High-Throughput Assays for Inhibitors of Understudied Bacterial Proteases

正在研究的细菌蛋白酶抑制剂的高通量测定

基本信息

批准号：
8817739
负责人：
Joshua A Kritzer
金额：
$ 27.96万
依托单位：
TUFTS UNIVERSITY MEDFORD
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：
2015-08-01 至 2018-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8817739
关键词：
Acute Disease Agglutination Anti-Bacterial Agents Anus Apoptosis Bacteria Bacterial Adhesion Bacterial Infections Bacterial Meningitis Bacterial Pneumonia Biochemical Biological Assay Biology Bronchitis Cell Survival Cells Cellular biology Chronic Chronic Disease Cleaved cell Clinical Communicable Diseases Development Diagnosis Drug Targeting Drug resistance Ensure Fluorescence Fluorescence Resonance Energy Transfer Fluorescent Probes Goals Gonorrhea Gram-Negative Bacteria Hemophilus Human IgA-specific serine endopeptidase Immune Immunoglobulins Immunologist In Vitro Infection Institutes Integration Host Factors LAMP-1 Laboratories Lead Libraries Life Measures Mediating Meningitis Mucous Membrane Otitis Media Peptide Hydrolases Peptides Phenotype Pneumonia Protease Inhibitor Proteins Reporting Research Role Site Streptococcus Streptococcus pneumoniae Surface Testing Therapeutic Tissues Tumor Necrosis Factor Receptor Virulence Virulence Factors Virulent Work base biophysical chemistry clinical application design ear infection experience follow-up high throughput screening inhibitor/antagonist innovation insight novel novel strategies pathogen prevent public health relevance screening small molecule tool trafficking

项目摘要

DESCRIPTION (provided by applicant): High-Throughput Assays for Inhibitors of Understudied Bacterial Proteases Bacteria that colonize the human mucosa can become virulent and invasive, causing chronic and acute diseases including drug-resistant gonorrhea, pneumonia, and meningitis. New drugs targeting colonization and invasion would be welcome additions to the antibacterial arsenal, but interactions among bacterial factors, host tissues, and host immune defenses are poorly understood. For instance, immunoglobulin A1 (IgA1) represents an important barrier to colonization of mucosal surfaces, and pathogenic strains of several Gram- positive and Gram-negative bacteria produce proteases that cleave IgA1. These include Neisserial, Haemophilus, and Streptococcus strains that cause chronic ear infections (otitis media), bronchitis, drug- resistant gonorrhea, bacterial pneumonia and bacterial meningitis. Despite a large body of evidence that IgAPs are virulence factors with diverse functions, they have been vastly understudied because there were few assays for measuring their activity and no selective inhibitors. Importantly, the potential for targeting IgAPs as an ani-virulence therapy remains untested, and will remain so until selective inhibitors are identified. The Kritzer lab uses synthesis, biophysical chemistry, and cell biology to develop inhibitors of difficult-to-target proteins. Recently, we designed fluorescence probes to quantitate IgAP activity from diverse human pathogens. Despite forty years of research into IgAP biology, these probes are the first and only of their kind and enable the first-ever high-throughput screens for IgAP inhibitors. The goal of this R01 proposal is to leverage these probes into high-throughput screens that will deliver selective inhibitors of IgAPs. We will accomplish this goal by: testing additional substrate sequences, developing the substrates into fluorescent probes, incorporating the probes into HTS assays for IgAP inhibitors, performing pilot screens, and testing hits in follow-up assays that measure their effects in vitro and on live bacteria. We have partnered with the Broad Institute to ensure smooth transitions from probe development to pilot assays to a full screening campaign. The assays developed in this project will be of general use to microbiologists and immunologists for quantifying IgAP activity. They could also find clinical applications in the diagnosis of bacterial meningitis and other infections. Most importantly, the inhibitors developed from high-throughput screening will allow us and others to investigate host-pathogen interactions involving IgAPs. The inhibitors will also represent starting points for evaluating IgAPs as drug targets for treating urgent infectious disease threats, including drug- resistant gonorrhea, bacterial pneumonia, and bacterial meningitis.

描述（由申请人提供）：针对人粘膜定植的细菌细菌细菌抑制剂的高通量测定可能变得有毒和侵入性，从而引起慢性和急性疾病，包括耐药性淋病，肺炎和脑膜炎。靶向定植和侵袭的新药物将受到抗菌武器库的补充，但细菌因素，宿主组织和宿主的免疫防御能力知之甚少。例如，免疫球蛋白A1（IgA1）代表了粘膜表面定植的重要障碍，而几种革兰氏阴性和革兰氏阴性细菌的致病菌株产生了裂解Iga1的蛋白酶。其中包括引起慢性耳朵感染（耳炎），支气管炎，耐药淋病，细菌性肺炎和细菌性脑膜炎的奈瑟氏菌，嗜血杆菌和链球菌菌株。尽管大量证据表明Igap是具有多种功能的毒力因素，但它们已经进行了大量研究，因为很少有测量其活性和没有选择性抑制剂的测定法。重要的是，将IgAP作为肛门病毒疗法的潜力仍未经过测试，并且在鉴定出选择性抑制剂之前一直保持下去。 Kritzer Lab使用合成，生物物理化学和细胞生物学来开发难以靶向蛋白质的抑制剂。最近，我们设计了荧光探针来定量IGAP活性来自多样化的人类病原体。尽管对IgAP生物学进行了四十年的研究，但这些探针是它们的第一个也是仅次的探针，并可以为IgAP抑制剂提供第一个高通量屏幕。该R01提案的目的是利用这些探针将这些探针用于高通量屏幕，以提供选择性的IGAP抑制剂。我们将通过：测试其他底物序列来实现此目标，将底物开发为荧光探针，将探针纳入IGAP抑制剂的HTS测定中，执行试验屏幕，并在随访测定中测试其在体外和实时细菌上的效果。我们已经与广大研究所合作，以确保从探测开发到试点测定到完整筛选活动的平稳过渡。该项目中开发的测定法将对微生物学家和免疫学家进行量化IgAP活性的普遍使用。他们还可以在诊断细菌性脑膜炎和其他感染中找到临床应用。最重要的是，由高通量筛选而开发的抑制剂将使我们和其他人可以研究涉及IGAP的宿主 - 病原体相互作用。抑制剂还将代表评估IGAP作为治疗紧急传染病威胁的药物靶标的起点，包括耐药性淋病，细菌性肺炎和细菌性脑膜炎。