The voltage-sensing domain of a hERG1 mutant is a cation-selective channel.

A cationic leak current known as an "omega current" may arise from mutations of the first charged residue in the S4 of the voltage sensor domains of sodium and potassium voltage-gated channels. The voltage-sensing domains (VSDs) in these mutated channels act as pores allowing nonspecific passage of cations, such as Li+, K+, Cs+, and guanidinium. Interestingly, no omega currents have been previously detected in the nonswapped voltage-gated potassium channels such as the human-ether-a-go-go-related (hERG1), hyperpolarization-activated cyclic nucleotide-gated, and ether-a-go-go channels. In this work, we discovered a novel omega current by mutating the first charged residue of the S4 of the hERG1, K525 to serine. To characterize this omega current, we used various probes, including the hERG1 pore domain blocker, dofetilide, to show that the omega current does not require cation flux via the canonical pore domain. In addition, the omega flux does not cross the conventional selectivity filter. We also show that the mutated channel (K525S hERG1) conducts guanidinium. These data are indicative of the formation of an omega current channel within the VSD. Using molecular dynamics simulations with replica-exchange umbrella sampling simulations of the wild-type hERG1 and the K525S hERG1, we explored the molecular underpinnings governing the cation flow in the VSD of the mutant. We also show that the wild-type hERG1 may form water crevices supported by the biophysical surface accessibility data. Overall, our multidisciplinary study demonstrates that the VSD of hERG1 may act as a cation-selective channel wherein a mutation of the first charged residue in the S4 generates an omega current. Our simulation uncovers the atomistic underpinning of this mechanism.

Assessing hERG1 Blockade from Bayesian Machine-Learning-Optimized Site Identification by Ligand Competitive Saturation Simulations.

Drug-induced cardiotoxicity is a potentially lethal and yet one of the most common side effects with the drugs in clinical use. Most of the drug-induced cardiotoxicity is associated with an off-target pharmacological blockade of K+ currents carried out by the cardiac Human-Ether-a-go-go-Related (hERG1) potassium channel. There is a compulsory preclinical stage safety assessment for the hERG1 blockade for all classes of drugs, which adds substantially to the cost of drug development. The availability of a high-resolution cryogenic electron microscopy (cryo-EM) structure for the channel in its open/depolarized state solved in 2017 enabled the application of molecular modeling for rapid assessment of drug blockade by molecular docking and simulation techniques. More importantly, if successful, in silico methods may allow a path to lead-compound salvaging by mapping out key block determinants. Here, we report the blind application of the site identification by the ligand competitive saturation (SILCS) protocol to map out druggable/regulatory hotspots in the hERG1 channel available for blockers and activators. The SILCS simulations use small solutes representative of common functional groups to sample the chemical space for the entire protein and its environment using all-atom simulations. The resulting chemical maps, FragMaps, explicitly account for receptor flexibility, protein-fragment interactions, and fragment desolvation penalty allowing for rapid ranking of potential ligands as blockers or nonblockers of hERG1. To illustrate the power of the approach, SILCS was applied to a test set of 55 blockers with diverse chemical scaffolds and pIC50 values measured under uniform conditions. The original SILCS model was based on the all-atom modeling of the hERG1 channel in an explicit lipid bilayer and was further augmented with a Bayesian-optimization/machine-learning (BML) stage employing an independent literature-derived training set of 163 molecules. BML approach was used to determine weighting factors for the FragMaps contributions to the scoring function. pIC50 predictions from the combined SILCS/BML approach to the 55 blockers showed a Pearson correlation (PC) coefficient of >0.535 relative to the experimental data. SILCS/BML model was shown to yield substantially improved performance as compared to commonly used rigid and flexible molecular docking methods for a well-established cohort of hERG1 blockers, where no correlation with experimental data was recorded. SILCS/BML results also suggest that a proper weighting of protonation states of common blockers present at physiological pH is essential for accurate predictions of blocker potency. The precalculated and optimized SILCS FragMaps can now be used for the rapid screening of small molecules for their cardiotoxic potential as well as for exploring alternative binding pockets in the hERG1 channel with applications to the rational design of activators.

Allosteric Coupling Between Drug Binding and the Aromatic Cassette in the Pore Domain of the hERG1 Channel: Implications for a State-Dependent Blockade.

Human-ether-a-go-go-related channel (hERG1) is the pore-forming domain of the delayed rectifier K+ channel in the heart which underlies the IKr current. The channel has been extensively studied due to its propensity to bind chemically diverse group of drugs. The subsequent hERG1 block can lead to a prolongation of the QT interval potentially leading to an abnormal cardiac electrical activity. The recently solved cryo-EM structure featured a striking non-swapped topology of the Voltage-Sensor Domain (VSD) which is packed against the pore-domain as well as a small and hydrophobic intra-cavity space. The small size and hydrophobicity of the cavity was unexpected and challenges the already-established hypothesis of drugs binding to the wide cavity. Recently, we showed that an amphipathic drug, ivabradine, may favorably bind the channel from the lipid-facing surface and we discovered a mutant (M651T) on the lipid facing domain between the VSD and the PD which inhibited the blocking capacity of the drug. Using multi-microseconds Molecular Dynamics (MD) simulations of wild-type and M651T mutant hERG1, we suggested the block of the channel through the lipid mediated pathway, the opening of which is facilitated by the flexible phenylalanine ring (F656). In this study, we characterize the dynamic interaction of the methionine-aromatic cassette in the S5-S6 helices by combining data from electrophysiological experiments with MD simulations and molecular docking to elucidate the complex allosteric coupling between drug binding to lipid-facing and intra-cavity sites and aromatic cassette dynamics. We investigated two well-established hERG1 blockers (ivabradine and dofetilide) for M651 sensitivity through electrophysiology and mutagenesis techniques. Our electrophysiology data reveal insensitivity of dofetilide to the mutations at site M651 on the lipid facing side of the channel, mirroring our results obtained from docking experiments. Moreover, we show that the dofetilide-induced block of hERG1 occurs through the intracellular space, whereas little to no block of ivabradine is observed during the intracellular application of the drug. The dynamic conformational rearrangement of the F656 appears to regulate the translocation of ivabradine into the central cavity. M651T mutation appears to disrupt this entry pathway by altering the molecular conformation of F656.

The Pore-Lipid Interface: Role of Amino-Acid Determinants of Lipophilic Access by Ivabradine to the hERG1 Pore Domain.

Abnormal cardiac electrical activity is a common side effect caused by unintended block of the promiscuous drug target human ether-à-go-go-related gene (hERG1), the pore-forming domain of the delayed rectifier K+ channel in the heart. hERG1 block leads to a prolongation of the QT interval, a phase of the cardiac cycle that underlies myocyte repolarization detectable on the electrocardiogram. Even newly released drugs such as heart-rate lowering agent ivabradine block the rapid delayed rectifier current IKr, prolong action potential duration, and induce potentially lethal arrhythmia known as torsades de pointes. In this study, we describe a critical drug-binding pocket located at the lateral pore surface facing the cellular membrane. Mutations of the conserved M651 residue alter ivabradine-induced block but not by the common hERG1 blocker dofetilide. As revealed by molecular dynamics simulations, binding of ivabradine to a lipophilic pore access site is coupled to a state-dependent reorientation of aromatic residues F557 and F656 in the S5 and S6 helices. We show that the M651 mutation impedes state-dependent dynamics of F557 and F656 aromatic cassettes at the protein-lipid interface, which has a potential to disrupt drug-induced block of the channel. This fundamentally new mechanism coupling the channel dynamics and small-molecule access from the membrane into the hERG1 intracavitary site provides a simple rationale for the well established state-dependence of drug blockade. SIGNIFICANCE STATEMENT: The drug interference with the function of the cardiac hERG channels represents one of the major sources of drug-induced heart disturbances. We found a novel and a critical drug-binding pocket adjacent to a lipid-facing surface of the hERG1 channel, which furthers our molecular understanding of drug-induced QT syndrome.

Lipid roles in hERG function and interactions with drugs.

Human-ether-a-go-go-related channel (hERG) is a voltage gated potassium channel (Kv11.1) abundantly expressed in heart and brain tissues. In addition to playing an important role in mediation of repolarizing K+ currents (IKr) in Action Potential (AP), hERG is notorious for its propensity to interact with various medications. The drug-induced block of K+ currents across hERG channel are strongly associated with dysrhythmic conditions collectively known as drug-induced long-QT-syndrome. The recent availability of the high-resolution Cryo-EM structures for the hERG channel has provided unique opportunity to resolve structural mechanisms involved into the process of voltage-gating of hERG channels, map various roles played by components of ventricular and neuronal membranes and then to connect it to cellular pathways through which diverse chemical compounds might be affecting function of the channel. Specifically, lipids and lipid derivatives such as polyunsaturated fatty acids (PUFAs), ceramides and steroids have been shown to directly interact with the lipid facing amino acids in various Kv channels including hERG. In this review, possible lipophilic pathways of hERG activators and blockers, together with the existence of fenestration windows and effects of PUFAs, ceramides and steroids are explored throughout different sections. Finally, the interplay between long QT inducing drugs and phospholipidosis is briefly discussed.