Chimeric Human-NavMs Sodium Channel Constructs for Structure/Function Studies of Selective Toxin Binding

电压门控钠通道是神经和心血管通道病变的主要原因，因此是当前和未来开发新型和高度特异性药物的重要靶点。该项目将结合分子生物学（膜蛋白的克隆、表达、纯化和表征）、结构生物学（晶体学、低温电镜）、结合研究（圆二色光谱、表面等离子体共振、微尺度热泳（MST））、生物信息学/计算生物学作为钠通道跨学科发现/设计项目的一部分，旨在确定钠通道电压传感器区域内的不同位点，这些位点通过不同的通道异构体对不同的自然产生的毒素赋予选择性和敏感性。九种钠通道同工异构体存在于人体的不同组织中，具有不同的生理作用，但具有较高的{~90-95%}序列同一性，是开发抗癫痫、抗心律失常和镇痛药物的靶点。这类药物必须考虑到重要的同源性，以防止不必要的副作用。镇痛作用对心脏抑制)。高度特异性的毒素，无论是天然的还是合成的，它们针对不同的同种异构体，是制药工业中使用的优秀工具，作为合理药物设计和开发新的靶向化合物（合成毒素或小分子）的基础。获得工业合作伙伴提供的新表征方法和新化合物将极大地增强该项目开发新型和有效的异构体特异性药物的潜力，并增强我们对钠通道结构和功能的理解。为了确定不同钠通道域中负责特异性结合的区域，我们将基于Wallace实验室最近确定的原核NavMs全长晶体结构构建嵌合体/突变体(1)，其中特定区域/残基被人类钠通道的同种异构体特异性区域取代。NavMs已被证明是人类钠通道药物结合特征的优秀且可获得的功能同源物(6)，基于人类同种异构体创建的嵌合/突变结构可以提供分子疾病相关区域的主要信息，这些区域可以靶向药物/同种异构体特异性毒素结合（1,2）。在电压传感器结构域和子结构域中结合差异的自然发生（和突变）毒素将被用作与选择性相关的目标区域/序列的指南，例如Nav1.7, 1.8和1.9人类通道。结合将首先使用生物物理方法进行评估，如SPR和热熔圆二色光谱（3,5），这些方法可在工业或学术现场使用，然后使用MST和Vertex用于筛选的其他内部方法进行扩展。一旦制备了合适的稳定的毒素通道复合物，就可以使用结构生物学技术通过任一/两个位点（晶体学，低温电镜）对其进行详细检查。因此，该项目将使学生有机会在学术和公司环境中进行和发展研究，使他们接触到一系列技术，化合物和与同事的互动，否则单独在一个地点无法获得；该项目还将进一步发展伙伴集团之间的联系。

Voltage-gated sodium channels are responsible for a wide range of neurological and cardiovascular channelopathies, and hence are important current and future targets for the development of novel and highly specific pharmaceutical drugs.This studentship will combine training and research in molecular biology (cloning, expression, purification and characterization of membrane proteins), structural biology (crystallography, cryoEM), binding studies (circular dichroism spectroscopy, surface plasmon resonance, microscale thermophoresis(MST)), and bioinformatics/computational biology as part of an interdisciplinary discovery/design project on sodium channels aimed at identifying different sites within the voltage sensor regions of sodium channels that confer selectivity and sensitivity by the different channel isoforms for different naturally-occurring toxins. The nine sodium channel isoforms are found in different tissues throughout the human body have different physiological roles, but have high {~90-95%} sequence identities and are targets for development of anti-epileptic, anti-arrhythmic, and analgesic drugs. Such drugs must take into account the significant homologies to prevent unwanted side effects (ie. analgesic effects on cardiac suppression). Highly specific toxsins, both natural and synthetic, which target different isoforms are excellent tools used in the pharmaceutical industry as the basis for rational drug design and development of new targeted compounds (either synthetic toxins or small molecules).The access to both novel methods for characterization and novel compounds provided by the industrial partner will greatly enhance the potential of this project for development of novel and potent isoform-specific drugs, as well as enhancing our understanding of sodium channel structure and function. In order to identify the regions within the different sodium channel domains that are responsible for specific binding, we will construct chimeras/mutants based on the prokaryotic NavMs full length crystal structure recently determined in the Wallace lab (1), in which specific regions/residues are replaced with isoform-specific regions of human sodium channels. NavMs has been shown to be an excellent and accessible functional orthologue for drug binding characteristics of human sodium channels (6) and chimeric/mutant structures created based on human isoforms can provide lead information on disease-related regions of the molecule that can be targeted for drug/isoform-specific toxin binding (1,2). Naturally-occurring (and mutant) toxins which bind differentially in the voltage-sensor domains and sub-domains will be used as guides to target regions/sequences associated with selectivity between, for example Nav1.7, 1.8, and 1.9 human channels. The binding will be initially assessed using biophysical methods such as SPR and thermal melt circular dichroism spectroscopy (3,5), available at either the industrial or academic site, and then extended using MST and other in house methods that Vertex uses for screening. Once suitable stable toxin-channel complexes are prepared, they can be examined in detail using structural biology techniques available via either/both sites (crystallography, cryoEM). This project will thus give the student the opportunity to undertake and develop studies in both an academic and company environment, giving them exposure to a range of techniques, compounds and interactions with colleagues otherwise not available at one of the sites alone; this project will also develop further ties between the partner groups.