Biomarker Screening of Essential Putative Glycoprotease Inhibitor

描述（由申请人提供）：鉴于众所周知的金黄色葡萄球菌致病性，耐甲氧西林金黄色葡萄球菌（MRSA）和耐万古霉素金黄色葡萄球菌（VRSA）的出现引起了越来越多的公共卫生关注。超过60%的金黄色葡萄球菌相关的医院获得性感染是由MRSA引起的，MRSA已经越来越多地传播到医疗机构之外，并已成为一种社区病原体。在美国，每年治疗住院患者的MRSA费用估计在32亿至42亿美元之间。由于治疗MRSA感染的药物选择有限，VRSA的威胁一直是深入研究的主题。因此，迫切需要开发更有效的预防和/或治疗药物来对抗这种病原体。我们已经证明葡萄球菌假定的糖蛋白酶（Gcp）是一种独特的抗菌靶点，因为它与细胞壁相关并分泌，对革兰氏阳性和革兰氏阴性病原体的生存都至关重要。然而，这种假定的糖蛋白酶的生物学功能尚不清楚。如果没有生化读出表型，我们很难开发针对这种新靶点的高通量抑制剂筛选。结合微阵列和蛋白质组学方法以及调控的RNA干扰技术，我们发现假定的糖蛋白酶的下调显著增加了金黄色葡萄球菌中其他酶的产生。因此，我们假设这些酶是替代标记物，监测这些生物标记物将为高通量筛选Gcp抑制剂提供强有力的方法。本提案的目的是开发一种基于全细胞的高通量测定方法，使用这些升高的蛋白作为Gcp的替代生物标志物。在具体目标1中，我们将在金黄色葡萄球菌中使用Gcp¿s替代标记构建生物发光报告系统。在具体目标2中，我们将使用生物发光报告系统对这种必要的假定的糖蛋白酶进行高通量筛选。在具体目标3中，我们将验证先导化合物以确定其作用模式、特异性和选择性。这些研究的成功完成将确定针对这种新颖独特的抗菌靶点Gcp的潜在抑制剂，并为针对其他功能未知靶点的高通量筛选化合物提供新的策略。因此，所提出的研究将为针对新药物靶点的化合物的高通量筛选提供一种新的策略，这将对感染的治疗干预和对抗新出现的耐抗生素金黄色葡萄球菌的医疗实践特别重要。

DESCRIPTION (provided by applicant): Given the well-known pathogenicity of Staphylococcus aureus, the emergence of methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) is causing increased public health concerns. More than 60% of S. aureus-associated hospital acquired infections are caused by MRSA, which has increasingly spread beyond healthcare facilities and has emerged as a community pathogen. The annual cost to treat MRSA in hospitalized patients in the U.S. is estimated to be between $3.2 billion to 4.2 billion. The threat of VRSA has been the topic of intensive research since there is a limited drug of choice for MRSA infections. Therefore, there is an urgent need to develop more efficient preventive and/or therapeutic agents to fight this pathogen. We have demonstrated that a staphylococcal putative glycoprotease (Gcp) is a unique antibacterial target since it is cell-wall associated and secreted, essential for survival for both Gram-positive and Gram-negative pathogens. However, the biological function of the putative glycoprotease is still unclear. Without a biochemical readout phenotype, it is difficult for us to develop a high-throughput screen of inhibitors against this novel target. Combined microarray and proteomic approaches with a regulated RNA interference technology, we found that the down-regulation of the putative glycoprotease significantly increases the productions of other enzymes in S. aureus. Therefore, we hypothesize that these enzymes are the surrogate markers and monitoring these biomarkers will provide a powerful approach to high-throughput screen inhibitors against Gcp. The objective of this proposal is to develop a whole cell-based high throughput assay using these elevated proteins as Gcp¿s surrogate biomarkers. In specific aim 1, we will construct bioluminescent reporter systems using Gcp¿s surrogate markers in S. aureus. In specific aim 2, we will perform high-throughput screening of novel chemical leads against this essential putative glycoprotease using the bioluminescent reporter systems. In specific aim 3, we will validate lead compounds to determine their mode of action, specificity, and selectivity. Successful completion of these studies will identify potential inhibitors against this novel, unique antibacterial target, Gcp, as well as provide a novel strategy for high-throughput screening of compounds against other functional unknown targets. Therefore, the proposed studies will provide a novel strategy for high-throughput screening of compounds against novel drug target that will be particularly important to therapeutic interventions for infections and medical practice to fight the emerging antibiotic-resistant S. aureus. PUBLIC HEALTH RELEVANCE: S. aureus is an important community and hospital acquired pathogen causing a wide variety of infections, ranging from superficial skin to life-threatening systemic infections, including pneumonia, endocarditis, septic arthritis and toxic shock syndrome. The recent emergence of multiple antibiotic- resistant strains of S. aureus, especially methicillin-resistant S. aureus and vancomycin-intermediate resistant S. aureus, is causing great concern in the public health community. There is an urgent need for new classes of antibiotics and potent vaccines to fight infections caused by S. aureus. The outcome of our proposed project will identify potential lead compounds against a unique, secreted antibacterial target, as well as provide a novel strategy for high-throughput screening of inhibitors against functional unknown targets.