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: 10729516
• 负责人: Emily May Teichman
• 金额: $ 4.77万
• 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/10729516
• 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; Physiological; Process; Resistance; Rodent; Rodent Model; Seminal; Series; Side; Structure; System; TLR4 gene; Techniques; Testing; Therapeutic; Transfection; Ventral Tegmental Area; analog; antidepressant effect; 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; neural; neuronal excitability; neuropsychiatric disorder; novel; pharmacologic; side effect; socioeconomics; structural determinants

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.

项目摘要 抑郁症是一种毁灭性的疾病，也是全球残疾的主要原因之一。然而，三分之一 的患者对目前可用的抗抑郁药有耐药性。因此，一种新的抗抑郁药， 与目前的抗抑郁药不同的药理学类别至关重要。针对HCN（超极化- 激活的，环核苷酸门控）通道通过非亚型选择性HCN抑制剂已经证明 小鼠的抗抑郁活性。然而，HCN通道在大脑和心脏中均存在; 因此，预期用于脑部疾病的非亚型选择性HCN抑制剂具有心脏毒性。到 为了解决这个问题，我的项目将寻求针对HCN 2通道，大脑中的主要亚型，而不是针对 HCN 4通道，心脏中的主要亚型。该项目的目的是确定HCN 2的选择性， 化合物对HCN 2的选择性和HCN 2通道的残基对选择性的贡献。我们的目标首先是 阐明与对HCN 2通道的选择性/优先性相关的化合物化学型， HCN 4频道这种方法将利用功能电生理学来测试不同化合物的效果 类似物对HCN 2和HCN 4转染的HEK 293细胞的Ih（超极化激活）电流的影响， 测试这些化合物对小鼠腹侧被盖区（VTA）中多巴胺神经元的生理作用。 小鼠脑（主要是HCN 2通道）和心肌细胞（主要是HCN 4）。此外，我们将采用 使用SplitLuc CETSA（细胞热位移测定）技术来测量这些化合物的结合亲和力的强度。 化合物对HCN 2的作用超过HCN 4通道。这些平行策略将有助于确定化合物化学型 与HCN 2通道的功能和结合选择性相关。我们的第二个目标是建立HCN 2 通道残基和与HCN 2相对于HCN 4的选择性相关的相应结合口袋。这 一种方法将利用系统诱变策略，将HCN 2和HCN 4不同的残基替换为一个 另一个，然后是功能和结合选择性测试（电生理学和CETSA）的影响， 探针7 G，HCN 2优先化合物，对这些突变体。在测定与以下物质相关的残留物时， HCN 2通道相对于HCN 4通道的7 G选择性，7 G与HCN 2通道的计算机对接 开放和封闭模型（以及HCN 4通道开放和封闭解析结构）将用于定义 装订口袋总体而言，该项目将提供HCN 2选择性的多方面表征， 对HCN通道的生长体进行了开创性的HCN通道亚型选择性实验， 文学此外，开发的化合物可用于阐明HCN 2通道在啮齿动物中的作用。 行为和脑功能/功能障碍，以及HCN 2选择性化学型的鉴定，结合 口袋，并且与7 G相比，对HCN 2相对于HCN 4具有增加的选择性比率的可能化合物可以 都为未来抗抑郁药物的发现提供了基础。