UC Davis

Many different types of drugs–from antibiotics to blood pressure medication–tend to interfere with the body’s ability to control heart rhythm by disrupting the proteins in heart cells that control the movement of charged atoms (ions) across the cell membrane known as voltage-gated ion channels (VGICs). These drugs can cause dangerous arrhythmias (abnormal heart rhythms) that can increase risk for heart failure, stroke, or death. Early and efficient assessment of cardiotoxicity is essential to the drug development process and to reducing drug development costs. Current methods for assessing safety are sensitive but not specific and can often result in false identification of unsafe treatments and the failure of potentially life-saving treatments to reach the public. Structural characterization of VGICs and their modulating interactions are necessary for rational design of safe therapeutics.

Human Ether-a-go-go-Related Gene (hERG) encodes a potassium-selective voltage-gated ion channel (KV11.1) essential for normal electrical activity in the heart. Genetic hERG mutations and blockage of the channel pore by drugs can cause long QT syndrome (LQTS). LQTS predisposes individuals to arrhythmia and puts them at risk for stroke or sudden cardiac arrest. A major problem in antiarrhythmic drug therapies is the proclivity for these drugs to promote fatal arrhythmias through hERG channel blockade. However, not all hERG channel blocking drugs are pro-arrhythmic, and their differential affinities to discrete channel conformational states and/or their state stability modulations have been suggested to contribute to arrhythmogenicity.

Voltage-gated calcium (CaV) channels play a key role in muscular contraction, neuronal excitation, gene expression regulation, and release of hormones or neurotransmitters. Dysregulation of CaV channels and the associated intracellular calcium homeostasis have been associated with various types of cardiac and neurological disorders. Found throughout the body, often as part of large complexes and/or clusters, the L-type CaV1.2 channel mediates the influx of Ca2+ into the cell in response to membrane depolarization. Mutations or blockage of the channel by drug molecules leading to altered functions of human CaV1.2 have been linked to cardiac arrhythmias, autism, bipolar disorder, and immunodeficiency. Many CaV channel blockers targeting the alpha-1-subunit of CaV1.2 are used to treat hypertension, coronary artery disease and other cardiovascular medical conditions. However, only a few drugs have been approved for clinical use due to severe side effects (including cardiotoxicity) or limited efficacy.

In this study, Rosetta electron density refinement and homology modeling protocols were used to build voltage sensing and pore domain structural models of wild-type hERG channel in open and closed states, open-state hERG mutant variants (Y652A, F656A, and Y652A/F656A double mutant) based on cryo-electron microscopy (cryo-EM) structures of hERG (PDBID: 5VA2) and EAG1 (PDBID: 5K7L) channels as well as open- and closed-state models of the wild-type CaV1.2 alpha-1-subunit using cryo-EM CaV1.1 (PDBID: 5GJV), and NaV1.4 (PDB ID: 6AGF) structures, respectively. The hERG channel models were developed as protein targets for Rosetta-based molecular docking studies of charged and neutral forms of amiodarone, nifekalant, dofetilide, d- and l-sotalol, flecainide, and moxifloxacin–a diverse set of pharmaceuticals chosen based on their different arrhythmogenic potentials and abilities to facilitate hERG current. The CaV1.2 models were used as targets for Rosetta docking studies with verapamil and amlodipine– representatives of two different calcium channel blocking classes: phenylalkylamines and dihydropyridines, respectively. We present here the results of our docking studies that provide structural insights into the molecular and state-dependent drug interactions with hERG and CaV1.2 channels that play a key role in differentiating safe and harmful ion channel blockers.

Key Findings:1. Pattern of hERG-drug interactions with the hydrophobic pocket is consistent with experimental data suggesting facilitating drugs may act as a wedge to bias hERG channel equilibrium towards the open state and increase hERG current amplitude in response to low-voltage depolarization. 2. Open-state WT hERG interface scores are lower than, or similar to, Y652A mutants suggesting that these poses are relevant for amiodarone, nifekalant, flecainide, moxifloxacin, d-sotalol, and dofetilide, based on comparison to existing experimental data. 3. Open-state WT hERG interface scores are not lower than the F656A mutants for nifekalant, neutral flecainide, neutral moxifloxacin, d-sotalol, l-sotalol, and dofetilide, suggesting limitations of our study using only two conformational states or limitations of Rosetta to model allosteric contributions of F656. 4. Percentage of poses remaining within the closed-state hERG channels suggest that closed channels can accommodate known trapped drugs (nifekalant, flecainide, d/l-sotalol, and dofetilide), but not amiodarone or moxifloxacin (non-trapped drugs). 5. Amlodipine and verapamil docked to rCaV1.2 models in open and closed states recapitulated known binding orientations and similar positioning within pore but did not reproduce known binding determinants necessitating revision of model development to include additional structural densities and increasing search radius in docking protocol in future studies.