Molecular mechanisms for small molecule compounds targeting SK/IK channels

 描述（由申请人提供）：Ca 2+激活钾通道，如小电导和中电导钾通道（SK和IK），广泛表达于可兴奋组织中。它们在调节Ca 2+对膜的兴奋性中起关键作用。与电压门控K+通道不同，SK/IK通道的激活仅由Ca 2+实现。钙调素（CaM），拴在通道的C末端，作为高亲和力的Ca 2+传感器。四个EF臂，两个位于CaM N端（N叶），另外两个位于C端（C叶），是高亲和力的Ca 2+结合位点。Ca 2+介导的CaM和CaM结合结构域（CaMBD）之间的相互作用激活了通道。除了它们的生理作用之外，SK/IK通道还与临床异常有关。因此，在学术界和工业界都投入了巨大的努力来开发靶向SK/IK通道的小分子。1-乙基-2-苯并咪唑啉酮（1-EBIO）是这样一种原型，其增强SK/IK通道活性并有效地降低膜兴奋性。研究表明，1-EBIO化合物在中枢神经系统和心血管系统的疾病模型中是有益的。然而，一般来说，1-EBIO化合物具有缺点，例如缺乏选择性，这阻碍了它们用于临床试验的潜力。一个关键的影响因素是缺乏对1-EBIO化合物如何与其结合位点相互作用并实现其对SK/IK通道的影响的了解。直到出版 在我们最近的论文中，尚不清楚这些化合物可能在何处与SK/IK通道相互作用。药物结合位点的分子性质仍不清楚，也不知道1-EBIO化合物如何工作，或者这些化合物如何实现其选择性。为了揭示这些化合物的药物结合位点的分子特性并了解1-EBIO化合物作用的分子机制，我们将使用结构生物学，分子生物学，生物化学生物物理学和电生理学的综合方法。具体而言，我们将重点讨论以下问题：（1）表征的 1-EBIO化合物的结合位点和有助于1-EBIO化合物对SK/IK通道的选择性的分子机制。1-EBIO在不同类型的SK/IK通道上复合。(2)1-EBIO化合物增强SK/IK通道的分子机制。(3)1-EBIO化合物抑制SK/IK通道的分子机制。拟议工作的结果将提供1-EBIO化合物药物结合位点的分子特性。这些结果将填补目前关于这些化合物如何调节SK/IK通道活性（增强和抑制）的知识空白。这些知识将有助于通过基于结构的药物设计/开发来开发未来几代靶向SK/IK通道的治疗药物。从广义上讲，我们的研究结果将有助于开发靶向参与Ca 2+依赖性信号传导的其他CaM靶蛋白复合物的化合物，以及靶向离子通道领域以外的膜脂质的新药。

 DESCRIPTION (provided by applicant): Ca2+‐activated potassium channels, such as small‐ and intermediate‐conductance K+ channels (SK and IK), are widely expressed in excitable tissues. They play pivotal roles in regulating membrane excitability by Ca2+. Unlike voltage‐gated K+ channels, activation of SK/IK channels is achieved exclusively by Ca2+. Calmodulin (CaM), tethered to the channel C‐terminus, serves as the high‐affinity Ca2+ sensor. Four EF‐hands, two located at the CaM N‐terminus (N‐lobe) and the other two at the C‐terminus (C‐lobe), are the high affinity Ca2+ binding sites. The Ca2+‐mediated interaction between CaM and the CaM binding domain (CaMBD) activates the channel. In addition to their physiological roles, SK/IK channels have been implicated in clinical abnormalities. Consequently, a tremendous effort has been devoted to developing small molecules targeting SK/IK channels in both academia and industry. 1‐ethyl‐2‐benzimidazolinone (1‐EBIO) is such a prototype that potentiates the SK/IK channel activity and effectively decreases the membrane excitability. Studies have shown that the 1‐ EBIO compounds are beneficial in disease models of the central nervous system and the cardiovascular system. In general, however, the 1‐EBIO compounds suffer from drawbacks, such as lack of selectivity, which hamper their potential for clinical trials. A key contributing factor is lack of knowledge of how the 1‐EBIO compounds interact with their binding site and achieve their effects on SK/IK channels. Until the publication of our recent papers, it was not known where these compounds might interact with SK/IK channels. The molecular properties of the drug binding site remain unclear and it is not known how the 1‐EBIO compounds work, or how these compounds achieve their selectivity. To uncover the molecular properties of the drug binding site for these compounds and understand the molecular mechanisms for the actions of the 1‐EBIO compounds, we will use integrated approaches of structural biology, molecular biology, biochemistry biophysics and electrophysiology. Specifically, we will focus on the following issues: (1) Characterization of the binding site for the 1‐EBIO compounds and molecular mechanisms that contribute to selectivity of the 1‐EBIO compounds for SK/IK channels. 1‐EBIO compounds over different types of SK/IK channels. (2) Molecular mechanisms by which the 1‐EBIO compounds potentiate SK/IK channels. (3) Molecular mechanisms by which the 1‐EBIO compounds inhibit SK/IK channels. Results from the proposed work will provide the molecular properties of the drug binding site for the 1‐EBIO compounds. The results will fill in the current knowledge gap regarding how these compounds modulate SK/IK channel activity (potentiation and inhibition). The knowledge will facilitate development of future generations of therapeutics targeting SK/IK channels by structure‐based drug design/development. Broadly, our results will help develop compounds targeting other CaM‐target protein complexes involved in Ca2+ dependent signaling as well as new drugs targeting membrane lipids beyond the ion channel field.