Developing Antivirals Targeting Proteases and Polymerases of Coronaviruses, Picornaviruses and Bunyavirales

PROJECT 2: DEVELOPING ANTIVIRALS TARGETING PROTEASES AND POLYMERASES OF CORONAVIRUSES, PICORNAVIRUSES AND BUNYAVIRALES SUMMARY The goal of this Project is to develop drug-like inhibitors of coronavirus (CoV), picornavirus (PV) and Bunyavirales (BV) replication by targeting essential protease (PR) and polymerase (Pol) enzymes encoded by the viruses. Molecular targets of the CoVs being pursued are the main PR (MPro), the papain-like PR (PLP), and the RNA- dependent RNA polymerase (RdRp). Molecular targets of the PVs are the enteroviral (EV) 2A and 3C PRs and RdRp, and for BV PLP and RdRp. In Aims 1 and 2 we will identify hits and conduct lead optimization, for proteases and RdRps, respectively. Mechanism of action and resistance potential will be explored for both aims, especially for inhibitors that target novel sites. The close evolutionary relationship between CoVs and PVs may also yield broad-spectrum inhibitors and feedback between both viral targets. The team of investigators have a long and successful track record of structure-guided drug design, including extensive targeting of PRs and Pols. We have established robust, scalable expression systems for producing reagent quantities of SARS-CoV-2 viral enzymes. High-throughput, sensitive assays for measuring PR and RdRp activity have been developed for SARS-CoV-2 MPro, PLP and EV 2A and have been used to discover both covalent and noncovalent low μM inhibitors for MPro, μM inhibitors for PLP, and a biologic activator of 2A. A 100,000-compound biochemical screen against Lassa virus RdRp has yielded numerous hits and a path to structure-guided optimization. We will develop robust high-throughput (HTP) PR and RdRp assays for related CoVs, PVs, and BVs. The substrate specificity of PRs will be profiled to inform substrate and inhibitor design, while cellular perturbations these inhibitors confer will be explored by proteomics and cellular tomography to understand mechanism of action. We will use large panels of recombinant viral and host PRs and Pols to rapidly evaluate hit and lead selectivity and specificity. These efforts will be supported by the activities of the eight Technology Cores. Efforts will focus on novel chemotypes, identified using a combination of structure-based docking, diverse and multi-technique fragment screens, and HTS. Compounds will be optimized to minimize eventual resistance. Mode of binding and quantitative structure-activity relationships (QSAR) will be established using X-ray crystallography, NMR spectroscopy, cryo-electron microscopy and viral replication assays. PR inhibitors and RdRp inhibitors will be tested together to identify additive or synergistic effects. This information will be used to direct the next round of screening and inhibitor improvement. Clinically relevant mutations identified in patients treated with PR or RdRp inhibitors will be introduced into the wild-type enzymes and characterized for their sensitivity to our novel chemotypes. Emerging from this work will be a diverse array of inhibitory chemotypes and structural scaffolds to facilitate development of highly effective drugs. While ambitious, extensive preliminary success supports the pragmatism of these aims.