Identification of potential drug binding sites within allosteric networks in cyclic nucleotide modulated channels

项目摘要 环核苷酸调节的离子通道是一类在许多生理过程中具有重要作用的蛋白质， 过程，包括心脏的调节，神经元信号传导和疼痛感知。发现新 由于许多原因，靶向不同离子通道的药物是众所周知的困难，因为传统的药物治疗方法的重点是治疗不同的离子通道。 正构激动剂和拮抗剂已经占主导地位。总的来说，这一提议将基因组、功能和 结构的方法来揭示如何具体的变构相互作用支配机械功能。检测能力 分离变构相互作用网络的功能可以提供一种更集中的设计方法， 用于环核苷酸调节通道的变构药物。在第一个目标中，我将识别和分类变构 网络使用协进化分析。突变和定量电生理学测量将 用于探测不同的残基位置如何在通道门控中对必需的能量耦合做出贡献。接下来， 我们将使用这种独特的信息来获得新的信道过渡态。这一目标的总体目标是 实验上定义了功能性调节环核苷酸调节离子通道的变构网络， 验证这些网络的功能，并使用这些信息来获得难以解析的构象状态。 在第二个目标中，我将寻求发现已知的环己基转移酶变构调节剂的未知结合位点。 核苷酸调节的离子通道。使用cryoEM，我们将确定通道的结构， 建立了变构调节剂，并在目标1中定义了它们与变构网络的相互作用。我们将 用诱变和双电极电压钳验证结合位点。这一目标将证明可行性 针对变构网络的药物设计策略。

PROJECT SUMMARY Cyclic nucleotide modulated ion channels are a class of proteins that have important roles in many physiological processes, including regulation of the heart, neuronal signaling, and pain perception. The discovery of new drugs targeting different ion channels is notoriously difficult for a host of reasons, as the focus on traditional orthosteric agonists and antagonists has been dominant. Overall, this proposal unifies genomic, functional, and structural methods to reveal how specific allosteric interactions govern mechanistic function. The ability to detect and isolate the function of networks of allosteric interactions can provide a more focused approach in the design of allosteric drugs for cyclic nucleotide modulated channels. In the first aim, I will identify and classify allosteric networks using coevolution analysis. Mutagenesis and quantitative electrophysiology measurements will be used to probe how different residue positions contribute to requisite energetic coupling in channel gating. Next, we will use this unique information to obtain novel channel transition states. The overall goal of this aim is to define allosteric networks that functionally regulate cyclic nucleotide modulated ion channels, experimentally validate these networks functionally, and use this information to obtain difficult-to-resolve conformational states. In the second aim, I will seek to uncover unknown binding sites for known allosteric modulators of cyclic nucleotide modulated ion channels. Using cryoEM, we will determine the structure of channels in complex with established allosteric modulators and define their interaction with the allosteric networks in aim 1. We will validate the binding site using mutagenesis and two electrode voltage clamp. This aim will demonstrate feasibility of drug design strategies targeting allosteric networks.