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

 描述(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抗药性正在增长，生物恐怖分子将熟悉的微生物武器化的可能性也在增加。抗菌剂发展缓慢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，以获得对传染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，使膜驻留蛋白在发病和防御中发挥中心作用。在过去的十年里，具有浸泡在膜内的活性位点的蛋白酶已经出现在不同病原体用来实现毒力的电路的核心。虽然这些广泛存在的微生物酶现在被认为是对抗传染病和/或耐药性的主要靶点，但从来没有分离出微生物膜内蛋白水解酶的有效抑制剂。到目前为止，这些反应一直无法在其自然的膜环境中进行直接筛选。我们通过开发一种系统来克服这一挑战，该系统允许对浸入膜内的菱形催化进行非侵入性和实时的控制和监测。为了回应NIH最近以PA-13-364重新发布的PA-10-213要求，我们建议利用这项创新来开发首次在自然膜环境中使用这些酶的新型HTS方法，并评估从我们的试点屏幕中出现的早期命中。成功的结果增加了最终为NIH的罕见或被忽视疾病的治疗(TRND)倡议做出贡献的令人兴奋的可能性。这项提议中首创的方法应该适用于其他膜浸泡酶。

 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.