P188G Mutation Enhances Pyrrolysyl-tRNA Synthetase Activity and Stabilizes Full-Length Protein and Discovering Drugs Against SARS-CoV-2 MPRO and PLPRO

Aggregation of pyrrolysyl-tRNA synthetase (PylRS) has proven problematic for expression of non-canonical amino acids (ncAAs) in proteins. C. M. alvus PylRS (CMaPylRS) is a highly soluble PylRS with a shorter N-terminal domain. While testing the orthogonality between CMaPylRS and M. mazei PylRS (MmPylRS), we found that tRNAPyl (PylT) from C. M. alvus stabilized MmPylRS and enhanced its activity 3-fold higher than its cognate tRNA by suppressing an amber codon within green fluorescent protein. We further found that MmPylRS was cleaved to two fragments, aa111-454 and aa190-454 when paired to CMaPylT or MmPylT. When the fragments were expressed separately, the MmPylRS activity was only 40%. We showed that a P188G mutation can stabilize MmPylRS and enhance its activity. Since 2020, millions have died from SARS-CoV-2 infection, and even more have suffered long-covid symptoms. As such, a cure is urgently in need. Main protease (Mpro) and papain-like protease (PLpro) are essential proteases in SARS-CoV-2 virology. We screened deubiquitinase inhibitor and cysteine protease inhibitor libraries against PLpro and identified 4 inhibitors: GRL-0617, TCID, SJB2-043 and PR-619 with IC50 under 10 µM using a peptide substrate, Z-LRGG-AMC. When the substrate was switched from Z-LRGG-AMC to Ubiquitin-AMC, the IC50 of SJB2-043 improved from 0.56 µM to 0.091 µM. The dramatic improvement suggests a binding mode for this inhibitor to PLpro distinct from other inhibitors. In vitro inhibition assays do not always match with in vivo assays due to mechanisms of cellular drug delivery. Because antiviral assays cannot be performed with SARS-CoV-2 in low-BSL laboratories, we developed a cellular assay to evaluate Mpro inhibitors to facilitate drug discovery. In this assay, a fusion protein of Mpro and eGFP in HEK293T cell was expressed and quantified via fluorescent flow cytometry. The cytotoxicity caused by Mpro results in low cell fluorescence, while a potent inhibitor rescues the cell (high fluorescence). Therefore, cellular potency of inhibitors can be quantified according to the inhibitor-dependent fluorescence enhancement. Using this assay, 30 known Mpro inhibitors were analyzed. MPI5-8 resulted in excellent antiviral effects and in vivo Mpro inhibition. This cellular assay reduces the BSL requirement for evaluating Mpro inhibitors and significantly facilitates the drug discovery process