Development of selective and potent protease inhibitors for corona and other pandemic viruses

摘要：冠状病毒和披膜病毒是世界范围内发病和死亡的主要原因， 准备引发未来致命的流行病这些病毒表达它们自己的半胱氨酸蛋白酶Mpro和 nsP 2 pro，在病毒生命周期的早期处理病毒蛋白。这些蛋白酶具有不同的结构特征， 它们可以被不带电的底物类似物抑制， 通过口服生物可利用的药物。最近启动的口服SARSCoV 2 Mpro抑制剂的临床试验验证了 这类目标。现在需要一组不同的蛋白酶抑制剂来对抗未来的耐药性， 为其他冠状病毒提供治疗。此外，没有蛋白酶抑制剂进入临床试验， 披膜病毒，包括人类病原体基孔肯雅病毒和委内瑞拉马脑炎病毒。 Lin、Bogyo和Einav实验室已经开发出了创造和测试病毒蛋白酶的创新方法 抑制剂，包括（i）可渗透细胞并特异性抑制病毒蛋白酶的新化学结构， (ii)活细胞中蛋白酶抑制的快速基于酶的测定，和（iii）独特的成人肺类器官 （ALO）研究药物对病毒复制和组织损伤的影响。这个团队已经产生了强大的， 选择性和口服生物可利用的SARSCoV 2 Mpro抑制剂，命名为ML 104 m和ML 1006 m。这些 化合物对SARSCoV 2复制的EC 50 < 1 μM，血浆和肝细胞稳定性高，口服10% 在小鼠中的生物利用度，口服给药后达到治疗范围内的血浆浓度。 该项目的主要目标是将我们的主要SARSCoV 2 Mpro抑制剂推进到IND使能阶段 同时将我们的方法推广到其他优先病毒。目标1将优化治疗指数（TI）， SARSCoV 2 Mpro抑制剂的药代动力学（PK）特征，并确定其治疗潜力。这包括 (A)优化用于SARSCoV 2 Mpro抑制的电流引线并产生酶降解衍生物， 项目4;（B）测试这些先导物单独和与来自以下的化合物组合的抗病毒功效： 项目3、4和6;（C）测试ALO中的疗效以及小鼠中的疗效、药代动力学和毒性;以及（D） 选择IND候选人。目标2将把我们的方法推广到其他冠状病毒和关键的披膜病毒， 研究抑制剂的作用机制这包括（A）优化MERSCoV的电流引线，（B）创建 CHIKV和VEEV nsP 2 pro的新抑制剂，以及（C）研究蛋白酶抑制剂在病毒生命期内何时起作用 周期和阻力如何出现。所有目标将利用翻译加速的活动， 流行病援助和SyneRx中心的结构生物学核心。 如果成功，该项目将迅速开发针对SARSCoV 2和其他冠状病毒的新型抑制剂， 已经准备好进入临床试验，并有潜力成为一流的。它还将证明 针对病毒蛋白酶的新方法的概念，开发其他冠状病毒和披膜病毒的抑制剂， 其没有治疗选择，并且表征蛋白酶抑制剂治疗晚期疾病的能力。

ABSTRACT: Coronaviruses and togaviruses are major causes of morbidity and mortality worldwide, or are poised to initiate future deadly epidemics. These viruses express their own cysteine proteases, Mpro and nsP2pro, to process viral proteins early in their life cycles. These proteases have structural features distinct from human proteases, and they can be inhibited with uncharged substrate analogs, making them targetable by orally bioavailable drugs. The recent initiation of clinical trials for an oral SARSCoV2 Mpro inhibitor validates this class of targets. A diverse set of protease inhibitors are now needed to counter future resistance and provide treatments for other coronaviruses. Further, no protease inhibitors have entered clinical trials for togaviruses, including the human pathogens Chikungunya and Venezuelean Equine Encephaitis Viruses. The Lin, Bogyo, and Einav Labs have developed innovative approaches to create and test viral protease inhibitors, including (i) novel chemical structures that are cell permeable and specifically inhibit viral proteases, (ii) a rapid luciferase-based assay of protease inhibition in living cells, and (iii) unique human adult lung organoids (ALOs) to study the effects of drugs on virus replication and tissue injury. This team has already generated potent, selective, and orally bioavailable inhibitors of SARSCoV2 Mpro, named ML104m and ML1006m. These compounds exhibit EC50 < 1 μM for SARSCoV2 replication, high plasma and hepatocyte stability, and 10% oral bioavailability in mice, achieving plasma concentrations after oral dosing in the therapeutic range. The main goal of this project is to advance our lead SARSCoV2 Mpro inhibitors into the IND-enabling stage while generalizing our approach to other priority viruses. Aim 1 will optimize the therapeutic index (TI) and pharmacokinetic (PK) profile of SARSCoV2 Mpro inhibitors, and define their therapeutic potential. This includes (A) optimizing current leads for SARSCoV2 Mpro inhibition and generating enzyme-degrading derivatives with Project 4; (B) testing these leads for antiviral efficacy individually and in combination with compounds from Projects 3, 4, and 6; (C) testing efficacy in ALOs and efficacy, pharmacokinetics, and toxicity in mice; and (D) selecting IND candidates. Aim 2 will generalize our methods to other coronaviruses and key togaviruses, and study our inhibitors’ mechanisms of action. This includes (A) optimizing current leads for MERSCoV, (B) creating new inhibitors of CHIKV and VEEV nsP2pro, and (C) studying when protease inhibitors act within the viral life cycle and how resistance emerges. All Aims will leverage the activities of the Translational Acceleration, Pandemic Assistance, and Structural Biology cores of the SyneRx Center. If successful, this project will rapidly develop novel inhibitors for SARSCoV2 and other coronaviruses that are ready to enter clinical trials and that have the potential to be best-in-class. It will also establish proof of concept for new ways to target viral proteases, develop inhibitors for other coronaviruses and togaviruses for which no treatment options exist, and characterize the ability of protease inhibitors to treat late-stage disease.