Structure, Function, and Therapeutic Potential of Clostridium difficile Caseinolytic Protease P (Duerfeldt)

项目概要（Duerfeldt项目） 艰难梭菌感染（CDI）是美国（500，000 2011年的病例），并代表了治疗发展的独特挑战，因为它既受到 临床上用传统抗生素治疗。无法有效治疗CDI产生了约50亿美元的过剩 每年的医疗保健费用，如果新的药物靶点和治疗策略能够 未识别。作为感染性细菌毒力的关键调节因子， 由于酪蛋白分解蛋白酶P（ClpP）是一种新的蛋白酶， 抗菌剂的开发事实上，ClpP代表独特的靶标，因为ClpP抑制和激活两者都是通过抑制ClpP来实现的。 具有治疗实用性，每种策略都会影响细菌致病性的不同方面。这提供 一个机会，以确定治疗潜力的两个正交战略对一个单一的目标，一个罕见的 药物发现中的现象。C.然而，艰难的，还没有被表征，因此， 阻碍了其作为治疗CDI的新靶点的追求。重要的是，C。艰难梭菌是典型的肠道微生物区系的独特之处， 它表达ClpP的两种亚型（ClpP 1和ClpP 2）。我们的初步数据和现有的分析 结构-功能分析表明，C. difficile不仅是独特的， 生物体，但也不同于迄今为止公开的任何致病系统。我们假设在C.艰难 ClpP保持其作为主要致病调节因子的进化保守作用，但表现出独特的结构， 和功能特性，为选择性治疗剂的开发提供了途径。一 我们需要采取多方面的方法来处理拟议的措施，我们在战略上 组建了一个由具有C语言专业知识的研究人员组成的合作网络。艰难微生物学，蛋白质化学， 蛋白质结晶/晶体学、分子生物学和药物化学。为了验证我们的假设，我们 建议1）表征载脂蛋白和调节剂结合的cdClpP复合物的结构独特方面; 2） 定义ClpP在C中的具体角色。艰难的发病机制;和3）整合筛选平台，以确定 用于cdClpP特异性调节的新引线。这项研究意义重大，因为需要新的药物靶点 有效治疗CDI我们希望我们的研究将揭示新的ClpP参与致病性， 揭示了C.难选的 调控此外，我们的研究将提供新的ClpP抑制剂和激活剂， 化学运动朝向选择性靶向该蛋白水解系统作为抗CDI策略。 因此，这项研究将大大推进对独特ClpP系统的基本理解，并使 以预防为重点的举措。

Project Summary (Duerfeldt Project) Clostridium difficile infection (CDI), is a leading cause of hospital-acquired illness in the United States (500,000 cases in 2011) and represents a unique challenge to therapeutic development, as it is both facilitated by and clinically managed with traditional antibiotics. The inability to effectively treat CDI produces ~$5B in excess healthcare costs annually, a number that will continue to rise if new drug targets and treatment strategies are not identified. As a key regulator of virulence in infectious bacteria, and given its roles in mediating protein turnover and bacterial homeostasis, caseinolytic protease P (ClpP) has emerged as a new target for antimicrobial development. Indeed, ClpP represents a unique target, as both ClpP inhibition and activation have therapeutic utility, with each strategy affecting different aspects of bacterial pathogenicity. This provides an opportunity to determine the therapeutic potential of two orthogonal strategies on a single target, a rare phenomenon in drug discovery. The ClpP system in C. difficile, however, has not been characterized, thus impeding its pursuit as a new target to treat CDI. Importantly, C. difficile is unique from typical gut microflora, in that it expresses two isoforms of ClpP (ClpP1 and ClpP2). Our preliminary data and analyses of existing structure-function profiles suggest that the ClpP system in C. difficile is not only unique from commensal organisms, but also distinct from any pathogenic system disclosed to date. We hypothesize that in C. difficile ClpP maintains its evolutionarily conserved role as a major pathogenic regulator but exhibits unique structural and functional characteristics that provide avenues for the development of selective therapeutic agents. A multi-dimensional approach is required to address the proposed initiatives and we have strategically assembled a collaborative network of researchers with expertise in C. difficile microbiology, protein chemistry, protein crystallization/crystallography, molecular biology, and medicinal chemistry. To test our hypothesis, we propose to 1) characterize the structurally unique aspects of apo- and modulator-bound cdClpP complexes; 2) define the specific roles of ClpP in C. difficile pathogenesis; and 3) integrate screening platforms to identify novel leads for cdClpP specific modulation. The research is significant because new drug targets are required to effectively treat CDI. We expect that our studies will uncover novel ClpP involvement in pathogenicity and reveal distinct structural and functional aspects of the ClpP system in C. difficile amenable to selective regulation. Additionally, our studies will provide novel ClpP inhibitors and activators that will enable medicinal chemistry campaigns towards the selective targeting of this proteolytic system as an anti-CDI strategy. Therefore, this research will significantly advance the basic understanding of a unique ClpP system and enable translationally focused initiatives.