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的致病性，耐甲氧西林金黄色葡萄球菌(MRSA)和万古霉素耐药金黄色葡萄球菌(VRSA)的出现正引起越来越多的公共卫生关注。超过60%的金黄色葡萄球菌医院获得性感染是由MRSA引起的，它已经越来越多地扩散到医疗机构之外，并已成为一种社区病原体。据估计，美国住院患者每年治疗MRSA的成本在32亿至42亿美元之间。VRSA的威胁一直是深入研究的主题，因为治疗MRSA感染的药物选择有限。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糖蛋白(GCP)是一个独特的抗菌靶点，因为它是细胞壁相关的和分泌的，对革兰氏阳性和革兰氏阴性病原体的生存都是必不可少的。然而，这种可能的糖蛋白水解酶的生物学功能仍不清楚。如果没有生化读出表型，我们就很难针对这个新的靶点开发高通量的抑制剂筛选。结合基因芯片和蛋白质组学方法以及受调控的RNA干扰技术，我们发现金黄色葡萄球菌中假定的糖蛋白水解酶的下调显著增加了其他酶的产量。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抑制GCP的药物提供强有力的途径。这项建议的目的是利用这些升高的蛋白质作为GCP和S的替代生物标志物，建立一种基于全细胞的高通量检测方法。在具体目标1中，我们将利用金黄色葡萄球菌中的GCP和S替代标记构建生物发光报告系统。在特定的目标2中，我们将使用生物发光报告系统高通量地筛选针对这种重要的假定糖蛋白水解酶的新的化学先导。在具体目标3中，我们将验证先导化合物，以确定它们的作用模式、特异性和选择性。这些研究的成功完成将确定针对这一新的、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化合物的高通量筛选提供一种新的策略。因此，拟议的研究将为针对新的药物靶点高通量筛选化合物提供一种新的策略，这对于感染的治疗干预和对抗新出现的耐药金黄色葡萄球菌的医疗实践将特别重要。 公共卫生相关性：金黄色葡萄球菌是一种重要的社区和医院获得性病原体，可引起多种感染，从浅表皮肤到危及生命的全身感染，包括肺炎、心内膜炎、感染性关节炎和中毒性休克综合征。近年来出现的多重耐药金黄色葡萄球菌，尤其是耐甲氧西林金黄色葡萄球菌和万古霉素中间耐药金黄色葡萄球菌，引起了公共卫生界的高度关注。迫切需要新的抗生素和有效的疫苗来对抗金黄色葡萄球菌引起的感染。我们提议的项目的结果将识别针对一个独特的、分泌的抗菌目标的潜在先导化合物，并为针对功能未知目标的高通量抑制剂筛选提供一种新的策略。

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.