Theoretical studies of ligand-receptor interactions in ion channels

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The human genome encodes over 400 proteins that form ion channels. Voltage-gated K+, Na+, and Ca2+ channels are drug targets for pain control and treatment of cardiovascular, neurological, autoimmune and other disorders. The channels exist in structurally different closed, open and inactivated states. Drugs often bind preferentially to certain states. Such state-dependent action is difficult to study by high-throughput ligand screening, the major experimental approach in drug discovery. Molecular modeling helps understand atomistic mechanisms of state-dependent drug action. In 2009-2013 we have published 13 NSERC-funded papers in PNAS USA, Chem Reviews, Trends Pharmacol Sci, J Biol Chem and other journals. We will continue theoretical studies of ion channels with drugs and toxins. Our approach includes 5 stages. 1) Building homology models of medicinally important ion channels using as templates available X-ray structures of K+ and Na+ channels in the open, closed, and inactivated states. 2) Analyzing published data on structure-activity relations of ligands and mutational analysis of ligand-binding sites. 3) Revealing paradoxes in the experimental data and focusing on data that lack structural interpretations or suggest questionable ones. 4) Docking representative ligands in the homology models with considering a possibility that ligands may directly interact with permeant cations (Zhorov & Tikhonov, 2013, Trends Pharmacol Sci). 5) Docking experiments usually predict an ensemble of energetically reasonable ligand-binding modes. We select those that are best consistent with experimental data. We will use the Monte Carlo-minimization method realized in the ZMM program, which I am elaborating over many years, as a major computational tool. ZMM has been validated, e.g., in (Garden & Zhorov, 2010). An advantage of the home-made software is that it can be adapted to address new problems. There is no shortage of published experimental data that need structural interpretations. Recent x-ray structures of bacterial Na+ channels provide reliable templates to model Na+ and Ca2+ channels with toxins and medically important drugs. I also propose a new direction for my research program. Mutational analysis is used to determine ligand-binding sites, but structural interpretation of results is difficult. Indeed, a ligand action may change due to mutation of a residue in the ligand-binding site (a direct effect) or far beyond this site (an allosteric effect). We will elaborate an approach to discriminate the direct and allosteric effects. The driving hypothesis is that mutations, which change state-depended contacts between ion channel segments, change populations of the open/closed/inactivated states and thus may allosterically affect drug action. To discriminating these effects we will partition the channel energy, select residues involved in strong intersegment contracts, explore how such contacts change in different channel states, and analyze mutational data in view of these results. This approach will help explain why mutations of conserved asparagines in Na+ and Ca2+ channels, which do not face the pore, affect action of many pore-targeting ligands. Mutational analyses of Kv1 channels block by structurally similar ligands PAP-1 and Psora-4 resulted in conflicting ligand-binding models (Zimin et al. 2010; Marzian et al. 2013). We will analyze underlying mutations and readdress the mechanism of Kv1 channel block by these highly potent ligands. Discriminating direct and allosteric effects of mutations will be important for building other ligand-channel models. Our studies will contribute to basic knowledge and development of new drugs that will help maintain and improve health of Canadians.