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

项目摘要(DuerFeldt项目) 艰难梭菌感染(CDI)是美国医院获得性疾病的主要原因(500,000 2011年的病例)，并对治疗发展提出了独特的挑战，因为它是由和 临床上用传统的抗生素治疗。无法有效地治疗CDI会产生约50亿美元的额外收入 每年的医疗费用，如果新的药物靶点和治疗策略 身份不明。作为感染性细菌毒力的关键调节因子，并考虑到其在调节蛋白质方面的作用 周转和细菌动态平衡，酪蛋白水解酶P(ClpP)已成为新的靶点 抗菌药物的开发。事实上，ClpP代表着一个独特的靶点，因为ClpP既有抑制作用，也有激活作用 具有治疗作用，每种策略影响细菌致病性的不同方面。这提供了 有机会确定同一靶点上两种正交策略的治疗潜力，这是一种罕见的机会 药物发现中的现象。然而，艰难梭菌中的ClpP系统还没有被表征，因此 阻碍其成为治疗CDI的新靶点。重要的是，艰难梭菌是典型的肠道微生物区系中独一无二的。 它表达ClpP的两种亚型(ClpP1和ClpP2)。我们的初步数据和现有的分析 结构-功能图谱表明艰难梭菌中的ClpP系统不仅不同于共生菌 生物体，但也有别于迄今披露的任何致病系统。我们假设在艰难梭菌中 ClpP作为一个主要的致病调节因子，在进化上保持了保守的作用，但具有独特的结构 以及为开发选择性治疗剂提供途径的功能特性。一个 需要多层面的方法来解决拟议的举措，我们已经从战略上 组建了一个由艰难梭菌微生物学、蛋白质化学、 蛋白质结晶/结晶学、分子生物学和药物化学。为了检验我们的假设，我们 建议1)表征脱氧核糖核酸和调节剂结合的cdClpP复合体的独特结构；2) 明确ClpP在艰难梭菌致病机制中的具体作用；以及3)整合筛选平台以识别 用于cdClpP特定调制的新型导线。这项研究意义重大，因为需要新的药物靶点 以有效地治疗CDI。我们期望我们的研究将发现新的ClpP参与致病和 揭示艰难梭菌ClpP系统的不同结构和功能 监管。此外，我们的研究将提供新的ClpP抑制剂和激活剂，使药物 化学运动以选择性靶向这种蛋白分解系统作为抗CDI的策略。 因此，这项研究将极大地促进对独特的CLPP系统的基本理解，并使 以翻译为重点的倡议。

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.