Biomarker Screening of Essential Putative Glycoprotease Inhibitor

必需的假定糖蛋白酶抑制剂的生物标志物筛选

• 批准号: 8071522
• 负责人: Yinduo Ji
• 金额: $ 37.37万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-15 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8071522
• 关键词: Accounting; Acetolactate Synthase; Acids; Acute suppurative arthritis due to bacteria; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotics; Bacteria; Biochemical; Biological Assay; Biological Markers; Biological Process; Cell Wall; Cells; Chemicals; Communities; Community Hospitals; DNA; Development; Down-Regulation; Drug Delivery Systems; Drug Industry; Endocarditis; Enzymes; Genes; Goals; Growth; Haemophilus influenzae; Health care facility; Homologous Gene; Hydro-Lyases; In Vitro; Infection; Intermediate resistance; Lead; Life; Luciferases; Medical; Metabolic Pathway; Molecular Target; Monitor; Multi-Drug Resistance; Mycoplasma genitalium; Nosocomial Infections; Outcome; Pathogenicity; Patients; Pharmaceutical Preparations; Phenotype; Plasmids; Pneumonia; Preventive; Production; Proteins; Proteomics; Public Health; RNA Interference; Reporter; Research; Resistance; Rice; Screening procedure; Set protein; Skin; Specificity; Staphylococcus aureus; Streptococcus pneumoniae; Surrogate Markers; System; Systemic infection; Technology; Testing; Therapeutic Agents; Therapeutic Intervention; Threonine Dehydratase; Toxic Shock Syndrome; Toxic effect; Vaccines; Validation; Vancomycin; Vancomycin-resistant S. aureus; antimicrobial; base; cellular targeting; cost; fighting; high throughput screening; inhibitor/antagonist; methicillin resistant Staphylococcus aureus; novel; novel strategies; novel therapeutics; pathogen; promoter; public health relevance; resistant strain; therapeutic target; tool

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.

描述（由申请人提供）：鉴于金黄色葡萄球菌的致病性众所周知，耐甲氧西林的S。金黄色葡萄球菌（MRSA）和万古霉素耐药的S.金黄色葡萄球菌（VRSA）引起越来越多的公共卫生问题。 60%以上的S.金黄色葡萄球菌相关的医院获得性感染是由MRSA引起的，MRSA已经越来越多地扩散到医疗机构之外，并且已经成为社区病原体。 在美国，每年治疗住院患者MRSA的费用估计在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/或治疗剂来对抗这种病原体。 我们已经证明，葡萄球菌推定的糖蛋白酶（GCP）是一个独特的抗菌靶标，因为它是细胞壁相关和分泌的，对革兰氏阳性和革兰氏阴性病原体的生存至关重要。 然而，推测的糖蛋白酶的生物学功能仍然不清楚。 如果没有生化读出表型，我们很难开发针对这种新靶点的抑制剂的高通量筛选。 结合微阵列和蛋白质组学方法与调控RNA干扰技术，我们发现，下调推定的糖蛋白酶显着增加其他酶的生产在S。金黄色。 因此，我们假设这些酶是替代标志物，监测这些生物标志物将提供一种强有力的方法来高通量筛选Gcp抑制剂。 该提案的目的是开发一种基于全细胞的高通量检测方法，使用这些升高的蛋白质作为Gcp的替代生物标志物。 在具体目标1中，我们将使用Gcp的替代标记在S.金黄色。 在具体目标2中，我们将使用生物发光报告系统对这种基本的推定糖蛋白酶进行高通量筛选新的化学先导物。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型独特抗菌靶标Gcp的潜在抑制剂，并为针对其他功能未知靶标的化合物的高通量筛选提供新策略。 因此，拟议的研究将提供一种新的策略，用于针对新的药物靶点的化合物的高通量筛选，这对于感染的治疗干预和医学实践以对抗正在出现的耐药性S.金黄色。 公共卫生相关性：S.金黄色葡萄球菌是一种重要的社区和医院获得性病原体，其引起各种各样的感染，从浅表皮肤到危及生命的全身感染，包括肺炎、心内膜炎、脓毒性关节炎和中毒性休克综合征。近年来出现的多重耐药的沙门氏菌.金黄色葡萄球菌，尤其是耐甲氧西林的S.金黄色葡萄球菌和万古霉素中间耐药的S.金黄色葡萄球菌引起了公共卫生界的极大关注。目前迫切需要新的抗生素和有效的疫苗来对抗由沙门氏菌引起的感染。金黄色。我们提出的项目的结果将确定潜在的先导化合物对一个独特的，分泌的抗菌目标，以及提供一种新的策略，高通量筛选抑制剂对功能未知的目标。