Selective inhibition of Helicobacter pylori methionine aminopeptidase by azaindole hydroxamic acid derivatives: Design, synthesis, in vitro biochemical and structural studies.

Methionine aminopeptidases (MetAPs) are an important class of enzymes that work co-translationally for the removal of initiator methionine. Chemical inhibition or gene knockdown is lethal to the microbes suggesting that they can be used as antibiotic targets. However, sequence and structural similarity between the microbial and host MetAPs has been a challenge in the identification of selective inhibitors. In this study, we have analyzed several thousands of MetAP sequences and established a pattern of variation in the S1 pocket of the enzyme. Based on this knowledge, we have designed a library of 17 azaindole based hydroxamic acid derivatives which selectively inhibited the MetAP from H. pylori compared to the human counterpart. Structural studies provided the molecular basis for the selectivity.

Strategic enzymatic enantioselective desymmetrization of prochiral cyclohexa-2,5-dienones.

Asymmetric desymmetrization through the selective reduction of one double bond of prochiral 2,5-cyclohexadienones is highly challenging. A novel method has been developed for synthesizing chiral cyclohexenones by employing an ene-reductase (Bacillus subtilis YqjM) enzyme that belongs to the OYE family. Our strategy demonstrates high substrate scope and enantioselectivity towards substrates containing all-carbon as well as heteroatom (O, N)-containing quaternary centers. The mechanistic studies (kH/D = ∼1.8) indicate that hydride transfer is probably the rate-limiting step. Mutation of several active site residues did not affect the stereochemical outcomes. This work provides a convenient way of synthesizing various enantioselective γ,γ-disubstituted cyclohexanones using enzymes.

Development of 2-chloroquinoline based heterocyclic frameworks as dual inhibitors of SARS-CoV-2 MPro and PLPro.

Evolution of new variants of SARS-CoV-2 warrant the need for the continued efforts in identifying target-oriented new drugs. Dual targeting agents against MPro and PLPro not only overcome the incomplete efficacy but also the drug resistance, which is common problem. Since both these are cysteine proteases, we designed 2-chloroquinoline based molecules with additional imine moiety in the middle as possible nucleophilic warheads. In the first round of design and synthesis, three molecules (C3, C4 and C5) inhibited (Ki < 2 µM) only MPro by binding covalently to C145 and one molecule (C10) inhibited both the proteases non-covalently (Ki < 2 µM) with negligible cytotoxicity. Further conversion of the imine in C10 to azetidinone (C11) improved the potency against both the enzymes in the nanomolar range (820 nM against MPro and 350 nM against PLPro) with no cytotoxicity. Conversion of imine to thiazolidinone (C12), reduced the inhibition by 3-5 folds against both the enzymes. Biochemical and computational studies suggest that C10-C12 bind in the substrate binding pocket of MPro and in the BL2 loop of the PLPro. Since these dual inhibitors have least cytotoxicity, they could be further explored as therapeutics against the SARS-CoV-2 and other analogous viruses.

A chiral selectivity relaxed paralog of DTD for proofreading tRNA mischarging in Animalia.

D-aminoacyl-tRNA deacylase (DTD), a bacterial/eukaryotic trans-editing factor, removes D-amino acids mischarged on tRNAs and achiral glycine mischarged on tRNAAla. An invariant cross-subunit Gly-cisPro motif forms the mechanistic basis of L-amino acid rejection from the catalytic site. Here, we present the identification of a DTD variant, named ATD (Animalia-specific tRNA deacylase), that harbors a Gly-transPro motif. The cis-to-trans switch causes a "gain of function" through L-chiral selectivity in ATD resulting in the clearing of L-alanine mischarged on tRNAThr(G4•U69) by eukaryotic AlaRS. The proofreading activity of ATD is conserved across diverse classes of phylum Chordata. Animalia genomes enriched in tRNAThr(G4•U69) genes are in strict association with the presence of ATD, underlining the mandatory requirement of a dedicated factor to proofread tRNA misaminoacylation. The study highlights the emergence of ATD during genome expansion as a key event associated with the evolution of Animalia.