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

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

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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（由适用提供）：病原体不断威胁人类健康，问题令人担忧；普遍或新兴剂的耐药性正在增长，生物恐怖主义者对熟悉的微生物进行武器化的潜力也在增长。抗菌剂的发育减慢，加剧了这一挑战的幅度。但是，高通量筛查（HTS）的创新已经使复合发现更加系统地，有望获得药物原型和研究问题，以获得对传染病分子基础的关键见解。膜是宿主和入侵病原体之间战斗的第一个区域，使膜居民蛋白质中心蛋白在发病机理和防御方面。在过去的十年中，浸入膜内的活性位点的蛋白质已经出现在潜水员病原体用于实现病毒的电路核心上。尽管这些宽度的微生物酶现在被认为是打击意外疾病和/或耐药性的主要靶标，但从未将潜在的抑制剂用于微生物内膜内蛋白酶。到目前为止，这些反应无法在其自然膜环境中进行直接筛查。我们通过开发一个系统来克服这一挑战，该系统允许浸入膜内的无创和实时菱形催化，以无创和实时控制。为了回应最近重新发行为PA-13-364的NIH请求PA-10-213，我们建议在自然膜环境中首次使用这些酶来开发新型HTS方法，并评估我们的试验筛网中出现的早期命中。成功的结果提出了最终为NIH罕见或被忽视疾病（TRND）倡议做出贡献的令人兴奋的可能性。此提案中率先进行的方法应适用于其他膜脱落酶。