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General structure-free energy relationships of hERG blocker binding under native cellular conditions

Hongbin Wan1, Kristina Spiru1, Sarah Williams1, Robert A. Pearlstein1•
Novartis1
09 Oct 2021-bioRxiv (Cold Spring Harbor Laboratory)-
TL;DR: In this paper, the authors showed that aqueous non-covalent barriers arise from solute-induced perturbation of the H-bond network of solvating water relative to bulk solvent.
Abstract: We proposed previously that aqueous non-covalent barriers arise from solute-induced perturbation of the H-bond network of solvating water ("the solvation field") relative to bulk solvent, where the association barrier equates to enthalpic losses incurred from incomplete replacement of the H- bonds of expelled H-bond enriched solvation by inter-partner H-bonds, and the dissociation barrier equates to enthalpic + entropic losses incurred during dissociation-induced resolvation of H-bond depleted positions of the free partners (where dynamic occupancy is powered largely by the expulsion of such solvation to bulk solvent during association). We analyzed blockade of the ether-a-go-go-related gene potassium channel (hERG) based on these principles, the results of which suggest that blockers: 1) project a single rod-shaped R-group (denoted as "BP") into the pore at a rate proportional to the desolvation cost of BP, with the largely solvated remainder (denoted as "BC") occupying the cytoplasmic "antechamber" of hERG; and 2) undergo second-order entry to the antechamber, followed by first-order association of BP to the pore. In this work, we used WATMD to qualitatively survey the solvation fields of the pore and a representative set of 16 blockers sampled from the Redfern dataset of marketed drugs spanning a range of pro-arrhythmicity. We show that the highly non-polar pore is solvated principally by H-bond depleted and bulk-like water (incurring zero desolvation cost), whereas blocker BP moieties are solvated by variable combinations of H-bond enriched and depleted water. With a few explainable exceptions, the blocker solvation fields (and implied desolvation/resolvation costs) are qualitatively well-correlated with blocker potency and Redfern safety classification.

Summary (3 min read)

Jump to: [Introduction] – [Binding free energy is contributed principally by solvation, and the implications thereof for hERG blockade] – [Materials and methods] – [5) Bright blue = 100% H visits.] – [Results] – [Discussion hERG blockers undergo atypical binding] – [General hERG safety criteria suggested by our findings] – [A general hERG mitigation strategy suggested by our findings] and [Conclusion]

Introduction

  • And remedies for, inadvertent blockade of the hERG potassium channel by chemically diverse low molecular weight (LMW) hits, leads, preclinical/clinical candidates, and drugs, this liability remains one of the many unsolved problems in pharmaceutical R&D that are typically addressed via black box trial-anderror optimization.
  • The lack of significant progress toward the development of reliable hERG avoidance and mitigation strategies may be attributed to: 1) Poor general understanding of aqueous non-covalent binding between cognate partners, including drugs and targets/off-targets (described below).
  • (which was not certified by peer review) is the author/funder.
  • The authors postulate that blocker promiscuity results from the lack of H-bond enriched "gatekeeper" solvation within the pore, thereby relegating the association free energy barrier to steric size/shape complementarity and induced-fit costs, together with pore-mediated blocker desolvation cost (noting that binding is largely nonspecific in the absence of H-bond enriched "gatekeeper" solvation).

Binding free energy is contributed principally by solvation, and the implications thereof for hERG blockade

  • In several of their previous works, the authors postulated that non-covalent binding under aqueous conditions is governed principally by the solvation free energy contribution, and in particular, the desolvation and resolvation costs incurred during association and dissociation, respectively.
  • The authors further postulate that: 1) Non-covalent free energy is released from binding interfaces during association, principally via mutual desolvation of H-bond depleted solvation (i.e., where such water is replaced by non-polar or weakly polar solute groups).
  • 3) Permeability and solubility (neglecting the dissolution component), both of which indirectly affect hERG blocker occupancy, likewise depend on desolvation and resolvation costs, as follows: a) Low solubility equates to high resolvation (which was not certified by peer review) is the author/funder.
  • The copyright holder for this preprint this version posted October 9, 2021.

Materials and methods

  • Molecular dynamics (MD) simulations are used extensively for predicting intra-and intermolecular structural rearrangements of proteins and other biomolecules [14] [15] [16] [17] .
  • Their simulations are focused on water exchanges between solvation and bulk solvent (which the authors refer to as "solvation dynamics (SD) simulations"), for which they believe that MD approaches are best suited.
  • The counts per voxel are always distributed in a Gaussian-like fashion around the mean H and O counts, which correspond to bulk-like solvation (normalized for the 2 H/O ratio).
  • The copyright holder for this preprint this version posted October 9, 2021.
  • ; https://doi.org/10.1101/2021.10.07.463585 doi: bioRxiv preprint Alternatively, white HOVs may be indicative of mixed donor/acceptor voxel environments or water molecules that are trapped within non-polar cavities (depending on the context).

5) Bright blue = 100% H visits.

  • The LMW SD protocol differs from the HMW protocol described in reference [2] in that LMW structures are fully restrained during the simulations (which would otherwise distribute over a large number of conformations in proportion to their force-field-calculated energies), whereas HMW structures are fully unrestrained (self-limited to high frequency rearrangements among the side chains and loops).
  • All blocker structures were generated using the Build Tool of Maestro release 2021-2 (Schrodinger, LLC), and minimized using the default minimization protocol.
  • Each structure was then simulated using AMBER 20 PMEMD CUDA (GAFF and ff99sb force-fields) for 100 ns in a box of explicit TIP3P water molecules, and the last 10 ns of each trajectory (40,000 frames) was processed into voxel counts via WATMD, and visualized as spheres using PyMol 2.4.1 (Schrodinger, LLC).
  • (which was not certified by peer review) is the author/funder.
  • A subset of the hERG blockers and data compiled by Redfern et al. selected for their study.

Results

  • The authors postulated previously that pore occupancy by non-trappable blockers builds and decays transiently during each action potential (AP) cycle, whereas that of trappable blockers accumulates across APs [5, 6] .
  • The authors postulate that blockers project their BP moieties into P at a rate governed by the full and partial desolvation costs of BP and BC, respectively, as reflected qualitatively in the sizes of the HOVs surrounding those moieties.
  • The copyright holder for this preprint this version posted October 9, 2021.
  • (which was not certified by peer review) is the author/funder.
  • The calculated solvation field within the blocker-accessible region of P is predicted to consist principally of H-bond depleted solvation , in agreement with their previously reported WaterMap results [5] (consistent with the largely non-polar lining of P).

Discussion hERG blockers undergo atypical binding

  • The copyright holder for this preprint this version posted October 9, 2021.
  • 1) Steric constraints on the passage of the bulky, chemically diverse BC moiety of most blockers through the pore entrance, which can be reasonably assumed to depend on timeconsuming induced-fit rearrangements.
  • The fact that blocker association is limited to the open state serves as further evidence for equilibrated channel populations in the cryo-EM preparations.

General hERG safety criteria suggested by our findings

  • The holistic optimization of primary target activity, permeability, solubility, and mitigation of hERG and other off-target activities, depends on the correct understanding of blocker structurekinetic and structure-free energy relationships leading to the arrhythmic tipping point of hERG occupancy under native cellular conditions.
  • The copyright holder for this preprint this version posted October 9, 2021.
  • Arrhythmic occupancy by trappable blockers depends on blocker free Cmax relative to hERG IC50 (accumulating to the 50% level at exposures ≈ the true blocker IC50 [5, 6] ), where the rate of fractional occupancy buildup depends on the slower of the antechamber and pore association steps.
  • ; https://doi.org/10.1101/2021.10.07.463585 doi: bioRxiv preprint 1) Free intracellular blocker exposure > hERG IC50 (concentration-driven) and/or kb approaching the rate of channel activation (kb-driven), where kb depends largely on the cost of expelling H-bond enriched blocker solvation in the absence of polar pore replacements.
  • (C) Distribution of the ratios of the minimum reported hERG IC50/maximum reported Cmax.

A general hERG mitigation strategy suggested by our findings

  • Potent hERG blockers reside within a Goldilocks zone of H-bond depleted and enriched solvation corresponding to optimal solubility, permeability, and hERG binding.
  • Successful hERG mitigation depends on the extent to which drug-hERG and drug-target Goldilocks zones are separable.
  • 2) Disruption of blocker trappability via incorporation of a bulky group within the putative constriction zone in the closed channel state (as the authors reported previously [4] ).
  • 3) Disruption of P-BP shape compatibility (e.g., putatively exemplified by ebastine versus terfenadine).
  • 5) Exploring structure-solubility and permeability relationships, which are likewise governed by solvation free energy and membrane desolvation/resolvation costs.

Conclusion

  • This work is a follow-on to their previous work in which the authors postulated that hERG blockers are first captured by the intracellular antechamber contained within the CNBH and C-linker domains of the channel, followed by the projection of a single R-group (BP) into the open pore (P) [4] .
  • Blocker occupancy is powered primarily by desolvation of H-bond depleted solvation of P, BP, the pore-facing surface of BC, and the peri-pore region of the antechamber (which slows k-b).
  • The key determinants of hERG blockade consist of: 1) Steric size/shape complementarity between BP and P, where BP is typically a nonpolar/weakly polar rod-shaped moiety of almost any chemical composition.
  • P is predicted by both WaterMap and WATMD to contain H-bond depleted and bulk-like solvation, which incurs no desolvation cost during association and a resolvation cost during dissociation equal to the total free energy of H-bond depleted solvation expelled during association.
  • 4) A basic group residing somewhere within BP that is typically used to improve solubility (traded off against a higher desolvation cost).

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Figures (6)

Content maybe subject to copyright    Report

General structure-free energy relationships of hERG blocker binding under native cellular
conditions
Hongbin Wan, Kristina Spiru, Sarah Williams, Robert A. Pearlstein
Novartis Institutes for BioMedical Research, 181 Massachusetts Avenue, Cambridge, MA 02139
Corresponding author: Robert A. Pearlstein, Ph.D.
Phone: +1 617-871-7293
Email: robert.pearlstein@novartis.com
Keywords: arrhythmia, solvation free energy, solvation field, binding free energy, binding
dynamics, cardiosafety, occupancy, channel gating, Na
v
1.5, cryo-EM structures
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted October 9, 2021. ; https://doi.org/10.1101/2021.10.07.463585doi: bioRxiv preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted October 9, 2021. ; https://doi.org/10.1101/2021.10.07.463585doi: bioRxiv preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted October 9, 2021. ; https://doi.org/10.1101/2021.10.07.463585doi: bioRxiv preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted October 9, 2021. ; https://doi.org/10.1101/2021.10.07.463585doi: bioRxiv preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted October 9, 2021. ; https://doi.org/10.1101/2021.10.07.463585doi: bioRxiv preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted October 9, 2021. ; https://doi.org/10.1101/2021.10.07.463585doi: bioRxiv preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted October 9, 2021. ; https://doi.org/10.1101/2021.10.07.463585doi: bioRxiv preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted October 9, 2021. ; https://doi.org/10.1101/2021.10.07.463585doi: bioRxiv preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted October 9, 2021. ; https://doi.org/10.1101/2021.10.07.463585doi: bioRxiv preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted October 9, 2021. ; https://doi.org/10.1101/2021.10.07.463585doi: bioRxiv preprint
Abstract
We proposed previously that aqueous non-covalent barriers arise from solute-induced perturbation
of the H-bond network of solvating water (“the solvation field”) relative to bulk solvent, where the
association barrier equates to enthalpic losses incurred from incomplete replacement of the H-
bonds of expelled H-bond enriched solvation by inter-partner H-bonds, and the dissociation barrier
equates to enthalpic + entropic losses incurred during dissociation-induced resolvation of H-bond
depleted positions of the free partners (where dynamic occupancy is powered largely by the
expulsion of such solvation to bulk solvent during association). We analyzed blockade of the ether-
a-go-go-related gene potassium channel (hERG) based on these principles, the results of which
suggest that blockers: 1) project a single rod-shaped R-group (denoted as “BP”) into the pore at a
rate proportional to the desolvation cost of BP, with the largely solvated remainder (denoted as
“BC”) occupying the cytoplasmic “antechamber” of hERG; and 2) undergo second-order entry to
the antechamber, followed by first-order association of BP to the pore. In this work, we used
WATMD to qualitatively survey the solvation fields of the pore and a representative set of 16
blockers sampled from the Redfern dataset of marketed drugs spanning a range of pro-
arrhythmicity. We show that the highly non-polar pore is solvated principally by H-bond depleted
and bulk-like water (incurring zero desolvation cost), whereas blocker BP moieties are solvated
by variable combinations of H-bond enriched and depleted water. With a few explainable
exceptions, the blocker solvation fields (and implied desolvation/resolvation costs) are
qualitatively well-correlated with blocker potency and Redfern safety classification.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted October 9, 2021. ; https://doi.org/10.1101/2021.10.07.463585doi: bioRxiv preprint
Introduction
Despite many years of intensive investigation into the possible causes of, and remedies for,
inadvertent blockade of the hERG potassium channel by chemically diverse low molecular weight
(LMW) hits, leads, preclinical/clinical candidates, and drugs, this liability remains one of the many
unsolved problems in pharmaceutical R&D that are typically addressed via black box trial-and-
error optimization. However, this approach is challenged by the convoluted nature of target/off-
target potency (including hERG), solubility, permeability, and pharmacokinetics (PK), in which
modulation of one property or behavior can positively or negatively affect one or more of the
others. Lead optimization often culminates in residual hERG activity at the clinical candidate
stage, resulting in potential no-go decisions or mandated clinical thorough QT (TQT) studies,
depending on the benefit/risk ratio. The lack of significant progress toward the development of
reliable hERG avoidance and mitigation strategies may be attributed to:
1) Poor general understanding of aqueous non-covalent binding between cognate partners,
including drugs and targets/off-targets (described below).
2) Consideration of ion channel blockade as a typical binding process, when in fact, it is
highly atypical due to:
a) The absence of native binding function of the ion conduction pathway, which serves
as the binding site for all known blockers. We attribute native binding function, in
general, to:
i. Complementarity between cognate binding partners in both steric
size/shape.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted October 9, 2021. ; https://doi.org/10.1101/2021.10.07.463585doi: bioRxiv preprint
ii. The positions/H-bond propensities of polar groups vis-à-vis H-bond
enriched solvation and non-polar groups vis-à-vis H-bond depleted
solvation [1–3].
We postulate that blocker promiscuity results from the lack of H-bond enriched
“gatekeeper” solvation within the pore, thereby relegating the association free
energy barrier to steric size/shape complementarity and induced-fit costs, together
with pore-mediated blocker desolvation cost (noting that binding is largely non-
specific in the absence of H-bond enriched “gatekeeper” solvation).
b) Two-step binding, consisting of:
i. The capture of a single solvated blocker copy within the large cytoplasmic
cavity of the channel adjoining the pore entrance (denoted as the
“antechamber”), which is lined by the C-linker (denoted as “C”) and cyclic
nucleotide binding homology (CNBH) domains. The on-rate is described
by k
c
∙ [free antechamber] ∙ [free blocker] (typical second-order binding, in
which k
on
is capped at the 10
9
M
-1
s
-1
diffusion limit), where k
c
denotes the
blocker-antechamber rate constant. The blocker-bound antechamber
concentration builds and decays with free cytoplasmic concentration, which
in turn, builds and decays with cardiac tissue uptake and plasma clearance,
respectively.
ii. Projection of a single quasi-rod-shaped blocker moiety (denoted as “BP”)
into the open pore (denoted as “P”) [4]. The on-rate is described by k
b
∙
[antechamber-bound blocker] (atypical first-order binding), where k
b
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted October 9, 2021. ; https://doi.org/10.1101/2021.10.07.463585doi: bioRxiv preprint
denotes the BP-P association rate constant. The blocker off-rate is described
by k
-b
∙ [bound blocker], where k
-b
is the dissociation rate constant of BP.
3) The lack of differentiation between trappable and non-trappable blockers, together with
poor understanding of structure-trappability relationships. Non-trappable blocker
occupancy builds and decays during each channel gating cycle (the peak magnitude of
which occurs at the intracellular C
max
, where C
max
is the peak exposure during a given
dosing cycle), whereas trappable blockers accumulate to their maximum fractional
occupancy (given by the Hill equation: free C
max
/(free C
max
+ IC
50
)), which decays during
the clearance phase of the pharmacokinetic (PK) curve (noting that k
-b
is not usurped by
channel closing). The highest maximum occupancy of non-trappable blockers is achieved
when k
b
≈ the channel opening rate and k
-b
≈ the channel closing rate (i.e., noting that k
-b
is usurped by the rate of channel closing), whereas trappable blockers accumulate to their
maximum occupancy over multiple channel gating cycles as the intracellular C
max
builds
to n ∙ IC
50
, where n is occupancy multiplier (n = 1 equates to 50% occupancy, n = 19 equates
to 95% occupancy, etc.).
4) Measurement of hERG blockade under equilibrium conditions in status quo hERG assays,
and the use of such data for generating hERG structure-activity relationship (SAR) models
when native binding between hERG and non-trappable blockers is, in practice, highly non-
equilibrium in nature. Typical native drug-target systems operate on far longer timescales
than the ~350 ms open channel time window of hERG, commensurate with significantly
slower requirements for k
on
and k
off
. In our previous works, we simulated hERG blockade
in the context of the cardiac AP using a modified version of the O’Hara-Rudy model of the
undiseased human ventricular cardiomyocyte [5,6]. Occupancy of hERG by certain
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted October 9, 2021. ; https://doi.org/10.1101/2021.10.07.463585doi: bioRxiv preprint
Citations
More filters
Posted Content•DOI•
Solvation dynamics-powered structure and function of multi-molecular cellular systems exemplified by non-equilibrium cereblon-degrader-CK1α ternary complex formation

[...]

Hongbin Wan1, Vibhas Aravamuthan, Sarah Williams, C. Wartchow, José S. Duca, Robert A. Pearlstein •
Novartis1
31 Oct 2021-bioRxiv
TL;DR: In this article, the structural dynamics of the COVID main protease were derived from the surface composition/topology and solvation field properties for both high and low molecular weight (HMW and LMW) solutes.
Abstract: Cellular functions are executed via a form of analog computing that is based on the switchable covalent and non-covalent states of multi-molecular fluxes (i.e., time-dependent species/state concentrations) operating in the non-linear dynamics regime. We and others have proposed that the non-covalent states and state transitions of aqueous fluxes are powered principally by the storage and release of potential energy to/from the anisotropic H-bond network of solvating water (which we refer to as the "solvation field"), which is a key tenet of a first principles theory on cellular structure and function (called Biodynamics) that we outlined previously. This energy is reflected in water occupancy as a function of solute surface position, which can be probed computationally using WATMD software. In our previous work, we used this approach to deduce the structural dynamics of the COVID main protease, including substrate binding-induced enzyme activation and dimerization, and product release-induced dimer dissociation. Here, we examine: 1) The general relationships between surface composition/topology and solvation field properties for both high and low molecular weight (HMW and LMW) solutes. 2) The general means by which structural dynamics are powered by solvation free energy, which we exemplify via binding between the E3 ligase CUL4A/RBX1/DDB1/CRBN, LMW degraders, and substrates. We propose that degraders organize the substrate binding surface of cereblon toward complementarity with native and neo substrates, thereby speeding the association rate constant and incrementally slowing the dissociation rate constant. 3) Structure-activity relationships (SAR) based on complementarity between the solvation fields of cognate protein-ligand partners exemplified via LMW degraders.

2 citations

Posted Content•DOI•
Improving the preclinical and clinical success rates of LMW drugs depends on radical revisions to the status quo scientific foundations of medicinal chemistry: a case study on COVID Mpro inhibition

[...]

Robert A. Pearlstein, Hongbin Wan, Sarah Williams
25 Apr 2023-bioRxiv
TL;DR: In this paper , the authors argue that the limit of understanding of cellular function/dysfunction and pharmacology based on empirical principles (observation/inference) has been reached, and that further progress depends on understanding these phenomena at the first principles theoretical level.
Abstract: The poor preclinical and clinical success rates of low molecular weight (LMW) compounds can be partially attributed to the inherent trial-and-error nature of pharmaceutical research, which is limited largely to retrospective data-driven, rather than prospective prediction-driven human relevant workflows stemming from: 1) inadequate scientific understanding of structure-activity, structure-property, and structure-free energy relationships; 2) disconnects between empirical models derived from in vitro equilibrium data (e.g., Hill and Michaelis-Menten models) vis-à-vis the native non-equilibrium cellular setting (where the pertinent metrics consist of rates, rather than equilibrium state distributions); and 3) inadequate understanding of the non-linear dynamic (NLD) basis of cellular function and disease. We argue that the limit of understanding of cellular function/dysfunction and pharmacology based on empirical principles (observation/inference) has been reached, and that further progress depends on understanding these phenomena at the first principles theoretical level. Toward that end, we have been developing and applying a theory (called “Biodynamics”) on the general mechanisms by which: 1) cellular functions are conveyed by dynamic multi-molecular/-ionic (multi-flux) systems operating in the NLD regime; 2) cellular dysfunction results from molecular dysfunction; 3) molecular structure and function are powered by covalent/non-covalent forms of free energy; and 4) cellular dysfunction is corrected pharmacologically. Biodynamics represents a radical departure from the status quo empirical science and reduction to practice thereof, replacing: 1) the interatomic contact model of structure-free energy and structure-property relationships with a solvation free energy model; 2) equilibrium drug-target occupancy models with dynamic models accounting for time-dependent drug and target/off-target binding site buildup and decay; and 3) linear models of molecular structure-function and multi-molecular/-ionic systems conveying cellular function and dysfunction with NLD models that more realistically capture the emergent non-linear behaviors of such systems. Here, we apply our theory to COVID Mpro inhibition and overview its implications for a holistic, in vivo relevant approach to drug design.
References
More filters
Journal Article•DOI•
Relationships between preclinical cardiac electrophysiology, clinical QT interval prolongation and torsade de pointes for a broad range of drugs : Evidence for a provisional safety margin in drug development

[...]

William S. Redfern1, Leif Carlsson1, A.S Davis1, W G Lynch2, I MacKenzie3, S Palethorpe1, Peter K. S. Siegl4, I. Strang1, A.T Sullivan5, Rob Wallis6, A. J. Camm7, Tim Hammond1 •
AstraZeneca1, Loughborough University2, Covance3, United States Military Academy4, GlaxoSmithKline5, Pfizer6, St George's Hospital7
01 Apr 2003-Cardiovascular Research
TL;DR: The dataset confirms the widely-held belief that most drugs associated with TdP in humans are also associated with hERG K(+) channel block at concentrations close to or superimposed upon the free plasma concentrations found in clinical use.
Abstract: Objective To attempt to determine the relative value of preclinical cardiac electrophysiology data (in vitro and in vivo) for predicting risk of torsade de pointes (TdP) in clinical use. Methods Published data on hERG (or I(Kr)) activity, cardiac action potential duration (at 90% repolarisation; APD(90)), and QT prolongation in dogs were compared against QT effects and reports of TdP in humans for 100 drugs. These data were set against the free plasma concentrations attained during clinical use (effective therapeutic plasma concentrations; ETPC(unbound)). The drugs were divided into five categories: (1) Class Ia and III antiarrhythmics; (2) Withdrawn from market due to TdP; (3) Measurable incidence/numerous reports of TdP in humans; (4) Isolated reports of TdP in humans; (5) No reports of TdP in humans. Results Data from hERG (or I(Kr)) assays in addition to ETPC(unbound) data were available for 52 drugs. For Category 1 drugs, data for hERG/I(Kr) IC(50), APD(90), QTc in animals and QTc in humans were generally close to or superimposed on the ETPC(unbound) values. This relationship was uncoupled in the other categories, with more complex relationships between the data. In Category 1 (except amiodarone), the ratios between hERG/I(Kr) IC(50) and ETPC(unbound) (max) ranged from 0.1- to 31-fold. Similar ranges were obtained for drugs in Category 2 (0.31- to 13-fold) and Category 3 (0.03- to 35-fold). A large spread was found for Category 4 drugs (0.13- to 35700-fold); this category embraced an assortment of mechanisms ranging from drugs which may well be affecting I(Kr) currents in clinical use (e.g. sparfloxacin) to others such as nifedipine (35700-fold) where channel block is not involved. Finally, for the majority of Category 5 drugs there was a >30-fold separation between hERG/I(Kr) activity and ETPC(unbound) values, with the notable exception of verapamil (1.7-fold), which is free from QT prolongation in man; this is probably explained by its multiple interactions with cardiac ion channels. Conclusions The dataset confirms the widely-held belief that most drugs associated with TdP in humans are also associated with hERG K(+) channel block at concentrations close to or superimposed upon the free plasma concentrations found in clinical use. A 30-fold margin between C(max) and hERG IC(50) may suffice for drugs currently undergoing clinical evaluation, but for future drug discovery programmes, pharmaceutical companies should consider increasing this margin, particularly for drugs aimed at non-debilitating diseases. However, interactions with multiple cardiac ion channels can either mitigate or exacerbate the prolongation of APD and QT that would ensue from block of I(Kr) currents alone, and delay of repolarisation per se is not necessarily torsadogenic. Clearly, an integrated assessment of in vitro and in vivo data is required in order to predict the torsadogenic risk of a new candidate drug in humans.

1,411 citations

Journal Article•DOI•
Simulation of the Undiseased Human Cardiac Ventricular Action Potential: Model Formulation and Experimental Validation

[...]

Thomas O'Hara1, László Virág2, András Varró2, András Varró3, Yoram Rudy1 •
Washington University in St. Louis1, University of Szeged2, Hungarian Academy of Sciences3
26 May 2011-PLOS Computational Biology
TL;DR: A model for the undiseased human ventricular action potential (AP) which reproduces a broad range of physiological behaviors is developed and experiments for rate dependence of Ca2+ (including peak and decay) and intracellular sodium ([Na+]i) in undISEased human myocytes were quantitatively reproduced by the model.
Abstract: Cellular electrophysiology experiments, important for understanding cardiac arrhythmia mechanisms, are usually performed with channels expressed in non myocytes, or with non-human myocytes. Differences between cell types and species affect results. Thus, an accurate model for the undiseased human ventricular action potential (AP) which reproduces a broad range of physiological behaviors is needed. Such a model requires extensive experimental data, but essential elements have been unavailable. Here, we develop a human ventricular AP model using new undiseased human ventricular data: Ca2+ versus voltage dependent inactivation of L-type Ca2+ current (ICaL); kinetics for the transient outward, rapid delayed rectifier (IKr), Na+/Ca2+ exchange (INaCa), and inward rectifier currents; AP recordings at all physiological cycle lengths; and rate dependence and restitution of AP duration (APD) with and without a variety of specific channel blockers. Simulated APs reproduced the experimental AP morphology, APD rate dependence, and restitution. Using undiseased human mRNA and protein data, models for different transmural cell types were developed. Experiments for rate dependence of Ca2+ (including peak and decay) and intracellular sodium ([Na+]i) in undiseased human myocytes were quantitatively reproduced by the model. Early afterdepolarizations were induced by IKr block during slow pacing, and AP and Ca2+ alternans appeared at rates >200 bpm, as observed in the nonfailing human ventricle. Ca2+/calmodulin-dependent protein kinase II (CaMK) modulated rate dependence of Ca2+ cycling. INaCa linked Ca2+ alternation to AP alternans. CaMK suppression or SERCA upregulation eliminated alternans. Steady state APD rate dependence was caused primarily by changes in [Na+]i, via its modulation of the electrogenic Na+/K+ ATPase current. At fast pacing rates, late Na+ current and ICaL were also contributors. APD shortening during restitution was primarily dependent on reduced late Na+ and ICaL currents due to inactivation at short diastolic intervals, with additional contribution from elevated IKr due to incomplete deactivation.

1,012 citations

Journal Article•DOI•
New insights about HERG blockade obtained from protein modeling, potential energy mapping, and docking studies

[...]

Ramy Farid1, Tyler Day1, Richard A. Friesner2, Robert A. Pearlstein1•
Schrödinger1, Columbia University2
01 May 2006-Bioorganic & Medicinal Chemistry
TL;DR: A homology model of the homo-tetrameric pore domain of HERG is created using the crystal structure of the bacterial potassium channel, KvAP, as a template and key aromatic groups of the blockers are predicted to form multiple simultaneous ring stacking and hydrophobic interactions among the eight aromatic residues lining the pore.

413 citations

Journal Article•DOI•
Cryo-EM Structure of the Open Human Ether-a-go-go-Related K(+) Channel hERG.

[...]

Weiwei Wang1, Roderick MacKinnon1•
Howard Hughes Medical Institute1
20 Apr 2017-Cell
TL;DR: The molecular structure of hERG is determined to 3.8 Å using cryo-electron microscopy, and the central cavity has an atypically small central volume surrounded by four deep hydrophobic pockets, which may explain hERG's unusual sensitivity to many drugs.

374 citations

Journal Article•DOI•
Toward a pharmacophore for drugs inducing the long QT syndrome: insights from a CoMFA study of HERG K(+) channel blockers.

[...]

Andrea Cavalli1, Elisabetta Poluzzi1, Fabrizio De Ponti1, Maurizio Recanatini1•
University of Bologna1
25 Jul 2002-Journal of Medicinal Chemistry
TL;DR: A pharmacophore for QT-prolonging drugs, along with a 3D QSAR (CoMFA) study for a series of very structurally variegate HERG K(+) channel blockers, and a theoretical screening tool able to predict whether a new molecule can interact with the HERG channel and eventually induce the long QT syndrome.
Abstract: In this paper, we present a pharmacophore for QT-prolonging drugs, along with a 3D QSAR (CoMFA) study for a series of very structurally variegate HERG K(+) channel blockers. The blockade of HERG K(+) channels is one of the most important molecular mechanisms through which QT-prolonging drugs increase cardiac action potential duration. Since QT prolongation is one of the most undesirable side effects of drugs, we first tried to identify the minimum set of molecular features responsible for this action and then we attempted to develop a quantitative model correlating the 3D stereoelectronic characteristics of the molecules with their HERG blocking potency. Having considered an initial set of 31 QT-prolonging drugs for which the HERG K(+) channel blocking activity was measured on mammalian transfected cells, we started the construction of a theoretical screening tool able to predict whether a new molecule can interact with the HERG channel and eventually induce the long QT syndrome. This in silico tool might be useful in the design of new drug candidates devoid of the physicochemical features likely to cause the above-mentioned side effect.

354 citations

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Frequently Asked Questions (13)
Q1. What have the authors contributed in "General structure-free energy relationships of herg blocker binding under native cellular conditions" ?

The authors proposed previously that aqueous non-covalent barriers arise from solute-induced perturbation of the H-bond network of solvating water ( “ the solvation field ” ) relative to bulk solvent, where the association barrier equates to enthalpic losses incurred from incomplete replacement of the Hbonds of expelled H-bond enriched solvation by inter-partner H-bonds, and the dissociation barrier equates to enthalpic + entropic losses incurred during dissociation-induced resolvation of H-bond depleted positions of the free partners ( where dynamic occupancy is powered largely by the expulsion of such solvation to bulk solvent during association ). The authors analyzed blockade of the ethera-go-go-related gene potassium channel ( hERG ) based on these principles, the results of which suggest that blockers: 1 ) project a single rod-shaped R-group ( denoted as “ BP ” ) into the pore at a rate proportional to the desolvation cost of BP, with the largely solvated remainder ( denoted as “ BC ” ) occupying the cytoplasmic “ antechamber ” of hERG ; and 2 ) undergo second-order entry to the antechamber, followed by first-order association of BP to the pore. In this work, the authors used WATMD to qualitatively survey the solvation fields of the pore and a representative set of 16 blockers sampled from the Redfern dataset of marketed drugs spanning a range of proarrhythmicity. The authors show that the highly non-polar pore is solvated principally by H-bond depleted and bulk-like water ( incurring zero desolvation cost ), whereas blocker BP moieties are solvated by variable combinations of H-bond enriched and depleted water. ( which was not certified by peer review ) is the author/funder. 

The key determinants of hERG blockade consist of:1) Steric size/shape complementarity between BP and P, where BP is typically a non-polar/weakly polar rod-shaped moiety of almost any chemical composition. 

Hbond enriched solvation is further enhanced by basic groups, which additionally speed kb (as a function of increasing pKa) due to electrostatic attraction with the negative field within P. Since blocker potency is typically enhanced by basic groups, it follows that the electrostatic kb-speeding contribution of such groups typically outweighs the kb-slowing desolvation contribution. 

2) Permeability is proportional to the desolvation cost of H-bond enriched blocker solvation,which additionally depends on polar groups for replacing the H-bond enriched solvation of membrane phospholipid head groups (the rationale underlying the Pfizer Rule of 5). 

The highest maximum occupancy of non-trappable blockers is achieved when kb ≈ the channel opening rate and k-b ≈ the channel closing rate (i.e., noting that k-b is usurped by the rate of channel closing), whereas trappable blockers accumulate to their maximum occupancy over multiple channel gating cycles as the intracellular Cmax builds to n ∙ IC50, where n is occupancy multiplier (n = 1 equates to 50% occupancy, n = 19 equates to 95% occupancy, etc.). 

2) The canonical association barrier consists principally of the total desolvation cost of thebinding interface (contributed by H-bond enriched solvation), and canonical dissociation barrier consists principally of the cost of resolvating H-bond depleted solvation positions expelled during association [1–3,9–12]. 

The overall asymmetric distribution of HOVs among BP and BC blocker moieties isconsistent with their proposed binding mode, in which BP (exhibiting the lower desolvation cost/lower association free energy barrier) projects into the pore, while BC (exhibiting the higher desolvation cost/higher association free energy barrier) remains within the solvated antechamber. 

Molecular dynamics (MD) simulations are used extensively for predicting intra- and intermolecular structural rearrangements of proteins and other biomolecules [14–17]. 

Lead optimization often culminates in residual hERG activity at the clinical candidate stage, resulting in potential no-go decisions or mandated clinical thorough QT (TQT) studies, depending on the benefit/risk ratio. 

Optimal positioning and pKa of a basic group on BP used for solubility (noting thatincreased solubility as a f(pKa) results in higher desolvation cost, but also speeds kb electrostatically). 

All blocker structures were generated using the Build Tool of Maestro release 2021-2 (Schrodinger, LLC), and minimized using the default minimization protocol. 

The t-butyl acid group located on the distal end of BP explains the weak hERG activity of fexofenadine, whereas the weaker potencies of propafenone and desipramine can be explained by the more distal position of the basic group on BP relative to that of Class 1-2 blockers. 

The t-butyl acid and hydroxymethyl groups of the BP moiety in fexofenadine (a metabolite of terfenadine) are spanned by numerous HOVs (Figures 3G and H), consistent with higher BP desolvation cost and the Class 4 designation of this drug.