PROTAC-based approach to develop broad-spectrum antiviral agents triggering the proteolysis of the major viral protease

In 2020, with the spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, the PROteolysis TArgeting Chimeras (PROTACs) strategy to defeat coronaviruses (CoV) disease was also investigated (1). Indeed, PROTAC degraders acting against the major viral protease 3-chymotrypsin-like protease (3CLPro) were hypothesized to be next-generation anti-CoV drugs (2,3). 3CLPro (also named nsp5 or main protease) is shared by all coronavirus genera as well as by members of the large genus Enterovirus in the Picornavirus family (4). Combining NMR spectroscopy and X-ray crystallography, we characterize the interaction between the α, β-unsaturated peptidomimetic-based PROTAC (FT235) with 3CLPro from SARS-COV-2 and 3CPro from Coxsackievirus B3 (CVB3), the latter being a cardiotropic virus belonging to enterovirus genus (5). The results are compared with those obtained with the precursor molecule (FT234). We demonstrate that PROTAC molecule bind to the active site of both enzymes and the residues involved in the interaction are identified by solution NMR experiments. Furthermore, the crystal structures of SARS-CoV-2 3CLPro in complex with both FT234 and FT235 molecules are obtained. For both ligands the Cβ carbon of the α, β-unsaturated amide moiety forms a covalent bond with the catalytic Cys145. Moreover, we obtain the crystal structure of 3CPro from CVB3 in complex with FT234 molecule, relieving the covalent binding with Cys147. Finally, preliminary cellular studies show that the synthetized PROTAC molecule drastically reduces protein levels of SARS-CoV-2 3CLPro in cultured cells. From this work it therefore appears that PROTAC technology is a promising strategy and it opens up new possibilities for creating cutting-edge antiviral treatments. References: 1. Desantis J, Goracci L. Proteolysis targeting chimeras in antiviral research. Future Med Chem. 2022;14(7):459-462. 2. Liu Y, Liang C, Xin L, et al. The development of Coronavirus 3C-Like protease (3CLpro) inhibitors from 2010 to 2020. Eur J Med Chem. 2020;206:112711. 3. Shaheer M, Singh R, Sobhia ME. Protein degradation: a novel computational approach to design protein degrader probes for main protease of SARS-CoV-2. J Biomol Struct Dyn. 2022;40(21):10905-10917. 4. Zhang L, Lin D, Kusov Y, et al. α-Ketoamides as Broad-Spectrum Inhibitors of Coronavirus and Enterovirus Replication: Structure-Based Design, Synthesis, and Activity Assessment. J Med Chem. 2020;63(9):4562-4578. 5. Massilamany C, Gangaplara A, Reddy J. Intricacies of cardiac damage in coxsackievirus B3 infection: implications for therapy. Int J Cardiol. 2014;177(2):330-339.