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Catalysis In the Membrane: Developing Direct Assays for High-Throughput Screening

膜催化：开发高通量筛选的直接测定方法

基本信息

批准号：
9197598
负责人：
SINISA URBAN
金额：
$ 40.5万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-01-01 至 2018-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9197598
关键词：
Acquired Immunodeficiency Syndrome Active Sites Address Adhesions Amoeba genus Antibiotics Antiparasitic Agents Bacterial Infections Biological Biological Assay Biophysics Catalysis Cause of Death Characteristics Chemicals Communicable Diseases Cryptosporidium Crystallization Dangerousness Detection Detergents Development Disease Disease Resistance Drug Targeting Drug resistance Dysentery Ensure Environment Enzymes Eukaryotic Cell Family Goals HIV Protease Hand Health Human Infection Invaded Investments Kinetics Libraries Malaria Membrane Membrane Proteins Methods Microbe Modernization Molecular Mycobacterium tuberculosis Outcome Parasites Parasitic infection Pathogenesis Pathogenicity Patients Peptide Hydrolases Pharmaceutical Preparations Production Property Protease Inhibitor Proteins Protozoa Pseudomonas aeruginosa Reaction Reagent Research Research Infrastructure Robotics Series Serine Protease Serine Proteinase Inhibitors Structure System Technology Testing Therapeutic Ticks Time Toxin Toxoplasma United States National Institutes of Health Vibrio cholerae Virulence acquired drug resistance antimicrobial drug assay development base cellular engineering combat high throughput screening inhibitor/antagonist innovation insight light scattering microbial microcalorimetry neglect next generation non-invasive monitor novel novel therapeutics pathogen pathogenic bacteria protease E protein E prototype public health relevance reconstitution response rhomboid rhomboid catalysis screening weapons

项目摘要

DESCRIPTION (provided by applicant): Pathogens constantly threaten human health, and the problem is worsening; drug resistance by prevalent or emerging agents is growing, as is the potential for weaponization of familiar microbes by bioterrorists. Slowed development of antimicrobial agents has exacerbated the magnitude of this challenge. However, innovations in high-throughput screening (HTS) have made the path to compound discovery more systematic, promising both drug prototypes and research probes for gaining key insights into the molecular basis of infectious disease. Membranes are the first zones of combat between host and invading pathogens, making membrane-resident proteins central players in pathogenesis and defense. Over the past decade, proteases with active sites immersed inside the membrane have emerged at the core of circuits that diverse pathogens use to achieve virulence. Although these widespread microbial enzymes are now considered prime targets for combating infectious disease and/or drug resistance, potent inhibitors have never been isolated for microbial intramembrane proteases. Until now, these reactions have been inaccessible to direct screening in their natural membrane environment. We overcome this challenge by developing a system that allows controlling and monitoring non-invasively and in real-time rhomboid catalysis immersed inside the membrane. In response to NIH request PA-10-213, recently reissued as PA-13-364, we propose to use this innovation to develop novel HTS methods with these enzymes for the first time in their natural membrane setting, and to evaluate early hits emerging from our pilot screens. Successful outcomes raise the exciting possibility of ultimately contributing to the NIH's therapeutics for rare or neglected diseases (TRND) initiative. Methods pioneered in this proposal should be applicable to other membrane-immersed enzymes.

描述（由申请人提供）：病原体不断威胁人类健康，问题正在恶化;流行或新兴病原体的耐药性正在增长，生物恐怖分子将熟悉的微生物武器化的可能性也在增长。抗菌剂的缓慢发展加剧了这一挑战的严重性。然而，高通量筛选（HTS）的创新使化合物发现的途径更加系统化，有望获得药物原型和研究探针，以获得对感染性疾病分子基础的关键见解。膜是宿主和入侵病原体之间的第一个战斗区域，使膜驻留蛋白在致病和防御中发挥核心作用。在过去的十年中，具有浸入膜内的活性位点的蛋白酶已经出现在各种病原体用于实现毒力的电路的核心。尽管这些广泛分布的微生物酶现在被认为是对抗感染性疾病和/或耐药性的主要靶标，但从未分离出微生物膜内蛋白酶的有效抑制剂。到目前为止，这些反应还无法在其天然膜环境中直接筛选。我们通过开发一种系统来克服这一挑战，该系统允许非侵入性地和实时地控制和监测浸没在膜内的菱形催化。为了响应NIH的要求PA-10-213，最近重新发布为PA-13-364，我们建议使用这一创新来开发新的HTS方法，这些酶首次在其自然膜环境中使用，并评估从我们的试点筛选中出现的早期命中。成功的结果提高了最终为NIH的罕见或被忽视疾病治疗（TRND）计划做出贡献的可能性。该提案中开创的方法应该适用于其他膜浸酶。