Chemically Interrogating Clostridium difficile Glucosylating Toxin Activation

描述（由申请人提供）：在本提案中，我描述了我的计划，以阐明自身蛋白水解调节细菌毒素功能的机制。到目前为止，在我的博士后工作中，我发现了一种独特的蛋白酶激活机制，并描绘了半胱氨酸蛋白酶结构域（CPD）在自动处理一种新发现的细菌毒素家族中的作用。我已经确定了这种新型蛋白酶家族的第一个化学抑制剂，并开始将这些抑制剂开发成化学探针，以研究蛋白酶底物的特异性和功能。在proposal的指导阶段，我将在斯坦福大学Matthew Bogyo博士的实验室继续学习如何合成小分子抑制剂和探针来监测和干扰CPD的激活。这一阶段将依赖于Bogyo实验室研究的深度和多样性，以及斯坦福大学优秀的研究环境。我将以表面等离子体共振课程来补充我的机构环境，并访问未来合作者的实验室（俄克拉荷马州立大学的Jimmy Ballard博士），在那里我将学习如何培养艰难梭菌并纯化其糖基化毒素。在斯坦福大学，我将继续利用医学院优秀的职业发展资源，参加旨在帮助博士后过渡到学术职位的讲座。我将继续咨询我的导师Bogyo博士和一个非正式的指导委员会，该委员会由斯坦福大学的John Boothroyd博士和K. Christopher Garcia博士组成，以帮助我成功地在学术就业市场中导航，申请资金，并建立自己的研究小组。一旦我在研究机构获得独立职位，我将应用在指导阶段开发的探针和抑制剂来研究自身蛋白水解在调节细胞内毒素功能和中毒动物模型中的功能。从长远来看，我将运用这种新的方法来成像蛋白酶的功能，并结合我在细菌遗传学和生物化学方面的背景，研究不同系统中的细菌蛋白酶。6. b。研究计划大多数分泌的细菌毒素是作为无活性前体产生的，在遇到真核细胞时被蛋白水解激活。而真核生物蛋白酶通常激活这些毒素，一组毒素被内部半胱氨酸蛋白酶结构域（CPD）自蛋白水解激活。艰难梭菌糖基化毒素的细胞毒功能是由CPD激活的，CPD本身在与真核特异性小分子肌醇六磷酸（InsP6）结合时被激活。尽管自动加工对毒素激活至关重要，但InsP6如何激活CPD，以及CPD在细胞中的何时何地切割这些毒素，目前尚不清楚。本提案概述了使用小分子来解决关于cpd介导的艰难梭菌糖基化毒素激活的这些重要问题的计划。在初步研究中确定的小分子抑制剂将发展成探针，使体外、宿主细胞内和动物体内的CPD激活可视化。这些探针将允许毒素激活的时间和位置通过共价标记激活的CPD实时监测。结合结构方法，这些探针还将有助于分析CPD底物的特异性和功能，从而为抑制艰难梭菌糖基化毒素功能的策略提供信息。

DESCRIPTION (provided by applicant): In this proposal, I describe my plans to elucidate the mechanism by which autoproteolysis regulates bacterial toxin function. In my postdoctoral work thus far, I have discovered a unique mechanism of protease activation and delineated a role for a cysteine protease domain (CPD) in autoprocessing a newly recognized family of bacterial toxins. I have identified the first chemical inhibitors of this novel protease family and have begun developing these inhibitors into chemical probes to study protease substrate specificity and function. In the mentored phase of the proposal, I will continue to learn how to synthesize small molecule inhibitors and probes to monitor and perturb CPD activation in the lab of Dr. Matthew Bogyo at Stanford University. This phase will rely on the depth and diversity of research within the Bogyo lab and the excellent research environment at Stanford. I will supplement my institutional environment with a course in surface plasmon resonance and a visit to a future collaborator's lab (Dr. Jimmy Ballard at Oklahoma State University) where I will learn how to culture Clostridium difficile and purify its glucosylating toxins. At Stanford, I will continue to capitalize on the excellent career development resources within the School of Medicine by attending lectures directed at helping postdoctoral fellows transition into academic positions. I will continue to consult with my advisor, Dr. Bogyo, and an informal mentoring committee, consisting of Dr. John Boothroyd and Dr. K. Christopher Garcia at Stanford, to help me successfully navigate the academic job market, apply for funding, and set-up my own research group. Once I acquire an independent position at a research institution, I will apply the probes and inhibitors developed during the mentored phase to study the function of autoproteolysis in regulating toxin function within cells and in animal models of intoxication. In the long-term, I will apply this new method for imaging protease function, along with my background in bacterial genetics and biochemistry, to study bacterial proteases in diverse systems. 6.B. Research Plan Most secreted bacterial toxins are produced as inactive precursors that become proteolytically activated upon encountering a eukaryotic cell. Whereas eukaryotic proteases typically activate these toxins, a select group of toxins are autoproteolytically activated by an internal cysteine protease domain (CPD). The cytotoxic function of Clostridium difficile glucosylating toxins is activated by the CPD, which itself is activated upon binding the eukaryotic-specific small molecule inositol hexakisphosphate (InsP6). Although autoprocessing is essential for toxin activation, how InsP6 activates the CPD, and when and where in the cell the CPD cleaves these toxins, is unknown. This proposal outlines plans to use small molecules to address these important questions regarding CPD-mediated activation of C. difficile glucosylating toxins. Small molecule inhibitors identified in preliminary studies will be developed into probes that allow CPD activation to be visualized in vitro, within host cells, and in animals. These probes will permit the timing and location of toxin activation to be monitored in real-time by covalently tagging activated CPD. Combined with structural methods, the probes will also facilitate analyses of CPD substrate specificity and function, which will in turn inform strategies directed at inhibiting C. difficile glucosylating toxin function. PUBLIC HEALTH RELEVANCE: Clostridium difficile is the leading cause of nosocomial diarrhea worldwide, and the glucosylating toxins TcdA and TcdB are the primary factors responsible for C. difficile-associated disease. Thus, understanding how the CPD regulates C. difficile toxin activation will inform strategies to alleviate the symptoms of this toxin-mediated disease. By targeting virulence rather than bacterial viability, chemical inhibition of the CPD may reduce the recurrence of antibiotic-induced nosocomial diarrhea.