentrations of compounds (without cells) were included to\ncorrect for non-specific background absorbance.”\nLectin fluorescence-based cell imaging – Please provide in detail the methods of cell treatment\nreferred to in the first sentence – Please indicate controls and standard curves used in the\nexperiments.\nResponse: We have now added the requested details on the cell treatment (Page 20, Lines 6–\n15).\nThe revised text now reads:\n“GAK or A375 cells were seeded into black, clear-bottom 96-well plates (Greiner) at a\ndensity of 5×103 cells per well and cultured overnight under standard conditions (37 °C, 5%\nCO₂). After attachment, cells were treated with CAIF at concentrations ranging from 0 to 300\nµM (from a 100× DMSO stock diluted in medium) for 72 hours. An equivalent volume of\nDMSO was added to the control wells. Following treatment, cells were washed three times\nwith PBS and fixed with 4% paraformaldehyde (PFA) at room temperature for 20 min. After\n14\nfixation, cells were blocked with 5% (w/v) BSA in PBS at room temperature for 2 h. For\nfucosylation detection, FITC-conjugated AAL or LCA lectins were diluted in PBS containing\n2.5% (w/v) BSA to final concentrations of 2 µg/mL and 5 µg/mL, respectively, and incubated\nwith the cells for 1 h at room temperature in the dark.”\nThe manuscript presents a novel allosteric site on FUT8 and the development of CAIF, a\ncovalent inhibitor, through structure-based drug design. The work integrates high-throughput\nscreening, crystallography, and cellular assays to demonstrate CAIF’s efficacy in suppressing\ncore fucosylation and cancer cell invasion. While the structural and mechanistic insights are\ncommendable, critical gaps in validation, selectivity, and translational relevance significantly\nweaken the study’s impact.\nResponse: We sincerely appreciate the reviewer's thoughtful evaluation of our manuscript\nand their recognition of the novel structural and mechanistic insights regarding FUT8\nallostery. We thank the reviewer for identifying areas where additional validation could\nstrengthen our study's impact. We provide detailed responses to each of the specific concerns\nraised.\nBelow are detailed concerns from this reviewer.\nMajor Concerns\n1. CAIF’s low cytotoxicity and anti-invasive effects are demonstrated only in cellular assays.\nWithout animal model data (e.g., tumor growth/metastasis suppression, pharmacokinetics, or\ntoxicity profiles), the therapeutic potential of CAIF remains speculative. The absence of in\nvivo experiments undermines the clinical relevance of the findings.\nResponse: We fully acknowledge the reviewer's valid concern regarding the need for in vivo\nvalidation of CAIF's therapeutic potential. While we agree that animal studies would\nstrengthen the clinical relevance, we would like to clarify that the primary focus of this\nmanuscript is to: (1) Report the discovery of a previously unrecognized druggable allosteric\npocket on FUT8, and (2) Establish proof-of-concept for a novel inhibition strategy targeting\nthe critical lysine residue adjacent to this pocket.\nWe have now: (1) More clearly framed this scope in the revised discussion (Page 13,\nLines 24–28); (2) Added explicit statements about current limitations regarding drug\ndevelopment (Page 14, Line 27– Page 15, Line 2); (3) Outlined specific plans for future\nmedicinal chemistry optimization and in vivo studies (Page 15, Lines 2–5). We believe these\n16\nclarifications better position the work as a foundational discovery that enables, rather than\nconcludes, subsequent therapeutic development. We sincerely appreciate the reviewer’s\nexpertise in highlighting this key consideration, which will undoubtedly strengthen our\nfollow-up studies.\n2.FUT8 belongs to a family of fucosyltransferases with shared structural motifs. While the\nauthors claim CAIF’s selectivity via its allosteric site, no data are provided to confirm that\nCAIF does not inhibit other FUT enzymes. Selectivity assays against FUT1–FUT7/9 or\nhomology modeling to compare binding sites are essential to validate specificity claims.\nResponse: We thank the reviewer for emphasizing the importance of selectivity, which we\nagree is a critical aspect of characterizing any novel inhibitor. We have directly addressed this\nconcern in our revision by conducting experimental selectivity profiling against the most\nrelevant off-targets.\nIn response to a similar comment from another reviewer, we performed extensive\ndose-response analyses against two more fucosyltransferases: FUT3 and FUT4. Our new data,\nnow presented in Supplementary Figure 8, demonstrate compelling selectivity: CAIF shows\nno inhibition of FUT4 at concentrations up to 300 µM. CAIF exhibits only very weak activity\nagainst FUT3 (IC > 689 µM). This results in a >114-fold selectivity for FUT8 (IC ~ 6 µM)\n50 50\nover FUT3.\nWe have added these results to the Results section (Page 12, Lines 8–12) and believe\nthey substantially strengthen our specificity claims.\nSupplementary Figure 8 | Selectivity of CAIF and its dependence on the\nallostericsite of FUT8. b) Dose-response inhibition curve of CAIF against the closely\nrelated fucosyltransferase FUT3. CAIF exhibited very weak activity against FUT3 (pIC50<\n3.2, IC50>689 µM). c) Dose-response inhibition curve of CAIF against the closely related\nfucosyltransferase FUT4. No inhibitory activity was observed.\n17\n3.The unresolved loops (e.g., residues 77–104) and conformational changes in the\n215NKG217 loop are not rigorously analyzed. A correlation between B-factors\n(crystallography) and RMSF values (MD simulations) would clarify dynamic contributions to\nallostery.\nResponse: We thank the reviewer for raising this point. The loop comprising residues 77–104\nis a region of high intrinsic disorder in FUT8 and, consistent with prior structural studies (e.g.,\nJBC 2020, Nature Communications 2020), has remained unresolved in all reported crystal\nstructures due to its flexibility. As this region is not involved in catalytic regulation, it was\nexcluded from both our crystallization construct and simulation system, allowing us to focus\non well-ordered functional domains.\nIn direct response to the reviewer’s suggestion, we performed an analysis between the\nB-factors from our crystal structure and the per-residue RMSF values from MD simulations.\nThe results show a strong agreement (now included as Supplementary Figure 6c), confirming\nthat the dynamic profiles derived from crystallography and simulations are consistent and\nreinforcing the reliability of our molecular dynamics model.\nRegarding the NKG loop, our simulations did not reveal significant RMSF changes\n215 217\nupon NH125 binding. These results indicate that the dynamic nature of the NKG loop is\n215 217\nan innate property of the FUT8 structure and is not induced or significantly altered by\nallosteric inhibitor binding. This suggests that the functional role of this loop may be related\nto its intrinsic conformational plasticity, which appears to be preserved rather than perturbed\nupon NH125 binding.\nWe have incorporated this interpretation into the revised manuscript (Page 9, Lines 17–\n23) to provide a more nuanced discussion of the loop's dynamics. We thank the reviewer for\nthis constructive feedback, which has helped us to better validate and present the dynamic\naspects of our work.\nabsence and presence of NH125. c) Per-residue profile comparing the crystallographic\nB-factors (orange) from the FUT8-NH125 complex structure and the root-mean-square\nfluctuation (RMSF) values (blue) derived from the MD simulation trajectory. The high\ndegree of correlation between the two profiles indicates that the dynamic motions\nsampled in the MD simulation are consistent with the flexibility observed in the crystalline\nstate. Error bars represent the SD of from three independently repeated simulations. d)\nPer-residue RMSF profile comparing the flexibility of FUT8 in the Apo state (red) and\nin the NH125-bound state (blue) derived from MD simulation trajectories. Error bars\nrepresent the SD of from three independently repeated simulations.\n4.The MD-derived free energy plots (Fig. 3c–f) lack quantitative descriptors (e.g., energy\nbarriers, metastable states). Markov state modeling (MSM) or residue-level hydrogen\nbonding/fluctuation analyses would strengthen mechanistic insights into how NH125/CAIF\nbinding alters FUT8 dynamics.\nResponse: We appreciate the reviewer's excellent suggestion to deepen our mechanistic\nanalysis. In direct response to the comment, we have performed several new analyses to\nprovide quantitative and residue-level insights into how NH125 binding alters FUT8\ndynamics.\n(FELs) by collectively aligning all six molecular dynamics trajectories (three independent\nreplicates each for the Apo and NH125-bound states) to a common reference structure prior to\nprincipal component analysis. This integrated approach, now presented in the new Fig. 3d,\nreveals that NH125 binding significantly reshapes the conformational energy landscape. We\nhave added quantitative descriptors of the metastable basins and their relative free energies in\nthe revised Fig. 3d and Results section (Page 10, Lines 1–19). Specifically, the Apo state\nexhibits four metastable basins with relative energies of 0.00 kJ/mol (upper-right, global\nminimum), 0.63 kJ/mol (upper-middle), 0.42 kJ/mol (central), and 9.15 kJ/mol\n(lower-middle). Upon NH125 binding, the landscape is reconfigured into four new basins at\n0.95 kJ/mol (upper-left), 0 kJ/mol (upper-middle, new global minimum), 9.78 kJ/mol\n(central), and 1.42 kJ/mol (lower-middle). This redistribution, including the stabilization of a\nnew global minimum and the destabilization of native basins, suggests that NH125 binding\nperturbs the thermodynamic preferences of FUT8, effectively trapping the enzyme in\nnon-productive states and disrupting functional conformational cycling.\nFurthermore, as recommended, we performed detailed residue-level analyses to elucidate\nthe dynamic changes underlying the allosteric mechanism. This included a correlation\nanalysis between crystallographic B-factors and MD-derived RMSF values, which showed\nstrong agreement (Supplementary Figure 6c), validating the reliability of our simulations.\nSubsequent flexibility analysis revealed that NH125 binding induces asymmetric changes\nacross the structure: key substrate-binding loops ( RTDKVGTEA and\n366 374\nSISWSAGLHNRYTENS ) showed pronounced increases in flexibility, while regions\n431 446\nsuch as parts of the SH3 domain exhibited slight decreases. Notably, the dynamic NKG\n215 217\nloop showed no significant change upon inhibitor binding, indicating its flexibility is an\ninnate property rather than an induced effect. These results are now included in the main text\n(Page 9, Lines 4–27) and Supplementary Figure 6d.\nWe believe these additions—particularly the quantitative FEL comparison and\nresidue-level dynamics—provide substantially deeper mechanistic insights into how NH125\n20\nreprograms FUT8 energy landscapes and internal dynamics to achieve allosteric inhibition.\nWe are grateful to the reviewer for these valuable suggestions.\nFig. 3 | Allosteric site binders alter the conformation and dynamics of FUT8. d)\nComparative conformational distribution of the Apo (red scatter plot) and NH125-bound\n(blue scatter plot) states, projected onto the first two principal components (PC1 and PC2)\nfrom a combined analysis of all six molecular dynamics trajectories aligned to a common\nreference. Encircled clusters represent metastable conformational states. The relative\nfree energy value (in kJ/mol) for each cluster is annotated, with the global minimum for\neach system designated as 0 kJ/mol. NH125 binding dramatically reorganizes the\nconformational ensemble, stabilizing a new global minimum state and altering the stability\nof functional basins, effectively trapping the enzyme in a non-productive conformation and\nillustrating the structural basis for its allosteric inhibitory mechanism.\n5.The K216-CAIF covalent interaction lacks orthogonal validation (e.g., mass spectrometry,\nactivity assays under reducing conditions). Mutagenesis (K216A) is also required to confirm\nits functional role in inhibitor binding.\ncovalent interaction with K216. We agree that orthogonal evidence is important, and we\nappreciate the opportunity to clarify the robust multi-faceted evidence we have gathered to\nsupport our conclusion.\n(1) High-Resolution Crystal Structure as Definitive Evidence: The most direct and\ndefinitive evidence for the covalent bond formation comes from our high-resolution (1.97 Å)\ncrystal structure (PDB: 9L65). The electron density map unambiguously shows a continuous\ncovalent bond between the inhibitor's warhead and the K216 side chain, leaving no doubt\nabout the nature of the interaction. Crystallography at this resolution is widely considered a\ngold-standard method for defining covalent ligand-protein interactions.\n(2) Validation by a Distinct Compound: As an orthogonal chemical validation, we\ndetermined the structure of FUT8 in complex with a different inhibitor SSO (PDB: 9L63,\n2.66 Å). This compound also forms a well-defined covalent bond with the same K216 residue.\nThe fact that two chemicals both target K216 provides strong, independent chemical\nvalidation of this residue's covalent addressability and reinforces the role of K216 as a key\nanchoring point for inhibitors.\n(3) Functional Role of K216 from Published Mutagenesis: The reviewer's suggestion to\nuse K216A mutagenesis to confirm the functional role is excellent. While we did not perform\nthis mutagenesis ourselves, its consequences have been definitively established in the\nfoundational study (Nature Communications 2020, JBC 2020). As cited in our manuscript,\nthey demonstrated that the K216A mutation completely abolishes FUT8’s catalytic activity.\nThey concluded that K216 is essential for acceptor substrate binding and catalysis. Our\nmechanism of action is in perfect agreement with this established finding: covalent\nmodification of K216 directly occludes and functionally mimics the effect of the K216\nmutation, thereby inhibiting FUT8 by preventing it from engaging its natural substrate.\nIn summary, our structural data provides direct visual proof of covalent binding, a\nsecond structure with a different compound offers orthogonal chemical validation, and\npreviously published functional mutagenesis data robustly confirms the critical functional role\n22\nof the K216 residue. We are therefore confident that the evidence collectively supports our\nmodel.\nWe have now revised the relevant section of the manuscript (Page 11, Lines 13–15) to\ninclude citations to the published studies, thereby strengthening the connection between our\nstructural findings and the established functional essentiality of K216.\n6.As a covalent inhibitor, CAIF’s potential for off-target modifications is not addressed.\nProteomic profiling or kinetic assays with unrelated lysine-dependent enzymes are needed to\nassess specificity.\nResponse: Thank you for raising this important point regarding the potential for off-target\nactivity. We agree that assessing the specificity of covalent inhibitors is crucial. In response,\nwe have conducted several key experiments to address this concern, which are now included\nin the supplementary information.\nFirstly, we performed a critical control experiment to rule out any direct interference of\nCAIF with our assay detection system itself. As shown in Supplementary Figure 8a, CAIF\nexhibited no inhibitory effect on the signal generation of the GDP detection kit. This result\nprovides direct experimental evidence that CAIF does not inhibit the lysine-dependent\nenzyme luciferase—a core component of the detection system—thereby mitigating concerns\nof promiscuous off-target reactivity in our assay context.\nFurthermore, to directly evaluate selectivity within the fucosyltransferase family, we\ntested CAIF against its two more fucosyltransferases, FUT3 and FUT4. As demonstrated in\nSupplementary Figure 8b and 8c, CAIF showed only very weak activity against FUT3 (IC₅₀ >\n689 µM) and no significant inhibition of FUT4 at the tested concentrations. This demonstrates\na clear and substantial selectivity for FUT8 over these highly related enzymes.\nWhile we acknowledge that a broader proteomic profiling study would be valuable for\nfuture translational work, we are confident that the combination of these experimental\nresults—showing no assay interference and high selectivity over the most relevant\noff-targets—supports the conclusion that the inhibitory activity observed in our study is\nspecifically mediated through FUT8.\nsite of FUT8. a) CAIF titration against a negative control assay, showing no inhibitory\nactivity. b) Dose-response inhibition curve of CAIF against the closely related\nfucosyltransferase FUT3. CAIF exhibited very weak activity against FUT3 (pIC < 3.2,\n50\nfucosyltransferase FUT4. No inhibitory activity was observed.\n7.CAIF’s long aliphatic chain raises concerns about solubility and bioavailability.\nPharmacokinetic studies (absorption, distribution, metabolism, excretion) are absent and\ncritical for translational evaluation.\nResponse: We thank the reviewer for raising this important point regarding the\nphysicochemical and pharmacokinetic properties of CAIF. We agree that the presence of a\nlong aliphatic chain often raises legitimate concerns about solubility and bioavailability.\nHowever, we would like to highlight that CAIF is an imidazolium salt—a positively charged\nspecies—which significantly enhances its aqueous solubility. This is evidenced\nexperimentally by our ability to prepare highly concentrated stock solutions of CAIF (100\nmM in DMSO), indicating no inherent solubility limitations for in vitro applications.\nWe fully acknowledge that excellent solubility in DMSO does not directly predict\nfavorable pharmacokinetic properties (ADME) or bioavailability in vivo. As we emphasized\nin our response to Concern 1, the current study is primarily focused on establishing the\nmechanistic principle of targeting this novel allosteric site and the adjacent catalytic lysine.\nProfiling ADME properties and in vivo efficacy is beyond the scope of this foundational work\nbut is a critical next step.\nIn direct response to the reviewer’s comment, we have now explicitly stated in the\nrevised discussion (Page 14, Line 27–Page 15, Line 5) that: “We acknowledge that the\ncurrent CAIF molecule, with its imidazolium salt character and aliphatic chain, primarily\nserves as a mechanistic probe. Its pharmacokinetic properties and overall drug-likeness\n24\nnecessitate further optimization in future translational studies. However, its well-defined\nmechanism of action and high selectivity provide a robust structural foundation and a\ncompelling starting point for such medicinal chemistry efforts. Future work will focus on\nbroader selectivity screening against other glycosyltransferases and proteomic off-target\nstudies to fully characterize its specificity, alongside medicinal chemistry refinements to\nimprove drug-likeness for in vivo applications.”\nWe are in complete agreement with the reviewer that these studies are essential for\ntranslational evaluation, and we sincerely appreciate this guidance, which will strongly shape\nour ongoing research program.\n8.The proposed steric clash between Y220 and Y250 (Fig. 3a) lacks experimental support.\nMutating Y220 to alanine and testing inhibitor efficacy would clarify its role in GDP ribose\nbinding and allosteric inhibition.\nResponse: We thank the reviewer for this excellent and constructive suggestion. We agree\nthat direct experimental evidence is crucial to validate the proposed steric clash mechanism\nbetween Y220 and Y250.\nAs the reviewer recommended, we generated the Y220A mutant protein, purified it, and\nperformed concentration-dependent inhibition assays with CAIF. The inhibitory activity of\nCAIF against the Y220A mutant is drastically reduced, with an IC₅₀ value exceeding 503 µM.\nThis represents a greater than 83-fold loss of potency compared to the wild-type protein (IC₅₀\n~ 6 µM). This is fully consistent with our proposed mechanism. This new result is included in\nnew Supplementary Figure 8d, and described in Page 12, Lines 21–26. We again thank the\nreviewer for prompting us to conduct this key experiment, which has significantly\nstrengthened the mechanistic conclusions of our study.\nsite of FUT8. d)Dose-response inhibition curve of CAIF against the FUT8 Y220A mutant.\nThe loss of potent inhibition highlights the critical role of Y220 for CAIF activity (pIC < 3.3,\n50\n9.The dimeric state of FUT8 under physiological conditions and its impact on inhibitor\nbinding (e.g., cooperative effects) are not discussed. Disruption of the dimer interface (via\nmutagenesis) could reveal its functional relevance.\nResponse: We thank the reviewer for raising this excellent and insightful point regarding the\npotential functional role of FUT8’s dimeric state and its implications for inhibitor binding.\nWe agree that understanding quaternary structure is crucial for fully elucidating the\nmechanism of action of therapeutic agents.\nIn our study, we identified a novel allosteric inhibitory site that is distal to the dimer\ninterface. Our structural analyses (e.g., by comparing the superpositions of the FUT8 dimer\nin the native- and inhibitor-bound states) revealed no significant conformational changes at\nthe dimer interface upon inhibitor binding (new Fig. 3b). Furthermore, our newly performed\nper-residue root-mean-square fluctuation (RMSF) analysis based on molecular dynamics\nsimulations also indicates that inhibitor binding induces minimal changes to the flexibility of\nresidues constituting the dimer interface (new Supplementary Figure 6d). This key\nobservation suggests that the inhibitor's mechanism of action is likely independent of dimer\ndisruption or cooperative effects propagated through the interface.\nWe note that the FUT8 dimer interface involves extensive interactions mediated by its\nstem and SH3 domains, forming a large and stable contact surface. As we now describe in the\nrevised introduction (Page 4, Lines 12-15), this extensive interface makes designing minimal,\ndisruptive point mutations to specifically dissociate the dimer without causing global\n26\nmisfolding a considerable challenge. While targeted mutagenesis of the dimer interface, as\nsuggested by the reviewer, would be a definitive approach to probe its functional role, our\ncurrent structural data indicate that our covalent allosteric inhibitor exerts its effect without\nperturbing the dimeric architecture. Therefore, we have focused our mechanistic studies on\ncharacterizing the direct consequences of ligand binding at the newly discovered allosteric\nsite.\nWe have now included this point in the revised manuscript to acknowledge the dimeric\nstate of FUT8 and to interpret our findings in this context (Page 9, Lines 24–27).\nFig. 3 | Allosteric site binders alter the conformation and dynamics of FUT8.\nb) The FUT8-NH125 (this study, blue) and FUT8 holo (PDB code: 6TKV, green)\nstructures are superimposed. The overall structures align closely, with no major\nconformational changes observed in the global fold upon NH125 binding. The allosteric\ninhibitor binding site (indicated by red ovals) is located distally from the extensive dimer\ninterface (indicated by a dashed box), indicating that the inhibition mechanism is\nindependent of quaternary structural perturbations.\nabsence and presence of NH125. d) Per-residue root-mean-square fluctuation\n(RMSF) of FUT8 in the Apo (red) and NH125-bound (blue) states, derived from molecular\ndynamics simulations. Error bars indicate the standard deviation (SD) across three\nindependent replicates.\nMinor\n1.Fig. 1a: Labels for positive/negative controls and hits are unclear; a legend is needed.\nResponse: We thank the reviewer for this suggestion. We have clarified the labels in Fig. 1a\nand updated the figure legend accordingly to explicitly define all data points and controls.\nThe revised legend now reads:\n“a) Results of the primary screening assay of approximately 2,300 compounds from the\nFDA-approved Drug Library. Data are shown for the tested compounds (concentration: 100\nµM, blue dots), positive control (experimental system without FUT8, orange dots), and\nnegative control (DMSO, black dots). Compounds demonstrating normalized values < 0.6\n(dashed line) were selected for further validation. Red dots indicate validated potential hits.”\n2.Fig. 3c–f: Axes for free energy landscapes are unlabeled; quantitative energy values should\nbe included.\nResponse: We thank the reviewer for this important observation. In the revised manuscript,\nwe have replaced the original panels Fig. 3c–f with a new, quantitatively detailed free energy\nlandscape (Fig. 3d) derived from a combined analysis of all replicate simulations aligned to a\n28\ncommon reference structure. The axes of the new Fig. 3d are now clearly labeled as the first\ntwo principal components (PC1 and PC2). Furthermore, we have explicitly annotated the\nrelative free energy values (in kJ/mol) for each metastable basin directly in the figure and\nprovided a detailed interpretation of these values in the figure legend and Results section\n(Page 10, Lines 1–19). We believe these revisions provide the necessary quantitative depth to\naccurately interpret the energetic changes induced by NH125 binding.\nFig. 3 | Allosteric site binders alter the conformation and dynamics of FUT8. d)\nComparative conformational distribution of the Apo (red scatter plot) and NH125-bound\n(blue scatter plot) states, projected onto the first two principal components (PC1 and PC2)\nfrom a combined analysis of all six molecular dynamics trajectories aligned to a common\nreference. Encircled clusters represent metastable conformational states. The relative\nfree energy value (in kJ/mol) for each cluster is annotated, with the global minimum for\neach system designated as 0 kJ/mol. NH125 binding dramatically reorganizes the\nconformational ensemble, stabilizing a new global minimum state and altering the stability\nof functional basins, effectively trapping the enzyme in a non-productive conformation and\nillustrating the structural basis for its allosteric inhibitory mechanism.\n29\n3.Tables: IC50 values for inhibitors are scattered; a consolidated table in supplementary\nmaterials would improve clarity.\nResponse: As suggested, we have consolidated all IC and pIC ± S.E. data for the\n50 50\ninhibitors into Supplementary Table 2 to improve clarity and accessibility.\nReviewer #4:\n[Comments integrated into Co-Reviewer's report]\nResponse: We thank the reviewer for their time and valuable comments.\nThe manuscript reports the discovery of a novel allosteric site on FUT8 and the development\nof a low-toxicity covalent inhibitor, CAIF (stearic acid-N-hydroxysuccinimide\nester-dimethylimidazolium bromide), through structure-based drug design. High-throughput\nscreening and crystallographic studies revealed that small molecules, such as NH125, bind to a\nchannel-like allosteric pocket, inducing conformational changes that disrupt FUT8 activity.\nLeveraging these insights, they designed CAIF to covalently target lysine K216 within the\nallosteric site of FUT8. CAIF exhibits minimal cytotoxicity and significantly inhibits core\nfucosylation and cancer cell invasion in cellular assays. This work establishes CAIF as a lead\ncompound for further optimization and development, offering a framework for targeting\nglycosyltransferases through allosteric and covalent inhibition strategies.\nThe manuscript is well written and has significant results and therefore can be considered for\npublication in Nature Communication after the responses to following comments are\nincorporated.\nResponse: We sincerely appreciate the positive assessment of our work and the constructive\ncomments provided. We are pleased that the reviewers recognize the significance of our\ndiscovery of a novel allosteric site on FUT8 and the development of our lead compound CAIF.\nBelow we provide point-by-point responses to all specific comments raised, with\ncorresponding revisions made to the manuscript.\n1. The authors claimed CAIF as a specific FUT8 inhibitor based on its interaction with\nallosteric site. However, as all 12 isoforms of FUTs share significant structural similarity and\nthus may possess similar allosteric sites as well. In this context, how authors hypothesized\nthat CAIF will specifically inhibit FUT8, and will not affect the other isoforms of FUTs.\nResponse: We thank the reviewer for raising this important question regarding the structural\nbasis for CAIF's specificity. Our hypothesis that CAIF would selectively inhibit FUT8 was\nbased on the unique structural features of FUT8’s allosteric site, which we have now\nvalidated.\n(1) Structural Uniqueness of the FUT8 Allosteric Site: As demonstrated in our new\ndistinct architecture not shared by other FUTs. Unlike FUT3 or FUT9, FUT8 contains an\nadditional Stem region and SH3 domain, resulting in a completely different spatial\narrangement of its Rossmann domains. The allosteric pocket targeted by CAIF is situated\nprecisely at the interface of these uniquely oriented domains—a region that is structurally\nabsent or highly divergent in other fucosyltransferases.\n(2) Experimental Validation of Selectivity: This structural uniqueness directly\ntranslates to functional selectivity. As also requested by other reviewers, we experimentally\ntested CAIF against two other FUTs: CAIF showed no inhibition of FUT4 (Supplementary\nFig. 8c). Only very weak activity was observed against FUT3 (IC > 689 µM)\n50\n(Supplementary Fig. 8b). This results in a >114-fold selectivity for FUT8 over FUT3,\nrobustly confirming that CAIF does not meaningfully inhibit other FUT isoforms.\nIn summary, the combination of our structural insights and enzymatic profiling confirms\nthat CAIF’s specificity arises from its engagement with an allosteric site unique to FUT8,\nthereby effectively overcoming the challenge of homology within the FUT family.\nSupplementary Figure 10. | Comparative structural analysis reveals the unique\narchitecture of FUT8’s allosteric site among fucosyltransferases. Overall structural\n32\ncomparison of FUT8 (orange, PDB: 9L65), FUT9 (green, PDB: 8D0O), and the\nAlphaFold-predicted model of FUT3 (cyan). Proteins are shown in cartoon representation.\nFUT8 contains the unique Stem region and SH3 domain in addition to the Rossmann\ndomains 1 and 2. Superposition of the central β-sheet of Rossmann domain 1 from FUT8,\nFUT9, and FUT3 highlights the dramatic divergence in the orientation and position of their\nrespective Rossmann domain 2. The FUT8 allosteric pocket pocket is situated at the\ninterface of Rossmann domains 1 and 2. This analysis demonstrates that the allosteric\npocket targeted by CAIF is a unique structural feature of FUT8, which is not conserved in\nother fucosyltransferases such as FUT3 or FUT9, thereby providing a molecular basis for\nits high selectivity.\nSupplementary Figure 8 | Selectivity of CAIF and its dependence on the\nallostericsite of FUT8. b) Dose-response inhibition curve of CAIF against the closely\nrelated fucosyltransferase FUT3. CAIF exhibited very weak activity against FUT3 (pIC50<\n3.2, IC50>689 µM). c) Dose-response inhibition curve of CAIF against the closely related\nfucosyltransferase FUT4. No inhibitory activity was observed.\n2. Authors mentioned that CAIF exhibits minimal cytotoxicity, while significantly inhibiting\nthe core fucosylation, and cancer cell invasion in cellular assays. This statement appears to be\nself-contradictory, and thus needs further explanation.\nResponse: We thank the reviewer for this insightful comment, which allows us to clarify an\nimportant aspect of FUT8 biology and the intended therapeutic strategy behind its inhibition.\nThe apparent contradiction arises from the distinct biological roles of FUT8 in cancer\nprogression.\nAs previously established in the literature (Cancer Cell 2017), FUT8 overexpression is\nnot a primary driver of tumor cell proliferation. Consequently, its genetic knockout or\ninhibition does not typically induce significant cytotoxicity or reduce cell viability. Instead,\nthe key oncogenic function of FUT8 overexpression, particularly in metastatic melanoma cell\n33\nlines as reported, is to potently drive cancer invasion and metastasis. This is corroborated by\nin vivo studies showing that FUT8 silencing suppresses metastasis without affecting primary\ntumor growth.\nTherefore, our goal was to develop a FUT8 inhibitor with precisely this profile: low\ncytotoxicity coupled with potent anti-invasive activity. We believe CAIF represents this class\nof inhibitor. Its minimal cytotoxicity is not a weakness but a highly desirable characteristic, as\nit indicates a wide therapeutic window. The compound selectively targets the pro-metastatic\nfunction of FUT8 (invasion) without incurring toxicity from inhibiting core cellular\nproliferation pathways. This strategy aims to translate into a therapy that specifically\nsuppresses metastasis, the primary cause of cancer mortality, while minimizing side effects on\nhealthy tissues.\nWe have added this information to Page 13, Lines 3–4 and Page 14, Lines 22–23 to\nensure this point is clear to readers.\n3. Various pro- and eukaryotic cell lines are mentioned in the manuscript. Authors needs to\nexplain the selection criteria of these cell lines with respect to FUT8 in the manuscript. The\ndetails of these cell lines also needs to be mentioned.\nResponse: We thank the reviewer for their comment regarding the cell lines used in our study.\nWe clarify our selection criteria and provide further details in the revised manuscript.\nOur study utilized exclusively human cell lines. Their selection was based on their\nexpression of FUT8 and their relevance to studying cancer biology and core fucosylation:\nA375: A human malignant melanoma cell line; GAK: A human renal cell adenocarcinoma\ncell line; HeLa: A human cervical adenocarcinoma cell line; HEK293T: A human embryonic\nkidney cell line.\nThe details regarding their source (ATCC) and culture conditions have now been\nexplicitly stated in the revised “Method” section to ensure clarity and reproducibility.\n4. Since, FUT8 is a bi-substrate enzyme and has two distinct donor and acceptor substrate\nbinding sites. The 2nd and 3rd paragraph in the introduction section should be merged and\n34\nreduced in a single paragraph. While a new paragraph needs to be added highlighting the key\nstructural details of both the binding sites of FUT8.\nResponse: We thank the reviewer for this insightful and constructive suggestion. We agree\nthat providing the structural context of FUT8 as a bi-substrate enzyme is crucial for a\ncomprehensive introduction and for framing our study on inhibitor development. We have\nthoroughly revised the Introduction section accordingly.\nSpecifically, we have: (1) Merged the original 2nd and 3rd paragraphs into a single,\nmore focused paragraph (now the 2nd paragraph of the revised Introduction). This paragraph\nsummarizes the multifaceted therapeutic value of inhibiting FUT8, encompassing its roles in\nanti-tumor immunity, drug resistance, apoptosis, viral replication, and biotherapeutic efficacy.\n(2) Added a new paragraph (now the 3rd paragraph of the revised Introduction) that\nhighlights the key structural details of FUT8. This new paragraph describes its domain\narchitecture, dimeric nature, and, most importantly, the catalytic mechanism and the distinct\nbinding sites for both the donor (GDP-Fuc) and acceptor (N-glycan) substrates, as elucidated\nby recent crystallographic studies. We have made sure to emphasize the critical residues and\nstructural features that define these two binding sites. We believe these revisions have\nsignificantly improved the logical flow of the Introduction. It now first establishes the\nbiological and therapeutic significance of FUT8, then provides the essential structural basis\nfor its function, and finally transitions into the challenges and progress in inhibitor\ndevelopment, which sets the perfect stage for stating the objective of our study.\nThe modified text now appears in the Introduction section (Page 4, Lines 8–28) of the\nrevised manuscript.\n5. Authors have not provided any details regarding the protein (FUT8) expression and\npurification in the Results Section. This needs to be incorporated in the revised manuscript.\nResponse: The detailed procedures for FUT8 expression and purification have been added to\nthe Results section (Page 15, Line 24 – Page 16, Line 14).\n6. They also need to mention the level of purity and yield of FUT8 protein.\nFUT8 protein (>90%) is now indicated in the legend of Supplementary Figure 1b, where the\nSDS-PAGE analysis of the purification process is shown. The typical yield (approximately\n1.5 mg per liter of culture) has been added to the “Protein Expression and Purification”\nsection (Page 16, Line 13) in the Methods.\n7. In in-vitro FUT8 inhibition assays, IC50 values are mentioned without Standard Error of\nMean (S.E.M). Authors need to incorporate the S.E.M with all mentioned IC50 values.\nResponse: As also suggested by Reviewers #1 and #3, we have presented the inhibition data\nas pIC ± S.E. to better reflect the statistical distribution of the values. The complete dataset,\n50\nincluding compounds, IC , and pIC ± S.E., is now provided in Supplementary Table 2.\n50 50\n8. 1H and 13C -NMR data of compound 2 is provided in supplementary materials. However,\nit is not clear which compound is compound 2?\nResponse: Compound 2 is an intermediate in the synthesis of CAIF, as labeled in the\nsynthetic scheme (Supplementary Information, Page 17). We have now further clarified its\nidentity in the revised text to avoid ambiguity.\n9. 13C-NMR chemical shift data of the CAIF is missing.\nResponse: We thank the reviewer for pointing out this oversight. The 13C-NMR chemical\nshift data of CAIF have now been added to the Supplementary Information (Page 18 and\nPage 20) in the revised version.\n10. Citations of figures need to be checked by the authors.\nResponse: We apologize for any inconsistencies. All figure citations have been thoroughly\nchecked and corrected in the revised manuscript to ensure accuracy.\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: We thank the reviewer for their time and valuable comments.\nManuscript ID: NCOMMS-25-06314-T\nManuscript Title: Exploiting human fucosyltransferase 8 allostery with a covalent inhibitor for\ncore fucosylation suppression\nReviewer #1 (Remarks to the Author):\nI am satisfied with the revisions made by the authors. However, there are four points of\nclarification needed in the following paragraph: “The screening quality was assessed by\ncalculating the Z'-factor using the formula: Z' = 1 − [3 × (sp + sn) / |μp − μn|], where μp and μn\nare the means, and sp and sn are the sample standard deviations (with Bessel’s correction, n−1\nin the denominator) of the positive (p) and negative (n) controls, respectively. Calculations were\nperformed using the STDEV.S function in Excel. A Z'-factor value > 0.5 was considered\nindicative of a robust screening system. Experimental data were normalized against control\nvalues, and compounds demonstrating normalized values < 0.6 were selected for further\nvalidation.”\n1. Zhang et al., 1999 J. Biomol. Screen. 4, 67-73 used the population standard deviations in the\ncalculation of Z’ (this has been criticized on statistical grounds, but nevertheless Z’ should be\ncalculated as described in this paper).\nResponse: We thank the reviewer for this important clarification. We have recalculated the Z'-\nfactor using the population standard deviation (employing the STDEV.P function in Excel) and\nhave updated the methodology section accordingly (Page 18, lines 21–24).\n2. The requirement for Z’ to be equal to or greater than 0.5 is given in Table 1 of Zhang et al.\n1999 (above). The authors may wish to cite this paper in the sentence “A Z'-factor value > 0.5\nwas considered indicative of a robust screening system.”\nResponse: We appreciate this suggestion. We have now inserted the appropriate citation to\nZhang et al., 1999 in this sentence to provide the necessary reference for the 0.5 cut-off criterion\n(Page 18, line 24).\n1\n3. Technically, Z’ is a measure of assay performance in the absence of any library compounds\n(the corresponding parameter in the presence of library compounds is Z).\nResponse: We have amended the text to specify that \"The Z'-factor is a measure of assay\nrobustness in the absence of any test compounds\" to ensure this distinction is clear to the reader\n(Page 18, lines 20–21).\n4. The final sentence in this paragraph is ambiguous in that it appears to relate to Z’. I think the\nauthors are saying that compounds which reduced enzymatic activity by 40% or more where\nselected for follow up. This needs to be updated to avoid ambiguity.\nResponse: We agree with the reviewer that the original sentence was ambiguous. The hit\nselection criterion was indeed based on a threshold of inhibition (40% reduction in activity),\nnot directly on the Z'-factor. We have rewritten the sentence to state clearly: \"Compounds that\nreduced FUT8 enzymatic activity by more than 40% (i.e., normalized signal < 0.6) were defined\nas hits and selected for further validation.\" (Page 18, lines 26–27)\nReviewer #2 (Remarks to the Author):\nThank you for the revised manuscript and for thoroughly addressing all referees' comments. I\nthink this version is significantly strengthened and easier to read. Below are suggestions to\nfurther improve the manuscript.\nRegarding my main concern on specificity claims, authors have shown experimentally that their\ninhibitor did not inhibit FUT3 and FUT4 although those are functional and not structural\nhomologs of FUT8. Authors may consider screening the human proteome computationally\n(such as using FoldSeek) for proteins that are structural homologs of FUT8 and test compound\nspecificity on those.\nResponse: We are grateful for this insightful suggestion to evaluate CAIF's activity against\nstructural homologs of FUT8. Accordingly, we performed a FoldSeek search of the human\nproteome, which identified POFUT1 and POFUT2 as the only significant structural homologs\ndespite low sequence identity (~11-12%). We subsequently expressed and purified both\nproteins and tested CAIF inhibition. Notably, CAIF showed no detectable activity against either\nthe revised manuscript (Page 12, line 17–27; Supplementary Figure 9d, e) and robustly\ndemonstrate CAIF's exceptional selectivity, validating the uniqueness of the targeted FUT8\nallosteric pocket. The corresponding methodological details have been updated in the \"Plasmid\nConstruction\", \"Protein Expression and Purification\", and \"Dose-Response Enzyme Activity\nInhibition Assays\" sections on Pages 16–20.\nSupplementary Figure 9. d) Dose-response inhibition curve of CAIF against POFUT1.\ne) Dose-response inhibition curve of CAIF against POFUT2.\nThe addition of the FEL adds an interesting aspect to the potential mechanism of action of the\ndesigned inhibitors and could be useful in further efforts to optimize the binder into a drug. The\nauthors may consider adding a structural representative of each metastable conformational state\nto shed some light on the residue level interactions that were altered when the protein is bound\nto NH125.\nResponse: We thank the reviewer for this excellent suggestion. Following the reviewer's advice,\nwe have now performed a detailed structural analysis of the representative conformations from\neach metastable state in the free energy landscape. To present these findings clearly, we have\ncreated a new supplementary figure (now Supplementary Figure 7) that integrates the\nstructural representatives, their superimposition, and a comparison with the substrate-bound\nholo state.\nSpecifically: Panel a) displays the four representative structures from the NH125-bound\nmetastable states, color-coded by their relative free energies. Panel b) shows their structural\n3\nsuperimposition, revealing high overall similarity and leading us to conclude that the free\nenergy differences likely stem from subtler effects (e.g., conformational entropy, solvation).\nPanels c) and d) compare these states with the active, substrate-bound holo structure (PDB:\n6TKV). This comparison reveals that NH125 binding induces a slight separation of the\nRossmann domains and causes distinct rearrangements in key substrate-binding loops (e.g.,\nloops 365-379 and 436-445 collapse the GDP pocket, while loops 213-220 and 290-300 retract\nfrom the acceptor sugar site).\nThe detailed findings and our interpretation are comprehensively described in the extended\ncaption of the new Supplementary Figure 7. We believe this new analysis significantly\nstrengthens our proposed mechanism by linking the altered free energy landscape to specific\nstructural changes. For the reviewer's convenience, we include this new figure and its detailed\ncaption below:\nSupplementary Figure 7 | NH125 Remodels the Conformational Landscape and Key\nFunctional Loops of FUT8.\n4\na) Representative structures of the four metastable states (I-IV) from the NH125-bound\nFUT8 free energy landscape. The relative free energies are: State I, 0 kJ/mol (global\nminimum), blue; State II, +0.95 kJ/mol, cyan; State III, +1.42 kJ/mol, green; State IV,\n+9.78 kJ/mol, red.\nb) Superimposition of the four representative structures, aligned on the GT-B fold (both\nRossmann domains). The high structural similarity suggests that the observed free\nenergy differences arise not from large conformational changes, but from subtler\neffects—such as conformational entropy, solvation, and minor rearrangements of non-\nbonded interactions—which are captured by the MD simulations but not evident from\nstatic structural alignment.\nc) Superimposition of the four NH125-bound metastable states with the substrate-bound\nholo-FUT8 structure (PDB: 6TKV, black). Aligned on the GT-B fold, the structures\nreveal that NH125 binding at the inter-domain interface induces a slight separation of\nthe Rossmann domains (indicated by arrows) compared to the catalytically competent\nholo state. This provides a structural basis for its allosteric inhibition, potentially\nmisaligning the active site.\nd) Conformational plasticity of loops 213-220, 244-254, 290-300, 365-379, and 436-445\nacross different states. Compared to the substrate-bound holo-FUT8 (PDB: 6TKV),\nthe NH125-bound states exhibit distinct rearrangements in these loops. Specifically,\nloops 365-379 and 436-445 collapse inward, narrowing the GDP-binding pocket, while\nloops 213-220 and 290-300 retract from the acceptor sugar-binding site.\nReviewer #3 (Remarks to the Author):\nThe authors have comprehensively addressed the majority of the concerns raised during the\ninitial review. The revisions significantly strengthen the manuscript, particularly through the\naddition of critical experimental data and enhanced mechanistic insights.\nResponse: We thank the reviewer for their positive feedback and for acknowledging that our\nrevisions have strengthened the manuscript.\nReviewer #5 (Remarks to the Author):\nThis study presents a highly significant advancement in the field of glycosylation-targeted\ntherapeutics by identifying an allosteric pocket in FUT8 and designing the first low-toxicity\ncovalent inhibitor (CAIF) that selectively disrupts FUT8 activity. The work is important\nbecause FUT8-mediated core fucosylation is a critical driver of cancer progression, immune\nevasion, and drug resistance, yet the lack of selective inhibitors has been a major obstacle due\nto the shared substrate specificity of fucosyltransferases. By combining structural insights,\n5\nhigh-throughput screening, and crystallographic validation, the authors not only provide\nevidence of allosteric modulation of FUT8 but also establish CAIF as a lead compound with\npromising biological efficacy in inhibiting cancer cell invasion. Beyond FUT8, this approach\noffers a broadly applicable framework for designing allosteric and covalent inhibitors against\nglycosyltransferases, thereby opening new therapeutic avenues in oncology and immune\nmodulation.\nThe manuscript presents significant results and has justified and supported their data with\nvarious techniques. All the suggested revisions have been incorporated. The support form\nadditional experimental data significantly improves the manuscript. Hence, the manuscript is\nacceptable to be published in the journal in its current form.\nResponse: We are grateful to the reviewer for their thorough and generous assessment of our\nwork. We appreciate their recognition of the significance and potential broad applicability of\nour findings.\nReviewer #6 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: We thank the reviewer for their time and valuable comments.\n6"}]

Decision letter