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The human ether-a-go-go-related voltage-gated cardiac ion channel (commonly known as hERG) conducts the rapid outward repolarizing potassium current in cardiomyocytes (IKr). Inadvertent blockade of this channel by drug-like molecules represents a key challenge in pharmaceutical R&D due to frequent overlap between the structure-activity relationships of hERG and many primary targets. Building on our previous work, together with recent cryo-EM structures of hERG, we set about to better understand the energetic and structural basis of promiscuous blocker-hERG binding in the context of Biodynamics theory. We propose a two-step blocker binding process consisting of: The initial capture step: diffusion of a single fully solvated blocker copy into a large cavity lined by the intra-cellular cyclic nucleotide binding homology domain (CNBHD). Occupation of this cavity is a necessary but insufficient condition for ion current disruption.The IKr disruption step: translocation of the captured blocker along the channel axis, such that: The head group, consisting of a quasi-rod-shaped moiety, projects into the open pore, accompanied by partial de-solvation of the binding interface.One tail moiety packs along a kink between the S6 helix and proximal C-linker helix adjacent to the intra-cellular entrance of the pore, likewise accompanied by mutual de-solvation of the binding interface (noting that the association barrier is comprised largely of the total head + tail group de-solvation cost).Blockers containing a highly planar moiety that projects into a putative constriction zone within the closed channel become trapped upon closing, as do blockers terminating prior to this region.A single captured blocker copy may conceivably associate and dissociate to/from the pore many times before exiting the CNBHD cavity. Lastly, we highlight possible flaws in the current hERG safety index (SI), and propose an alternate in vivo-relevant strategy factoring in: Benefit/risk.The predicted arrhythmogenic fractional hERG occupancy (based on action potential (AP) simulations of the undiseased human ventricular cardiomyocyte).Alteration of the safety threshold due to underlying disease.Risk of exposure escalation toward the predicted arrhythmic limit due to patient-to-patient pharmacokinetic (PK) variability, drug-drug interactions, overdose, and use for off-label indications in which the hERG safety parameters may differ from their on-label counterparts.

Abstract The ventricular action potential (AP) is subserved by an interdependent system of voltage-gated ion channels and pumps that both alter and respond (directly or indirectly) to the dynamic transmembrane potential (Δψm(t)) via voltage-dependent state transitions governing inward and outward ion currents. The native dynamic inward-outward current balance is subject to disruption caused by acquired or inherited loss or gain of function in one or more ion channels or pumps. Building on our previous work, we used a modified version of the O’Hara-Rudy (ORd) model of the undiseased human ventricular cardiomyocyte to study the pro-arrhythmic effects of three types of arrhythmia-inducing perturbations in midmyocytes (M cells): 1. Blockade of the human ether-a-go-go related gene (hERG) K+ channel introduced via a Markov state binding model. 2. Mutation-induced voltage shifts in hERG channel gating, resulting in faster inactivation or slowed recovery of both phosphorylated and non-phosphorylated forms of the channel (known as LQT2 syndrome). 3. Mutation-induced voltage shifts in Nav1.5 gating, resulting in slowed late inactivation of the phosphorylated and non-phosphorylated forms of the channel (known as LQT3 syndrome). We studied the relationships between ion current anomalies and AP morphology as a function of cycle length (CL) and perturbation type/level. The results are summarized as follows: 1. AP duration (APD) is governed directly by Kir2.1 activation (IK1), which is delayed when repolarization is slowed by abnormal net inward tipping of the dynamic inward-outward current balance (reflected in decreased d(Δψm(t))/dt during the late AP repolarization phase). In the case of hERG blockade by non-trappable compounds, the perturbation level consists of the dynamic fractional occupancy of the channel, which is governed by blocker kon relative to the rate of channel opening, pharmacokinetic exposure, and koff (in that order). 2. Arrhythmia progresses from prolonged paced APs → atypical APs (spontaneous and paced) → self-sustaining oscillations. Abrupt transitions between these regimes occur at CL- and perturbation-specific thresholds (denoted as T1, T2, and T3, respectively), whereas intra-regime progression proceeds in a graded fashion toward the subsequent threshold. APD and d(Δψm(t))/dt during the late repolarization phase varied significantly across the 200 APs of our simulations near the T1 threshold at CL = 1/35 min, reflecting increasing instability of the AP generation system. 3. Arrhythmic APs exhibit highly variable cycle-to-cycle morphologies, depending on the perturbation level, type, and phasing between the underlying ion channel states and pacing cycle. 4. Atypical APs may be triggered by typical or atypical depolarizations prior to the T3 threshold, depending on perturbation type/level and phasing relative to CL: 1. APD/CL resides outside of the Goldilocks zone: 1. APD/CL → 1 at shorter CL and/or longer APD, resulting in pro-arrhythmic “collisions” between successive paced APs (APi and APj) within a given cardiomyocyte. We studied this scenario at 60 and 80 beats per minute (BPM), equating to CL = 1/60 and 1/80 min. 2. APD/CL < 1 at longer CL results in spontaneous atypical depolarizations within prolonged paced APs at elevated takeoff Δψm(t) and increased channel phosphorylation levels. We studied this scenario at CL = 1/35 min. 5. APD and d(Δψm(t))/dt during the late repolarization phase become increasingly variable over successive APs on approach to the T1 threshold, which is the possible source of short-long-short sequences observed in the ECG preceding torsades de pointes arrhythmia (TdP). * All atypical depolarizations are solely Cav1.2 (ICa,L)-driven (Δψm(t) falls within the Nav1.5 inactivation window), whereas typical depolarizations are Nav1.5 (INa) + ICa,L-driven. Atypical depolarization versus typical repolarization occurrences are determined by the faster of Cav1.2 and Kir2.1 (IK1) activation (where IK1 becomes increasingly dampened as the minimum Δψm(t) drifts above the Kir2.1 activation window). * Cav1.2 inactivation gates reset to the open position (accompanied by recovery) synchronously with channel closing under control conditions, generating a small ICa,L window current in the process. This current grows toward a depolarizing spike when the lag time between recovery and closing grows above a threshold level. * APs undergo damped oscillatory Cav1.2 recovery/re-inactivation cycles above the T3 threshold, which are refreshed by subsequent pacing signals (nodal or reentrant in origin). Competing Interest Statement The authors have declared no competing interest.

Anti-yeast iso-1 cytochrome c (cyt. c) monoclonal antibodies 2-96-12 and 4-74-6 have closely related epitopes (antigenic determinants). However, while the specificity of 4-74-6 is stringent, 2-96-12 cross-reacts with many evolutionarily related cytochromes c. Such a marked difference in specificity of antibodies with overlapping epitopes may represent unique antibody immunodiversity. Thus, we constructed Fv fragment models consisting of the variable domains of the heavy and light chains of 2-96-12 and 4-74-6 and that of another anti-iso-1 cyt. c as a control to gain insight into the origin of this difference in specificity. Our models show that 4-74-6 and 2-96-12 contain five and two aromatic side chains, respectively, in or near the central area of the antigen-combining site. The side chains of Arg95H (heavy chain) in 2-96-12 and Arg91L (light chain) in 4-74-6 project toward the central area of the combining site in our model. Antigen docking to our Fv models, combined with previous immunological studies, suggests that iso-1 cyt. c Asp60 may interact with Arg95H in 2-96-12 and Arg91L in 4-74-6 and that both epitopes of 2-96-12 and 4-74-7 may include iso-1 cyt. c Leu58, Asp60, Asn62, and Asn63. The effect of the Arg95H to Lys mutation on the antigen binding is also in accord with our model. The difference in specificity may be partly explained by a greater degree of conformational flexibility in and around the central area of the combining site in 2-96-12 compared to 4-74-6 due to differences in aromatic side chain packing.

The poor preclinical and clinical success rates of low molecular weight compounds is partially attributable to the inherent trial-and-error nature of pharmaceutical research, which is limited largely to retrospective data-driven, rather than prospective prediction-driven 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-a-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 are developing and applying a theory on the 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. Our theory 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 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. Competing Interest Statement The authors have declared no competing interest.

We previously reported a first principles multi-scale theory called Biodynamics that attributes cellular functions to sets of coupled molecular and ionic fluxes operating in the non-equilibrium/non-linear dynamic regime. Fluxes build and decay over time and undergo dynamic non-covalent intra- and intermolecular state transitions powered principally by the storage and release of free energy to/from the H-bond networks of external and internal solvation (that we refer to as solvation dynamics) at rates governed by the desolvation and resolvation costs incurred during their entry and exit, respectively. We have thus far examined the functional state transitions of cereblon and COVID Mpro in this context, and now turn to the agonist-induced activating and deactivating state transitions of class A G-protein coupled receptors (GPCRs) and membrane potential-/dipole potential-induced activating and deactivating state transitions of voltage-gated cation channels (VGCCs). We analyzed crystal structures of the activated and deactivated forms of the human β2 adrenergic receptor (β2AR) and cryo-EM structures of the activated and deactivated forms of Nav1.7 channels. We postulate that activation and deactivation of the β2AR is conveyed by switchable changes in transmembrane helix (TMH) orientations relative to extracellular loop 2 (ECL2) and curvature of TMH6 and TMH7, all of which are powered by solvation free energy and kickstarted by agonist binding. The known activation and deactivation mechanisms of Nav1.7 consist of S4 translations toward and away from the extracellular membrane surface, respectively, resulting in S4-S5 linker repositioning, followed by rearrangements of the S5 and S6 helices. The latter TMH conveys channel opening and closing by respectively curving away from and toward the central pore axis. We postulate that all of these rearrangements are likewise powered by solvation free energy and kickstarted by changes in the membrane and dipole potentials. The results of our study may facilitate structure-based design of GPCR agonists/antagonists and mitigation of drug-induced ion channel blockade. Competing Interest Statement The authors have declared no competing interest.

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. Competing Interest Statement The authors have declared no competing interest.

The SARS-CoV-2 Main protease (Mpro) is of major interest as an anti-viral drug target. Structure-based virtual screening efforts, fueled by a growing list of apo and inhibitor-bound SARS-CoV/CoV-2 Mpro crystal structures, are underway in many labs. However, little is known about the dynamic enzyme mechanism, which is needed to inform both structure-based design and assay development. Here, we apply Biodynamics theory to characterize the structural dynamics of substrate-induced Mpro activation, and explore the implications thereof for efficacious inhibition under non-equilibrium conditions. The catalytic cycle (including tetrahedral intermediate formation and hydrolysis) is governed by concerted dynamic structural rearrangements of domain 3 and the m-shaped loop (residues 132-147) on which Cys145 (comprising the thiolate nucleophile and one-half of the oxyanion hole) and Gly143 reside (comprising the other half of the oxyanion hole). In particular: 1) Domain 3 undergoes dynamic rigid-body rotations about the domain 2-3 linker, alternately visiting two conformational states (denoted as Mpro(1) ↔ Mpro(2)). 2) The Gly143-containing crest of the m-shaped loop (denoted as crest B) undergoes up and down translations in concert with the domain 3 rotations (denoted as Mpro(1/down) ↔ Mpro(2/up)), whereas the Cys145-containing crest (denoted as crest A) remains statically in the up position. The crest B translations are driven by conformational transitions within the rising leg of the loop (Lys137-Asn142). We propose that substrates associate to the Mpro(1/down) state, which promotes the Mpro(2/up) state, dimerization (denoted as 2∙Mpro(2/up)-substrate), and catalysis. The structure resets to the dynamic monomeric form upon dissociation of the N-terminal product. We describe the energetics of the aforementioned state transitions, and address the implications of our proposed mechanism for efficacious Mpro inhibition under native-like conditions. Competing Interest Statement The authors have declared no competing interest.

The human ether-a-go-go-related voltage-gated cardiac ion channel (commonly known as hERG) conducts the rapid outward repolarizing potassium current in cardiomyocytes (IKr). Inadvertent blockade of this channel by drug-like molecules represents a key challenge in pharmaceutical R&D due to frequent overlap between the structure-activity relationships of hERG and many primary targets. Building on our previous work, together with recent cryo-EM structures of hERG, we set about to better understand the energetic and structural basis of promiscuous blocker-hERG binding in the context of Biodynamics theory. We propose a two-step blocker binding process consisting of: Diffusion of a single fully solvated blocker copy into a large cavity lined by the intra-cellular cyclic nucleotide binding homology domain (the initial capture step). Occupation of this cavity is a necessary but insufficient condition for ion current disruption.Translocation of the captured blocker along the channel axis (the IKr disruption step), such that: The head group, consisting of a quasi-linear moiety, projects into the open pore, accompanied by partial de-solvation of the binding interface.One tail moiety packs along a kink between the S6 helix and proximal C-linker helix adjacent to the intra-cellular entrance of the pore, likewise accompanied by mutual de-solvation of the binding interface (noting that the association barrier is comprised largely of the total head + tail group de-solvation cost).Blockers containing a highly planar moiety that projects into a putative constriction zone within the closed channel become trapped upon closing, as do blockers terminating prior to this region.A single captured blocker molecule may associate and dissociate from the pore many times before exiting the CNBHD cavity. Lastly, we highlight possible flaws in the current hERG safety index (SI) and propose an alternate in vivo-relevant strategy factoring in: Benefit/risk.The predicted arrhythmogenic fractional hERG occupancy (based on action potential simulations of the undiseased human ventricular cardiomyocyte).Alteration of the safety threshold due to underlying disease. Risk of exposure escalation toward the predicted arrhythmic limit due to patient-to-patient pharmacokinetic variability, drug-drug interactions, overdose, and use for off-label indications in which the hERG safety parameters may differ from their on-label counterparts.

Cellular function depends on heterogeneous dynamic intra-, inter-, and supramolecular structure-function relationships. However, the specific mechanisms by which cellular function is transduced from molecular systems, and by which cellular dysfunction arises from molecular dysfunction are poorly understood. We proposed previously that cellular function manifests as a molecular form of analog computing, in which specific time-dependent state transition fluxes within sets of molecular species ( molecular differential equations (MDEs)) are sped and slowed in response to specific perturbations (inputs). In this work, we offer a theoretical treatment of the molecular mechanisms underlying cellular analog computing (which we refer to as biodynamics ), focusing primarily on non-equilibrium (dynamic) intermolecular state transitions that serve as the principal means by which MDE systems are solved (the molecular equivalent of mathematical integration ). Under these conditions, bound state occupancy is governed by kon and koff, together with the rates of binding partner buildup and decay. Achieving constant fractional occupancy over time depends on: 1) equivalence between kon and the rate of binding site buildup); 2) equivalence between koff and the rate of binding site decay; and 3) free ligand concentration relative to koff/kon (n * Kd, where n is the fold increase in binding partner concentration needed to achieve a given fractional occupancy). Failure to satisfy these conditions results in fractional occupancy well below that corresponding to n * Kd. The implications of biodynamics for cellular function/dysfunction and drug discovery are discussed.

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. The general relationships between surface composition/topology and solvation field properties for both high and low molecular weight (HMW and LMW) solutes. 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. Structure-activity relationships (SAR) based on complementarity between the solvation fields of cognate protein-ligand partners exemplified via LMW degraders.