Structure Assisted Design of SK Channel Selective Activators

小电导(KCa2)和中电导(KCa3.1)钙激活的K+通道是电压依赖性的。 独立并共享共同的钙/钙调蛋白介导的门控机制。它们的电压不足- 依赖使KCa~(2+)/3通道在负膜电位下保持开放，该通道 因此，在需要超极化的生理过程中扮演着重要的角色。而三KCa2 通道，KCa2.1(SK1)，KCa2.2(SK2)和KCa2.3(SK3)最为人所知的是它们在神经元中的作用 超极化后，KCa3.1(IK)主要在免疫系统、血管内皮细胞和血管内皮细胞中被研究。 分泌上皮，其中通道参与激活、增殖和分泌过程，通过 钙离子内流事件的调节。小分子KCa2/3通道调节剂既构成有用的化学物质 生物探针以及潜在的治疗自身免疫性疾病、高血压和 各种神经紊乱，如共济失调、癫痫和酒精依赖。 多年来，我们实验室一直致力于KCa2/3通道的药理研究。在我们之后 最初开发了KCa3.1阻滞剂，如TRAM-34，后来我们发现了KCa3.1/3混合激活剂SKA-31 以及KCa3.1选择性激活剂SKA-121和SKA-111，它们对KCa3.1的选择性是40倍或100倍 通过钾离子通道。所有这些化合物都已被广泛用于探索生理和生理功能。 KCA通道的病理生理功能是用经典的药物化学方法设计的 没有任何结构性的投入。然而，使用最近解决的KCa2.2钙调蛋白的晶体结构 结合结构域(CaM-BD)与CaM的复合体来自我们的顾问苗张，我们产生了Rosetta KCa2.3和KCa3.1 CaM-BD/CaM络合物的同源模型，并发现广泛的 氢键网络稳定KCa3.1中的SKA-121是KCa3.1选择性的关键。使用这种原子化的尺度 对KCA通道亚型选择性的结构性洞察我们现在建议在AIM-1下切换选择性 并进行假说驱动的结构辅助药物设计--新型萘噻唑/恶唑型KCA 使用Rosetta配体和新的 Rosetta药物设计方法。在合成和实验测试KCA通道效力和 通过电生理的选择性，我们打算首先通过诱变来确定结合模式，然后将 在AIM-2下，新的KCa2激活剂成为科学界有用的药理探针，我们在那里 将确定对其他离子通道的选择性，并评估药代动力学特性和大脑 穿透力。 我们建议的创新有两个方面：1)这项工作将是假说驱动的第一次尝试 基于结构的小分子离子通道调节剂的药物设计；2)本工作将为 使用KCa2通道选择性门控调制器，这将是探索 钾通道的病理生理作用及其作为癫痫，共济失调， 物质依赖和创伤后应激障碍。

Small-conductance (KCa2) and intermediate-conductance (KCa3.1) calcium-activated K+ channels are voltage- independent and share a common Ca2+/calmodulin mediated gating mechanism. Their lack of voltage- dependence enables KCa2/3 channels to remain open at negative membrane potentials and the channels therefore play important roles in physiological processes that require hyperpolarization. While the three KCa2 channels, KCa2.1 (SK1), KCa2.2 (SK2) and KCa2.3 (SK3) are best known for their role in neuronal afterhyperpolarization, KCa3.1 (IK) has mostly been studied in the immune system, vascular endothelium and in secretory epithelia, where the channel is involved in activation, proliferation and secretion processes through modulation of Ca2+ influx events. Small molecule KCa2/3 channel modulators constitute both useful chemical biology probes as well as potential novel drugs for the treatment of autoimmune diseases, hypertension, and various neurological disorders such as ataxia, epilepsy, and alcohol dependence. Our laboratory has been working on the pharmacology of KCa2/3 channels for many years. After we initially developed KCa3.1 blockers such as TRAM-34, we later discovered the mixed KCa2/3 activator SKA-31 and the KCa3.1 selective activators SKA-121 and SKA-111, which display 40- or 100-fold selectivity for KCa3.1 over KCa2 channels. All these compounds, which have been widely used to probe the physiological and pathophysiological functions of KCa channels, were designed using classical medicinal chemistry approaches without any structural input. However, using the recently solved crystal structures of the KCa2.2 calmodulin binding domain (CaM-BD) in complex with CaM from our consultant Miao Zhang, we generated Rosetta homology models of the KCa2.3 and KCa3.1 CaM-BD/CaM complexes and discovered that an extensive hydrogen bond network stabilizing SKA-121 in KCa3.1 is key to its KCa3.1 selectivity. Using this atomistic scale structural insight into KCa channel subtype selectivity we are now proposing to switch selectivity under Aim-1 and perform hypothesis-driven structure-assisted drug design of novel napthothiazole/oxazole-type KCa activators that make unique contacts with KCa2-specific residues using the Rosetta Ligand and the new RosettaDrug Design approach. After synthesizing and experimentally testing KCa channel potency and selectivity by electrophysiology, we intend to first confirm the binding mode by mutagenesis and then turn the new KCa2 activators into a useful pharmacological probe for the scientific community under Aim-2, where we will determine selectivity over other ion channels and evaluate pharmacokinetic properties and brain penetration. The innovation in our proposal is twofold: 1) This work will be one of the first attempts at hypothesis-driven structure based drug design for a small molecule ion channel modulator; 2) This work will provide the scientific community with KCa2 channel selective gating modulators which will be useful tools to explore the pathophysiological role of KCa2 channels and their suitability as therapeutic targets for epilepsy, ataxia, substance dependence and post-traumatic stress disorder.