High-Throughput Assays for Inhibitors of Understudied Bacterial Proteases

描述(由申请人提供)：高通量测试未被研究的细菌蛋白酶的抑制物定植于人体粘膜的细菌可变得毒力强且具有侵袭性，可导致慢性和急性疾病，包括耐药淋病、肺炎和脑膜炎。针对定植和入侵的新药将是抗菌药物库中受欢迎的补充，但细菌因子、宿主组织和 对宿主的免疫防御知之甚少。例如，免疫球蛋白A1(IgA1)是黏膜表面定植的重要屏障，几种革兰氏阳性和革兰氏阴性细菌的病原菌产生裂解IgA1的蛋白酶。这些细菌包括奈瑟氏杆菌、嗜血杆菌和链球菌，可导致慢性耳部感染(中耳炎)、支气管炎、耐药淋病、细菌性肺炎和细菌性脑膜炎。尽管有大量证据表明，IgAP是具有不同功能的毒力因子，但由于几乎没有检测其活性的方法，也没有选择性的抑制剂，对它们的研究一直很少。重要的是，靶向免疫球蛋白作为抗病毒疗法的潜力仍未得到测试，而且在确定选择性抑制剂之前仍将如此。Kritzer实验室使用合成、生物物理化学和细胞生物学来开发难以靶向的蛋白质的抑制剂。最近，我们设计了荧光探针来定量测定IGAP的活性 来自不同的人类病原体。尽管对IGAP生物学进行了40年的研究，但这些探针是第一个也是唯一一个此类探针，能够实现对IGAP抑制剂的首次高通量筛选。这项R01提案的目标是利用这些对高通量筛选的探测，这些筛查将提供选择性的IgAP抑制剂。我们将通过以下方式实现这一目标：测试额外的底物序列，将底物开发成荧光探针，将探针纳入IGAP抑制剂的HTS检测，进行中试筛选，并在后续测试中测试HITS，以测量其在体外和对活细菌的影响。我们与博德研究所合作，确保从探针开发到中试分析再到全面筛查活动的平稳过渡。该项目中开发的分析方法将对微生物学家和免疫学家量化IGAP活性具有普遍意义。它们还可以在诊断细菌性脑膜炎和其他感染方面找到临床应用。最重要的是，从高通量筛选中开发的抑制剂将使我们和其他人能够研究涉及IgAP的宿主-病原体相互作用。这些抑制剂也将是评估IgAP作为治疗紧急传染病威胁的药物靶标的起点，包括耐药淋病、细菌性肺炎和细菌性脑膜炎。

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.