Development of ALG-097431 as a broad-spectrum 3CL pro inhibitor

项目摘要/摘要：ALG-097431作为广谱SARS-CoV-2 3CLpro的开发 抑制剂 用小分子药物抑制病毒蛋白酶是一种经证实成功的治疗方法， 目前用于治疗HCV和HIV感染。SARS-CoV-2主要的3-糜蛋白酶样半胱氨酸 蛋白酶（Mpro，3CLpro）代表了另一种有前途的病毒蛋白酶靶点，用于抗病毒治疗，而不需要人 同源物3CLpro的小分子、共价结合抑制剂的发现和开发是本领域的一个热点。 这个节目的焦点。与我们在药物设计和发现中心（CD 3）的合作者一起， 在比利时鲁汶大学的Rega研究所，我们已经开发了多种生物化学和基于细胞的检测方法， 分析化合物并获得SARS-CoV-2感染的仓鼠模型。通过基于结构的 优化，结合选择性和SARS-CoV-2 3CLpro生化酶法，我们有 鉴定了一系列高选择性和有效的3CLpro抑制剂。SARS-CoV-2复制的抑制 在表达人ACE-2受体的A549细胞中证实了这些化合物与化合物 表明低纳摩尔EC 50值。在A549细胞中，在高达 100 μM。这些化合物的活性扩展到其他人类冠状病毒，如α-冠状病毒 229 E和β-冠状病毒OC 43，表现出广谱抗冠状病毒活性。一名代表 将化合物ALG-097111引入体内功效模型，其中仓鼠用 SARS-CoV-2.在感染后两天，在感染后24小时， 病毒RNA拷贝和3.7 log 10（TCID 50/mg）的下降，在肺中的感染性病毒滴度观察到， ALG-097111给药动物。进一步的化合物优化导致ALG-097431的开发。 该化合物相对于3CLpro酶显示出4 nM的IC 50和39 nM的EC 50（选择性指数为1.5 nM）。 >1200）。此外，ALG-097431在原发性肿瘤中表现出强效活性。 人气道上皮细胞，气液界面三维培养系统（EC 90 = 12 nM）。该化合物证明 清洁的体外ADME-毒性特征，并在大鼠和犬中显示出良好的口服生物利用度。人体有效剂量- 预测表明，每天两次给药的潜力与250 - 600毫克剂量，而不需要共同的， 利托那韦的给药。该项目的目标是将ALG-097431推进到人体临床试验中， 同时优化其它化合物以增强抗病毒活性和PK性质。最初，这将 包括建立PK/PD相关性，并进行进一步的选择性分析和更广泛的PK 分析.在探索性安全药理学研究、GMP药物合成、制剂开发和 将启动生物分析方法的验证。在临床研究开始之前， 将在根据GLP法规进行的多项体外和体内研究中评价ALG-097431。

Project Summary/Abstract: Development of ALG-097431 as a Broad-Spectrum SARS-CoV-2 3CLpro Inhibitor Inhibition of viral proteases with small molecule drugs is a proven successful therapeutic approach and is currently used for the treatment of HCV and HIV infection. The SARS-CoV-2 main, 3-chymotrypsin-like cysteine protease (Mpro, 3CLpro) represents another promising viral protease target for antiviral therapy with no human homolog. The discovery and development of small molecule, covalent binding inhibitors of the 3CLpro is the focus of this program. Together with our collaborators at the Centre for Drug Design and Discovery (CD3) and the Rega Institute at KU Leuven, Belgium, we have developed multiple biochemical and cell-based assays to profile compounds and have access to the hamster model of SARS-CoV-2 infection. Through structure-based optimization, combined with selectivity and SARS-CoV-2 3CLpro biochemical enzymatic assays, we have identified a series of highly selective and potent 3CLpro inhibitors. The inhibition of replication of SARS-CoV-2 by these compounds was confirmed in A549 cells expressing the human ACE-2 receptor with compounds demonstrating low nanomolar EC50 values. No cytotoxicity was observed in A549 cells at concentrations up to 100 μM. The activity of these compounds extended to other human coronaviruses such as the alpha-coronavirus 229E and the beta-coronavirus OC43, demonstrating broad-spectrum anti-coronaviral activity. A representative compound, ALG-097111, was taken into an in vivo efficacy model in which hamsters were challenged with SARS-CoV-2. At two days post-infection, a robust and significant 3.5 log10 (RNA copies/mg) reduction of the viral RNA copies and 3.7 log10 (TCID50/mg) reduction in the infectious virus titers in the lungs was observed in ALG-097111-treated animals. Further compound optimization has resulted in the development of ALG-097431. This compound demonstrates an IC50 of 4 nM vs. the 3CLpro enzyme and an EC50 of 39 nM (selectivity index of >1200) in a A549-ACE2/B.1.1.7 cell-based assay. Further, ALG-097431 exhibits potent activity in a primary human airway epithelial cell, air-liquid interface 3D-culture system (EC90 = 12 nM). The compound demonstrates a clean in vitro ADME-tox profile and shows good oral bioavailability in rats and dogs. Human efficacious dose- projections indicate the potential for twice-daily dosing with a 250 – 600 mg dose without the need for co- administration with ritonavir. The goals of this project are to advance ALG-097431 into human clinical trials while simultaneously optimizing further compounds for enhanced antiviral activity and PK properties. Initially this will involve establishing PK/PD correlations and conducting further selectivity analysis and more extensive PK analysis. Following exploratory safety pharmacology studies, GMP drug synthesis, formulation development and validation of bioanalytical methods will be initiated. Prior to the initiation of clinical studies, the potential toxicity of ALG-097431 will be evaluated in multiple in vitro and in vivo studies conducted under GLP regulations.