Unveiling and Exploiting the Structural Determinants of HCN2 Channel Selectivity

揭示和利用 HCN2 通道选择性的结构决定因素

• 批准号: 10607061
• 负责人: Emily May Teichman
• 金额: $ 4.68万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-07 至 2025-08-21
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10607061
• 关键词: Affinity; Amaze; Antidepressive Agents; Behavior; Binding; Biological Assay; Biophysics; Brain; Brain Diseases; Carbon; Cardiac; Cardiac Myocytes; Cardiotoxicity; Cell physiology; Cells; Cyclic Nucleotides; Disease; Docking; Economic Burden; Electrophysiology (science); Ethers; Foundations; Functional disorder; Future; HCN4 gene; Hand; Heart; Heart Rate; In Vitro; Individual; Interdisciplinary Study; Ion Channel; Left; Length; Literature; Measures; Medical Economics; Mental Depression; Mental disorders; Modeling; Mus; Mutagenesis; Names; Neurons; Parents; Patients; Pharmaceutical Preparations; Pharmacology; Physiological; Process; Resistance; Rodent; Rodent Model; Seminal; Series; Side; Structure; System; TLR4 gene; Techniques; Testing; Therapeutic; Ventral Tegmental Area; analog; antidepressant effect; base; cyclic-nucleotide gated ion channels; disability; dopamine system; dopaminergic neuron; drug development; drug discovery; experimental study; heart function; hydroxyl group; in silico; inhibitor; mutant; nervous system disorder; neuronal excitability; neuropsychiatric disorder; novel; relating to nervous system; side effect; socioeconomics

PROJECT SUMMARY Depression is a devastating disease, and one of the leading causes of disability worldwide. However, one third of patients are treatment resistant to the current antidepressants available. Therefore, a new antidepressant in a different pharmacological class from the current antidepressants is vital. Targeting the HCN (hyperpolarization- activated, cyclic nucleotide-gated) channel via a non-subtype-selective HCN inhibitor has demonstrated antidepressant activity in mice. However, the HCN channel is critically present in both the brain and the heart; thus a non-subtype-selective HCN inhibitor for brain disorders would be expected to have cardiac toxicity. To solve this issue, my project will seek to target the HCN2 channel, the major subtype in the brain, without targeting the HCN4 channel, the major subtype in the heart. The aim of this project is to define HCN2 selectivity, both in compound selectivity towards HCN2 and the HCN2 channel’s residues contributing to selectivity. We first aim to elucidate the compound chemotype associated with selectivity/preferentiality towards the HCN2 channel over the HCN4 channel. This approach will utilize functional electrophysiology to test the effect of different compound analogs on the Ih (hyperpolarization-activated) currents of HCN2- and HCN4-transfected HEK293 cells, and to test the physiological effect of these compounds on dopamine neurons in the ventral tegmental area (VTA) of the mouse brain (majority HCN2 channels) and cardiomyocytes (majority HCN4). Further, we will employ the SplitLuc CETSA (cellular thermal shift assay) technique to measure the strength of the binding affinity of these compounds for HCN2 over HCN4 channels. These parallel strategies will help to identify compound chemotypes associated with functional and binding selectivity to the HCN2 channel. Our second aim is to establish the HCN2 channel residues and corresponding binding pocket associated with HCN2 selectivity versus HCN4. This approach will utilize a systematic mutagenesis strategy of replacing HCN2 and HCN4 differing residues with one another, followed by functional and binding selectivity testing (electrophysiology and CETSA) of the effect of the probe 7G, an HCN2-preferential compound, on these mutants. Upon determination of residues associated with 7G selectivity for the HCN2 channel over the HCN4 channel, in silico docking of 7G onto the HCN2 channel open and closed models (as well as the HCN4 channel open and closed solved structures) will be used to define the binding pocket. Overall, this project will provide a multifaceted characterization of HCN2 selectivity, contributing seminal HCN channel subtype-selectivity experiments to the growing body of HCN channel literature. Additionally, compounds developed can be utilized to elucidate the effect of HCN2 channels in rodent behavior and brain functions/dysfunctions, and the identification of an HCN2-selective chemotype, binding pocket, and possibly compounds with an increased selectivity ratio for HCN2 over HCN4 compared to 7G can all provide a basis for future antidepressant drug discovery.

项目总结