High-Throughput Assays for Inhibitors of Understudied Bacterial Proteases

描述（由申请方提供）：未充分研究的细菌蛋白酶抑制剂的高通量测定定植在人体粘膜上的细菌可能具有毒性和侵袭性，引起慢性和急性疾病，包括耐药淋病、肺炎和脑膜炎。靶向定殖和侵袭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 宿主免疫防御机制知之甚少。例如，免疫球蛋白A1（IgA 1）代表粘膜表面定殖的重要屏障，并且几种革兰氏阳性和革兰氏阴性细菌的致病菌株产生切割IgA 1的蛋白酶。这些包括奈瑟氏球菌、嗜血杆菌和链球菌菌株，它们可引起慢性耳部感染（中耳炎）、支气管炎、抗药性淋病、细菌性肺炎和细菌性脑膜炎。尽管有大量的证据表明IgAP是具有不同功能的毒力因子，但由于很少有测定其活性的测定方法，也没有选择性抑制剂，因此对它们的研究非常不足。重要的是，靶向IgAP作为抗毒力疗法的潜力尚未得到测试，并且将一直如此，直到鉴定出选择性抑制剂。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蛋白质的抑制剂。最近，我们设计了荧光探针来定量IgAP活性， 从不同的人类病原体。尽管对IgAP生物学的研究已有四十年，但这些探针是第一个也是唯一一个此类探针，并首次实现了IgAP抑制剂的高通量筛选。该R 01提案的目标是利用这些探针进行高通量筛选，以提供选择性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检测中，进行中试筛选，并在后续检测中测试命中率，以测量其在体外和活细菌上的影响。我们已与布罗德研究所合作，以确保从探针开发到中试检测再到全面筛查活动的平稳过渡。在这个项目中开发的检测方法将被微生物学家和免疫学家用于定量IgAP活性。它们还可以在细菌性脑膜炎和其他感染的诊断中找到临床应用。最重要的是，从高通量筛选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.