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

摘要：冠状病毒和冠状病毒是世界范围内发病率和死亡率的主要原因，或者说是 准备引发未来的致命流行病。这些病毒表达它们自己的半胱氨酸蛋白酶、MPRO和 NsP2pro，以在病毒蛋白质的生命周期早期处理它们。这些蛋白水解酶具有明显的结构特征 来自人类的蛋白酶，它们可以被不带电荷的底物类似物抑制，使它们成为靶向 通过口服生物利用药。最近开始的口服SARSCoV2 MPRO抑制剂的临床试验证实 这类目标。现在需要一系列不同的蛋白酶抑制剂来对抗未来的耐药性和 为其他冠状病毒提供治疗。此外，还没有蛋白水解酶抑制剂进入临床试验 托加病毒，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实验室已经开发出创新的方法来制造和测试病毒蛋白酶 抑制剂，包括(I)具有细胞渗透性和特异性抑制病毒蛋白水解酶的新型化学结构， (Ii)基于荧光素酶的活体细胞中抑制蛋白水解酶的快速检测，以及(Iii)独特的成人肺有机类物质 (ALOS)研究药物对病毒复制和组织损伤的影响。这支队伍已经产生了强有力的， 选择性口服生物可利用的SARSCoV2 MPRO抑制剂，命名为ML104m和ML1006m。这些 化合物表现出EC501μM对SARSCoV2复制，高血浆和肝细胞稳定性，以及10%口服 在小鼠体内的生物利用度，在治疗范围内口服后达到血药浓度。 该项目的主要目标是将我们的主要SARSCoV2 MPRO抑制剂推进到IND使能阶段 同时推广我们对其他优先病毒的方法。目标1将优化治疗指数(TI)和 SARSCoV2 MPRO抑制剂的药代动力学(PK)谱，并确定其治疗潜力。这包括 (A)优化SARSCoV2 MPRO抑制的电流引线，并产生酶降解衍生物 项目4；(B)单独测试这些先导化合物的抗病毒效果，并与下列化合物结合使用 项目3、4和6；(C)在ALOS中测试疗效以及在小鼠身上的疗效、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TOGA病毒，以及 研究我们的抑制剂的作用机制。这包括(A)优化MERSCoV的电流导线，(B)创建 CHIKV和VEEV nsP2pro的新抑制剂，以及(C)研究蛋白酶抑制剂在病毒寿命内的作用 循环以及阻力是如何出现的。所有AIMS将利用翻译加速的活动， 大流行援助和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SARSCoV2和其他冠状病毒的新型抑制剂 已经准备好进入临床试验，有可能成为同类中最好的。它还将建立证据证明 针对病毒蛋白酶的新方法的构想，开发其他冠状病毒和TOGA病毒的抑制剂 没有治疗选择，并表征了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.