potency/exposure relationships. Redfern et al. developed the following hERG SI based on potency, clinical PK data (including the highest reported Cmax), and reported TdP cases for 100 marketed drugs (including anti-arrhythmic hERG blockers), ranging from no reported cases to TdP-linked withdrawals [12] :
Upper safe human TFPCmax ≤ 1 30 • in vitro hERG IC50
(1)
The Redfern SI implicitly accounts for exposure escalation via a wide 30-fold margin between the in vitro hERG IC50 and TFPCmax, which as we demonstrate below, translates to effectively zero tolerated hERG occupancy at the maximum anticipated therapeutic exposure in humans. However, the safety margin for compounds exhibiting residual hERG activity at the projected TFPCmax cannot be assessed systematically or tailored to benefit/risk via the all-or-none Redfern criterion.
Considerable time and effort may be invested in hERG mitigation to the limit of detection, which is subject to the following caveats:
1) Constraints on chemical mitigation imposed by the typically high overlap among the structural and physico-chemical properties promoting hERG and primary target potency,
All calculations and visualizations were performed using Maestro 2019-1 (Schrodinger, LLC, Portland, OR) on a representative set of canonical hERG blockers taken from reference [12] (Table   1) , as well as trappable and non-trappable propafenone analogs taken from reference [25] ( Table   2 ). The structures were built using LigPrep and energy minimized using Macromodel (MMFF force-field, and default parameters). The hERG structure (PDB code 5VA2) [26] was prepared (hydrogen addition, Asn, Gln, His orientations) using PPrep. Blocker docking sites and the solvation properties thereof were characterized using SiteMap. Overlay models were generated via manual superposition on a template consisting of the GDN detergent crystallized with Nav1.4
(PDB code 6AGF) [27] , which we propose as the general intra-cellular binding mode for voltagegated ion channel blockers. We emphasize the theory/knowledge-, rather than computation-driven, underpinnings of this work, the core tenets of which include: 1) the dependence of dynamic occupancy on binding partner buildup and decay under non-equilibrium conditions in vivo,
wherein the applicability of equilibrium binding metrics (e.g. G, Kd, Ki, IC50, EC50) is limited primarily to the in vitro setting [17, 18] ; and 2) the predominant contribution of de-solvation and re-solvation costs to non-covalent kinetic barriers under aqueous conditions (a core tenet of Biodynamics theory [18, 23] ). Table 1 . Representative hERG blockers studied in this work [12] . Table 2 . Published trappable and non-trappable hERG blockers studied in this work [25] . Table 3 . Candidate COVID-19 therapies known to block hERG [28, 29] (noting that the HCQ IC50 was estimated from the reported percent inhibition (35%) at 3 M using the Hill equation).
We characterized the energetic, structural, and chemical drivers of blocker binding and trappability using modeled structures of known trappable and non-trappable compounds, together with a set of recently published cryo-EM structures of the full length open hERG channel (PDB codes 5VA1, 5VA2, 5VA3) [26] ) [26] . Our overall findings suggest that blocker binding is governed by the following contributions: 1) Steric shape and size complementarity between blockers and the pore binding region.
2) Blocker de-solvation rate (proportional to the de-solvation free energy cost) vis-à-vis the channel-opening rate, together with pore and blocker re-solvation rates (proportional to the re-solvation free energy cost) vis-à-vis the channel-closing rate.
3) Blocker basicity/pKa vis-à-vis the negative field within the pore, which speeds the association rate.
Next, we outline a more in vivo-relevant hERG mitigation strategy based on these findings. Lastly, we revisit the status quo hERG safety assessment protocol, and propose a more in vivo-relevant strategy centered on the putative relationship between dynamic occupancy, PK, and cellular arrhythmogenesis.
The hERG channel is a tetrameric protein comprised principally of Per-Arnt-Sim (PAS), transmembrane voltage sensing, transmembrane pore, C-linker, and intra-cellular cyclic nucleotide binding homology (CNBH) domains (one per monomer) ( Figure 1 ). We speculate that the CNBH domain serves to bind the independent tetrameric subunits together via the expulsion of intersubunit H-bond depleted solvation (noting that the absence of this domain in Nav1.5 and Cav1.2 is consistent with the uni-chain composition of those channels). A large intra-cellular tunnel-like cavity within the combined CNBH/C-linker domains (which we refer to as the "outer vestibule")
is observed in the cryo-EM structure, through which blockers must necessarily transit to reach the pore region. viewed along the pore axis from the intra-to extra-cellular direction. The pore (highlighted in cyan) and CNBH domain (highlighted in purple) cavities reside at the distal and proximal ends of the structure, respectively. The helical C-linker domains reside at the four corners of the channel (highlighted in orange), with the voltage-sensing domains (highlighted in light green) partitioned above in the membrane. The C-linker domains reside between the pore and CNBH domains, forming a continuous cavity with the latter (which we refer to as the "outer vestibule"). The PAS domain and linker were omitted from the crystallized protein construct. (B-C) Longitudinal cutaway views of the intra-cellular region of the ion conduction pathway, consisting of the Clinker lined cavity (highlighted in orange), which is sandwiched between the intra-cellular pore entrance (highlighted in green) and CNBH domain cavity (highlighted in magenta).
It is widely assumed that bound hERG blockers are fully buried within the pore domain based on mutagenesis data and in silico docking [30, 31] , which we had likewise assumed in our previous work [24] . However, this hypothesis is inconsistent with the large size/volume of many blockers, wherein binding would likely depend on an unreasonably high level of induced fit. A glycodiosgenin (GDN) detergent molecule ( Figure 2A ) bound to the closed state of the human voltagegated Nav1.4 channel observed in a recent cryo-EM structure (PDB code 6AGF) [27] offers a possible clue as to the general binding mode of cation channel blockers. GDN straddles the pore and pore entrance with its rigid polycyclic moiety buried within, and its two unresolved disaccharide moieties projecting out to the cytoplasm (reminiscent of a drain-plug) ( Figure 2B -C).
We proceeded to test whether hERG blockers could potentially bind in a similar "drain-plug-like" fashion using a ligand-based overlay model (noting that the cytoplasmic hERG blocker moiety would necessarily reside within the outer vestibule). We overlaid the hERG and Nav1.4 structures, and fit our reference set of hERG blockers (see Materials and methods) to the common pore-bound moiety of GDN ( Figure 3 ). According to our model, the typically Y-or L-shaped hERG blockers project a single quasi-linear/rod-shaped moiety into the pore. We assume that basic nitrogen-containing moieties, when present, reside within this region. Total burial within the pore is unlikely (other than relatively small quaternary alkylamines), given the diverse range of size, chemical composition, and structural variability of the known blocker chemical space. Our analysis suggests the existence of three primary blocker-hERG docking interfaces within the ion conduction pathway (Figure 4 ), as follows:
1) The region of the lumen enclosed by the four two-helix bundles of the C-linker residing adjacent to the intra-cellular pore entrance (denoted "C"). Land Y-shaped blockers likely project moieties (denoted "BC") into this region, whereas linear blockers may not.
2) The pore lumen spanning between the intra-cellular entrance and intra-cellular face of Tyr652 (denoted "P"). Blockers project a single quasi-linear moiety (denoted "BP") into this region. The occupied distance along the pore axis varies among blockers.
3) The upper region of the pore, adjacent to the side chains of Tyr652, which we refer to as "Y"). Blockers project the terminal BP group into this region (denoted "BY"), noting that only a subset of blockers terminate in this region (i.e. those that maximally occupy P). Terfenadine was docked manually into clusters of SiteMap site points (white spheres) described
in Materials and methods. The butterfly-shaped diphenylmethane tail moiety of terfenadine is complementary in shape to the C-linker helix (noting that butterfly-shaped bisaryl groups are relatively commonplace among hERG blockers [17] ).
Blockers necessarily translocate into the pore via the outer vestibule, relegating hERG blockade to a two-step process consisting of:
1) A capture step, in which a single solvated blocker copy diffuses from the cytoplasm into the CNBH domain cavity (which in and of itself is unlikely to block ion conduction into and through the pore).
2) A longitudinal translocation step, in which the captured copy shifts from the CNBHD cavity into the C-linker cavity and pore entrance ( Figure 6A The extent to which BP penetrates into the pore is determined by its length relative to that of P, or the steric size of BY relative to the diameter of the pore entrance. b) Positioning BC into C, accompanied by full or partial mutual de-solvation of C and BC (noting that BP insertion and BY binding are interdependent processes).
Putative H-bond enriched solvation of BC is represented as a red "bumper" in Figure 6C . BC is restricted to the C-linker cavity via steric clashing with side chains at the pore entrance (color-coded yellow in Figure 6 ), and as such, blockers containing BP moieties shorter than the longitudinal pore length necessarily terminate below the Y docking site.
The total mutual de-solvation costs of C-BC, P-BP, and Y-BY putatively serves as the overall blocker association barrier. The results of our previous WaterMap calculations suggest that P is solvated almost entirely by bulk-like and H-bond depleted water, thereby avoiding disruption of the negative field by ordered H-bond enriched water [17] . The outer vestibule was omitted in our WaterMap calculations, which were performed prior to determination of the cryo-EM structure.
H-bond depleted solvation is localized to the non-polar side chains of the pore, and most notably the intra-cellular facing surface of Tyr652 (the Y docking site) located adjacent to the selectivity filter at the distal end of P. We used SiteMap to characterize the solvation within the pore of the cryo-EM structure (data not shown). As expected, the results are consistent with those of our previous WaterMap calculations [17] . 
We proposed previously that non-covalent association and dissociation free energy barriers consist principally of H-bond enriched and depleted solvation free energy (relative to the free energy of bulk solvent), respectively [23] . The rate of blocker association depends on the total solvation free energy of H-bond enriched water expelled from the binding interface (i.e. the mutual blockerchannel de-solvation cost). The rate of blocker dissociation depends on the magnitude of the total free energy cost of re-solvating H-bond depleted positions within the dissociated binding interface (i.e. the total blocker and channel re-solvation cost). H-bond enriched solvation incurs zero resolvation cost during dissociation, whereas H-bond depleted solvation incurs zero de-solvation cost during association. In our previous work, we demonstrated that the pore in hERG is solvated almost exclusively by H-bond depleted and bulk-like water [17] corresponding to low de-solvation and high re-solvation costs, respectively. The rate of non-trappable blocker binding, therefore, depends largely on the de-solvation cost of the pore-binding blocker moiety, and the dissociation rate is proportional to the channel-closing rate or blocker dissociation rate, whichever is faster (minimally ~2 s -1 , which we refer to as the "koff floor" [17] ). The rate of pore insertion by trappable blockers is likewise proportional to the de-solvation cost of the pore-binding moiety, whereas the koff floor is ~0.7 s -1 [17] . It is therefore apparent that dynamic blocker occupancy is influenced heavily by the rates of channel opening and closing, which is zero in competition binding (where the channels are static), and typically sub-physiological in patch clamp assays. As such, binding measurements performed under non-physiological conditions do not translate reliably to the in vivo setting for non-trappable blockers exhibiting koff < the floor and kon < the rate of channel opening. Percent inhibition in all such cases is increasingly overestimated as kon decreases relative to the channel-opening rate (see [17, 18] ). Blocker binding kinetics can be qualitatively inferred from conventional structure-activity relationships (neglecting channel-gating dynamics), as follows:
1) Significant decrease in the percent inhibition/occupancy of a given blocker analog:
a) kon slowing due to increased blocker de-solvation cost incurred during association via the addition of, or increased polarity of, a polar BC, BP, or BY blocker group (especially BY). Examples of reduced hERG activity putatively due to increased polarity are available in [33] and [34] .
b) koff speeding due to decreased blocker re-solvation cost during dissociation via the deletion of, or decreased polarity of, a polar BC, BP, or BY blocker group (especially BY). c) Slowed kon due to reduced pKa of a basic group (or removal thereof) within the BP moiety.
2) Significant increase in the percent inhibition of a given blocker analog:
a) kon speeding due to decreased blocker de-solvation cost incurred during association via the deletion of, or decreased polarity of, a polar BC, BP, or BY blocker group (especially BY).
b) koff slowing due to increased blocker and/or hERG re-solvation cost during dissociation via the addition of, or decreased polarity of, a non-polar blocker group (especially in BY).
c) kon speeding due to increased pKa of a basic group (or addition thereof) within the BP moiety.
We inferred certain structure-kinetics relationships from the structure-activity relationships of two proprietary in-house datasets based on the aforementioned principles. A large activity cliff in dataset 1 is attributable to increased de-solvation cost of the pyridazyl versus pyridyl moieties predicted to bind in site C (Table 4 ) and the 1-versus 2-pyridyl combined with Cl versus F ( Table   5 ). The structure-property relationships underlying the solvation differences among these groups is unobvious. A large activity cliff in dataset 2 can be attributed to increased de-solvation cost of the oxadiazole versus sulfadiazole and oxapyrrole groups predicted to project into a cluster of Hbond depleted solvation in site Y (Table 6 ), which putatively manifests as slowed kon due to the loss of H-bonds of polar group solvation transferred to bulk solvent. We note that key local solvation effects may be masked in global changes in scalar logP and solubility. Non-trappable blocker-induced expulsion of this solvation is expected to slow koff, minimally to the rate of channel closing (below which channel closing becomes rate-determining). koff slowing in the case of shorter BP groups falling short of the Y site depends on the expulsion of H-bond depleted solvation from the C region (equating to an enthalpic re-solvation cost during dissociation), together with the entropic de-solvation contribution. 
The concept of blocker trappability was proposed by Starmer et al. [35] and Stork et al. [36] (referred to by Armstrong et al. [37] and Mitcheson et al. [38] as the "foot-in-the-door" model). Windisch et al. studied structure-trappability relationships for a series of analogs around the known trappable blocker, propafenone [25] . We set about to explain this relationship by overlaying and comparing the predicted conformational properties of these analogs vis-à-vis our proposed In summary, blocker binding is driven largely by:
1) Concurrent steric shape complementarity to the C (L-or Y-shaped blocker moieties), P (quasi-linear blocker moieties), and Y regions of the channel.
2) Principally blocker de-solvation costs during translocation from the CNBHD cavity into the C, P, and Y (optionally) regions of the channel.
3) Principally, channel re-solvation costs at the Y and C sites incurred during blocker-hERG dissociation.
group along the longitudinal axis of P (Figures 3 and 4 , right).
Trappable blockers either terminate within P prior to the putative constriction zone formed by Phe652 side chains, or project a planar group into the constriction zone. Trappable blockers accumulate occupancy over multiple channel gating cycles (i.e. heartbeats), peaking at the intracellular free Cmax, whereas non-trappable blocker occupancy builds and decays within the ventricular hERG population during each cycle. The maximum dynamic occupancy (corresponding to koff/kon) across all cycles within a dosing period peaks at the free intra-cellular Cmax for both trappable and non-trappable blockers. Trappable blocker occupancy is agnostic to gating frequency (i.e. on-rate merely governs the rate of buildup to the maximum occupancy)
whereas non-trappable blocker occupancy depends on kon and koff relative to the channel opening and closing rates, respectively.
The Redfern SI (equation 1) was derived from a comparison of measured hERG IC50, therapeutic Cmax, and maximum observed Cmax for 100 marketed drugs classified according to TdP propensity:
withdrawn drugs versus multiple reported cases, versus isolated cases, versus no reported cases, versus anti-arrhythmic hERG blocking drugs. We have identified the following potential deficiencies in the derivation and application of this metric: 
However, it is apparent that the TFPCmax equates to the therapeutic total intra-cellular Cmax 
Blocker occupancy (proportional to percent inhibition) is channel state-and time-dependent, and therefore out of the scope of equation 3: 
Status quo hERG mitigation and preclinical safety assessment is typically guided by high throughput in vitro IC50 data and animal PK data. hERG mitigation depends on guidance from accurate data capable of unambiguously resolving the true SAR [41] , which is difficult to acquire via high throughput in vitro measurements ( Figure 11 ) (noting the often flat hERG free energy landscape across primary target potency-sparing chemical series). Mitigation is aimed minimally at satisfaction of the Redfern SI, and maximally at total abrogation of hERG activity (i.e. IC50 > the detectability limit) among clinical drug candidates. However, total hERG mitigation combined with optimal primary target potency and other requirements, is rarely achieved in practice, 
quantitative hERG data (which may extend to clinical candidates in some cases). Instead, we propose the following approach:
1) Minimize TFPCmax via kinetically tuned drug-target binding, defined as the optimization of kon to the rate of binding site buildup (explained in detail in [17] and Selvaggio et al. [18] ).
2) Test for blocker trappability based on the structural criteria outlined above (Figures 7-8) ,
together with frequency independent percent inhibition using an appropriate patch clamp protocol [36] .
3) Perform trappable  non-trappable chemical transformations wherever possible based on the structural criteria outlined above (noting that non-trappable blocker occupancy does not accumulate over time).
4) SAR and safety assessment should be based on percent inhibition, rather than IC50 data (noting that error may be amplified in best-fit dose-response curves, secondly, that percent inhibition is a more direct measure of blocker-hERG occupancy at concentrations of interest, and thirdly, fractional hERG occupancy in vivo cannot be predicted from IC50 in the absence of TFICmax). Sufficient accuracy is needed to confirm critical SAR and inform safety assessment. Data accuracy can be gauged as follows:
a) Similar observed trends between orthogonal assays (e.g. patch clamp and radioligand binding).
b) Convergence of replicate runs in each assay. c) Self-consistency of hERG SAR (the existence of identifiable hERG structureactivity drivers across a given chemical series).
Overlay blocker scaffolds to our drain-plug model, and identify the BC, BP, and BY features. Increase the de-solvation cost (reflected in the polarity [33, 34] ) of these features to slow kon (especially BY, when present), while maintaining on-target activity. 
Chloroquine and its hydroxy derivative (HCQ) are currently of interest as potential SARS-CoV-2
anti-viral therapies [42] . The anti-viral efficacy of these drugs is unconfirmed (and is under considerable scrutiny [43] ), whereas both drugs are known to exhibit pro-arrhythmic and arrhythmic levels of hERG blockade in the clinical setting (based on data from the Federal Adverse Event Reporting System summarized in [44] , a recent COVID-19 patient cohort [45] , and reports of QT prolongation above 500 ms in COVID-19 patients [46] ). Furthermore, hERG is likely blocked as well by the principal metabolites of HCQ, which consist of desethylchloroquine, desethylhydroxychloroquine, and bisdesethylchloroquine [47] . The reported hERG IC50s for chloroquine and HCQ are 2.5 M [28] and ~5.6 M (the latter of which we estimated from the Hill equation based on 35% inhibition at 3.0 M reported in [29] ). Both drugs fit unambiguously to our hERG blocker overlay model (shown for chloroquine in Figure 13) IC50 ). Chloroquine and HCQ lack definitive preclinical hERG safety margins vis-à-vis the Redfern criterion, which is exacerbated by putative hERG trappability and low plasma protein binding (possibly traded against high lysosomal uptake [49] ). HCQ has nevertheless realized many prescription-years as an anti-malarial [50] and auto-immune therapy, the safety profile of which is well-understood for those specific indications [43] . All safety indices are context dependent, and can be exceeded (and the TI lost) in cases of significant exposure escalation above the established therapeutic level due to additive multi-drug effects, and/or the impact of underlying disease on drug clearance or the safety threshold. LQT monitoring and exposure control are essential for offlabel HCQ administration, given:
1) That arrhythmia can result from even transient excursions in exposure  the arrhythmic threshold. The high potential for cardiovascular impairment in COVID-19 patients may result in a downward shift of our predicted arrhythmic hERG occupancy level (which is based on the undiseased heart).
2) Co-administration with other pro-arrhythmic drugs, including azithromycin (hERG IC50 = 219 M [51] ) may lower the safe exposure level of HCQ (noting that reported cases of azithromycin-induced arrhythmia [44] have been attributed to intra-cellular Na + loading [51] ).
3) That HCQ metabolites likewise block hERG and exhibit long t1/2.
4) The high potential for impaired clearance due to renal compromise in COVID-19 patients.
5) The potential for DDIs in COVID-19 patients undergoing multi-drug therapies.
6) That intravenous HCQ administration results in up to 19-fold higher blood levels (2, 436 ng/ml [48] , translating to ~13 M) compared with oral administration. We note that this level is more than 2-fold above our estimated hERG IC50. 
We used non-equilibrium structure-free energy (Biodynamics) principles, combined with a threedimensional ligand-based alignment of a set of trappable and non-trappable hERG blockers and published cryo-EM structures of hERG and Nav1.4 [26, 27] , to understand the non-equilibrium structure-free energy relationships governing blocker-hERG binding. Specifically, we propose that mutual blocker-hERG de-solvation and re-solvation costs that respectively govern kon and koff are localized to a set of blocker-specific docking interfaces (denoted as C-BC, P-BP, and Y-BY) in the C-linker and pore cavities. This hypothesis is consistent with our previous ligand-based analysis of hERG blocker chemical space (based on the Redfern dataset [39] and internal data), including neutral bisaryl, basic bisaryl, and alkylamine-containing scaffolds [17] .
hERG safety assessment and mitigation are necessarily weighted toward the prevention of false negatives over false positives (erring on the side of caution), which is entirely justified given the acute, life-threatening implications of TdP, combined with: 1) Uncertainty in the true cause-effect relationship between measured hERG inhibition and arrhythmic risk.
2) The chicken-egg nature of hERG assessment/mitigation during the lead optimization stage, stemming from the lack of in vivo ECG and PK data needed to establish a human-relevant safety margin. Critical unknown parameters during this stage include:
a) The predicted TFPCmax in humans.
b) The predicted TFICmax at the TFPCmax in humans, noting that percent hERG inhibition cannot be assessed from IC50s in the absence of TFICmax information.
Furthermore, TFICmax may be influenced by potentially unquantifiable lysosomal trapping, membrane binding, primary target binding, and off-target binding.
c) Confirmed hERG IC50 and percent inhibition data, noting that such data is typically measured using less accurate high throughput techniques (Figure 11 ), and furthermore, may overestimate the dynamic occupancy of non-trappable blockers.
Simultaneous satisfaction of the highly stringent Redfern criterion (translating approximately to zero tolerated hERG inhibition at the TFPCmax) and therapeutic target criteria is extremely challenging, time-consuming, and failure prone. The question is whether absolute hERG safety at all possible exposures, and across all indications and patient populations is an acceptable tradeoff against slowed progress and failure among hERG-afflicted R&D programs addressing unmet medical needs (which our findings can only help inform, but not answer).
We predicted arrhythmogenic propensity in terms of fractional occupancy of the ventricular hERG channel population in the undiseased human heart at exposures ranging between the TFICmax and maximum FICmax expected from DDIs or overdose (noting that the possible need for dose escalation during clinical trials must be considered in establishing the hERG SI). We define the human-relevant SI in terms of the (exposure) between the true TFICmax (typically > the efficacious free Cmax, allowing for metabolic clearance) and the arrhythmic exposure, which may be significantly less in the presence of cardiac dysfunction. The actual safety margin depends on the maximum (exposure) due to PK variability, DDIs, overdose, or dose escalation ( Figure 14 ).
In contrast, the Redfern SI begins with near zero tolerated hERG inhibition at the TFPCmax, translating to a 100% safety margin relative to the putative arrhythmic inhibition level.
Furthermore, the Redfern SI appears biased toward potent trappable blockers, which based on our previous analysis [17] , demand a greater safety margin than non-trappables. Figure 14 . Our proposed safety margin is defined in terms of the fractional hERG occupancy at the therapeutic intra-cellular free Cmax relative to that at the predicted arrhythmic exposure level in the otherwise normal heart. This metric differs from the Redfern SI, in which hERG percent inhibition is restricted to effectively zero at the TFPCmax (corresponding to the bright green doubleheaded arrow in the lower part of the figure). The safe (exposure) depends on the benefit/risk of the disease indication, but should always correspond to hERG occupancy far below the arrhythmic level (assumed here as 50% of the open/conducting channel population [17] ), and for all practical purposes, QT < 500 ms.
In this work, we emphasize the need for human-relevant hERG safety prediction and mitigation criteria during the preclinical stages of drug discovery, accounting for the true relationships between chemical structure and in vivo-relevant dynamic hERG occupancy, and between dynamic occupancy and the pro-arrhythmic effects thereof on the otherwise normal human cardiac AP. Both relationships seem to be poorly understood under the conventional wisdom (including reliance on static equilibrium binding metrics and near zero tolerance for blockade at therapeutic exposures).
We propose that blocker association is driven by steric shape complementarity, blocker desolvation cost at key docking interfaces (together with electrostatic attraction in the case of basic compounds), and dissociation is driven largely by mutual blocker and binding site re-solvation costs (noting that the dissociation rate of non-trappable blockers under physiological conditions is necessarily faster than the rate of channel closing). We further propose a drain-plug-like canonical binding mode, in which blockers straddle the pore and C-linker cavities (analogous to the binding mode of GDN in the cryo-EM structure of Nav1.4 [27] ), projecting R-groups into non-bulk-like solvation sites in the C-linker (C/BC), and pore (P/BP, Y/BY) regions of the outer vestibule. We attribute trappability to a putative constriction zone that forms during channel closing, with which only planar blocker groups (or the absence of groups in this position) are sterically compatible.
Disruption of blocker binding is putatively achievable via trappable  non-trappable chemical transformations, together with incorporation of polar groups at BC, BP, and BY, thereby increasing the blocker de-solvation cost at those sites. We showed that the Redfern SI equates effectively to zero hERG occupancy at the TFPCmax, and is weighted heavily toward the prevention of false negative blockers from entering clinical trials at the possible expense of false positives. Our approach, guided by accurately measured in vitro percent inhibition and human-relevant in vivo PK data, may help to minimize trial-and-error mitigation, and lower uncertainty in human-relevant hERG safety predictions.