Development of PROTACs Targeting Papain-like Protease as Broad-Spectrum Anti-Coronavirus Therapeutics

PROJECT SUMMARY/ABSTRACT The current COVID-19 pandemic caused by SARS-CoV-2 has paralyzed much of the world. Encouragingly, a few COVID-19 vaccines have been developed and approved for human immunization. However, existing COVID-19 vaccines target the highly mutable membrane Spike protein of SARS-CoV-2. New viral strains with critical mutations in Spike have emerged such as alpha, beta, delta, and gamma variants that could make current vaccines less effective. To truly overcome the threat posed by SARS-CoV-2 and its emerging variants of concern, it is paramount to develop antiviral drugs that can combat COVID-19 and also be potentially repurposed to combat novel coronaviruses (CoVs) in the future. To address this urgent need, this proposal aims to interface papain-like protease (PLpro) inhibitors/binders and the emerging proteolysis targeting chimera (PROTAC) technology for the development of broad-spectrum anti-CoV PROTACs. PLpro, one of two essential SARS-CoV- 2 proteases, plays a dual role in promoting viral transcription and replication, and antagonizing host innate immune responses. PLpro is encoded by nonstructural protein 3 (nsp3) which is relatively conserved across various CoVs. Thus, PLpro is an attractive target for the development of broad-spectrum anti-CoV drugs. The proposed small-molecule PROTACs are bifunctional small molecules containing two active ligands connected via a chemical linker. One ligand binds specifically to a viral protein target PLpro while the other ligand selectively engages an E3 ubiquitin ligase. The recruitment of the E3 ligase to PLpro facilitates the formation of a ternary complex, leading to ubiquitination and ultimate degradation of PLpro by the ubiquitination-proteasome pathway. As PROTACs regulate protein function by degrading target proteins instead of inhibiting them, they offer many advantages over traditional occupancy-based inhibitors, including (i) sub-stoichiometric activity, (ii) high barrier to resistance, (iii) improved target selectivity, and (iv) that weak binders (no inhibition required) can become potent degraders. On this basis, the overall goal of this application is to validate the degradation of PLpro as a new strategy for the development of broad-spectrum antiviral therapeutics to combat CoVs. In Aim 1, to chemically optimize non-covalent, reversible covalent, and multiple binding-mode PROTACs by linking VHL E3 ligands and PLpro inhibitors/binders, and to evaluate their degradation potency of PLpro in cell-based assays. In Aim 2, to determine the anti-SARS-CoV-2 activity of developed PROTAC molecules. Potent PROTACs (EC50 < 500 nM) will be further tested for their antiviral activities against other CoVs. In vitro DMPK and cytotoxicity studies of potent PROTACs will be conducted. The successful completion of the proposed study will lead to potent anti-CoV PROTACs against COVID-19 that the mechanism of action is fundamentally different from existing antivirals. The developed PROTAC molecules will also have the potential to be repurposed to contain future coronavirus outbreaks.