Unveiling and Exploiting the Structural Determinants of HCN2 Channel Selectivity

项目总结 抑郁症是一种毁灭性的疾病，也是全世界导致残疾的主要原因之一。然而，三分之一的人 的患者对目前可用的抗抑郁药物治疗耐药。因此，一种新的抗抑郁药物在中国 与目前的抗抑郁药不同的药理类别至关重要。瞄准HCN(超极化- 激活的、环核苷酸门控的)通道通过非亚型选择性的HCN抑制剂已经证明 对小鼠的抗抑郁活性。然而，HCN通道在大脑和心脏中都存在； 因此，治疗脑部疾病的非亚型选择性HCN抑制剂预计会有心脏毒性。至 解决这个问题，我的项目将寻求以大脑中的主要亚型--hcn2通道为目标，而不是目标 HCN4通道，心脏的主要亚型。该项目的目标是定义HCN选择性，这两项都是 化合物对HCN_2和HCN_2通道残基的选择性有助于选择性。我们首先的目标是 阐明与对HCN_2通道的选择性/偏好有关的化合物化学类型。 HCN4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 HCN_2和HCN_4转染人HEK293细胞超极化激活电流的类似物 测试这些化合物对大鼠腹侧被盖区(VTA)多巴胺神经元的生理影响 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 SplitLuc CETSA(细胞热漂移分析)技术测量这些分子结合亲和力的强度 用于HCN4通道上的HCN的化合物。这些平行策略将有助于识别化合物化学类型。 与对HCN_2通道的功能和结合选择性有关。我们的第二个目标是建立hcn2 与HCN4相比，通道残基和相应的与HCN2选择性相关的结合口袋。这 该方法将利用一种系统的突变策略，将不同的HCN_2和HCN_4残基替换为一个 其次是功能和结合选择性测试(电生理学和CETSA)的影响 在这些突变体上，探针7G，一个对HCN有偏好的化合物。在测定与以下物质相关的残留物时 7G在HCN4通道上的7G选择性，在7G对接到HCN2通道上 将使用开放和封闭模型(以及HCN4通道开放和封闭求解结构)来定义 捆绑口袋。总体而言，该项目将提供对HCN选择性的多方面表征， 精液HCN通道亚型选择性实验对HCN通道生长体的贡献 文学。此外，所开发的化合物可用于阐明啮齿动物体内hcn2通道的作用。 行为和脑功能/功能障碍，以及对HCN-2选择性化学类型、结合的鉴定 Pocket，可能与7G相比对HCN_2和HCN_4具有更高选择性比的化合物可以 所有这些都为未来抗抑郁药物的发现提供了基础。

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.