Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp13 helicase.

The coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global public health challenge. While the efficacy of vaccines against emerging and future virus variants remains unclear, there is a need for therapeutics. Repurposing existing drugs represents a promising and potentially rapid opportunity to find novel antivirals against SARS-CoV-2. The virus encodes at least nine enzymatic activities that are potential drug targets. Here, we have expressed, purified and developed enzymatic assays for SARS-CoV-2 nsp13 helicase, a viral replication protein that is essential for the coronavirus life cycle. We screened a custom chemical library of over 5000 previously characterized pharmaceuticals for nsp13 inhibitors using a fluorescence resonance energy transfer-based high-throughput screening approach. From this, we have identified FPA-124 and several suramin-related compounds as novel inhibitors of nsp13 helicase activity in vitro. We describe the efficacy of these drugs using assays we developed to monitor SARS-CoV-2 growth in Vero E6 cells.

Discovery of MK-8742: an HCV NS5A inhibitor with broad genotype activity.

The NS5A protein plays a critical role in the replication of HCV and has been the focus of numerous research efforts over the past few years. NS5A inhibitors have shown impressive in vitro potency profiles in HCV replicon assays, making them attractive components for inclusion in all oral combination regimens. Early work in the NS5A arena led to the discovery of our first clinical candidate, MK-4882 [2-((S)-pyrrolidin-2-yl)-5-(2-(4-(5-((S)-pyrrolidin-2-yl)-1H-imidazol-2-yl)phenyl)benzofuran-5-yl)-1H-imidazole]. While preclinical proof-of-concept studies in HCV-infected chimpanzees harboring chronic genotype 1 infections resulted in significant decreases in viral load after both single- and multiple-dose treatments, viral breakthrough proved to be a concern, thus necessitating the development of compounds with increased potency against a number of genotypes and NS5A resistance mutations. Modification of the MK-4882 core scaffold by introduction of a cyclic constraint afforded a series of tetracyclic inhibitors, which showed improved virologic profiles. Herein we describe the research efforts that led to the discovery of MK-8742, a tetracyclic indole-based NS5A inhibitor, which is currently in phase 2b clinical trials as part of an all-oral, interferon-free regimen for the treatment of HCV infection.

Discovery of vaniprevir (MK-7009), a macrocyclic hepatitis C virus NS3/4a protease inhibitor.

A new class of HCV NS3/4a protease inhibitors which contain a P2 to P4 macrocyclic constraint was designed using a molecular-modeling derived strategy. Exploration of the P2 heterocyclic region, the P2 to P4 linker, and the P1 side chain of this class of compounds via a modular synthetic strategy allowed for the optimization of enzyme potency, cellular activity, and rat liver exposure following oral dosing. These studies led to the identification of clinical candidate 35b (vaniprevir, MK-7009), which is active against both the genotype 1 and genotype 2 NS3/4a protease enzymes and has good plasma exposure and excellent liver exposure in multiple species.

Orally efficacious NR2B-selective NMDA receptor antagonists.

A novel series of benzamidines was synthesized and shown to exhibit NR2B-subtype selective NMDA antagonist activity. Compound 31 is orally active in a carrageenan-induced rat hyperalgesia model of pain and shows no motor coordination side effects.