ABSTRACT
The SARS-CoV-2 main protease (Mpro) is critical for the production of functional viral proteins during infection and, like many viral proteases, can also target host proteins to subvert their cellular functions. Here, we show that the human tRNA methyltransferase TRMT1 can be recognized and cleaved by SARS-CoV-2 Mpro. TRMT1 installs the N2,N2-dimethylguanosine (m2,2G) modification on mammalian tRNAs, which promotes global protein synthesis and cellular redox homeostasis. We find that Mpro can cleave endogenous TRMT1 in human cell lysate, resulting in removal of the TRMT1 zinc finger domain required for tRNA modification activity in cells. Evolutionary analysis shows that the TRMT1 cleavage site is highly conserved in mammals, except in Muroidea, where TRMT1 may be resistant to cleavage. In primates, regions outside the cleavage site with rapid evolution could indicate adaptation to ancient viral pathogens. We determined the structure of a TRMT1 peptide in complex with Mpro, revealing a substrate binding conformation distinct from the majority of available Mpro-peptide complexes. Kinetic parameters for peptide cleavage showed that the TRMT1(526-536) sequence is cleaved with comparable efficiency to the Mpro-targeted nsp8/9 viral cleavage site. Mutagenesis studies and molecular dynamics simulations together indicate that kinetic discrimination occurs during a later step of Mpro-mediated proteolysis that follows substrate binding. Our results provide new information about the structural basis for Mpro substrate recognition and cleavage that could help inform future therapeutic design and raise the possibility that proteolysis of human TRMT1 during SARS-CoV-2 infection suppresses protein translation and oxidative stress response to impact viral pathogenesis.
ABSTRACT
More effective delivery of non-steroidal anti-inflammatory drugs (NSAIDs) to the brain could treat the underlying inflammatory pathology of a range of CNS diseases and conditions. Use of a blood-brain barrier shuttle such as the N-benzylamide moiety, which has been largely unexplored for this purpose, could improve the brain bioavailabilities of NSAIDs. A series of novel N-benzylamide NSAID conjugates was synthesized via a three-step process with a microwave-assisted bimolecular nucleophilic substitution as the final step. We explored conditions to promote substitution over a competing elimination reaction, which was successfully suppressed with isopropyl alcohol solvent. All molecules exhibit physicochemical properties consistent with those of brain-penetrant molecules. Furthermore, they exhibit long (>48â¯h) half-lives in phosphate-buffered saline (PBS; pH 7.4) and short to moderate half-lives in human plasma. N-Benzylamide NSAID conjugates represent promising CNS drug discovery leads.
