Starting from the MLPCN probe compound ML300, a structure-based optimization campaign was initiated against the recent severe acute respiratory syndrome coronavirus (SARS-CoV-2) main protease (3CL pro ). X-ray structures of SARS-CoV-1 and SARS-CoV-2 3CL pro enzymes in complex with multiple ML300-based inhibitors, including the original probe ML300, were obtained and proved instrumental in guiding chemistry toward probe compound 41 (CCF0058981). The disclosed inhibitors utilize a noncovalent mode of action and complex in a noncanonical binding mode not observed by peptidic 3CL pro inhibitors. In vitro DMPK profiling highlights key areas where further optimization in the series is required to obtain useful in vivo probes. Antiviral activity was established using a SARS-CoV-2-infected Vero E6 cell viability assay and a plaque formation assay. Compound 41 demonstrates nanomolar activity in these respective assays, comparable in potency to remdesivir. These findings have implications for antiviral development to combat current and future SARS-like zoonotic coronavirus outbreaks.

Type IV secretion systems are multiprotein complexes that mediate the translocation of macromolecules across the bacterial cell envelope. In Helicobacter pylori a type IV secretion system encoded by the cag pathogenicity island encodes 27 proteins and most are essential for virulence. We here present the identification and characterization of inhibitors of Cagα, a hexameric ATPase and member of the family of VirB11-like proteins that is essential for translocation of the CagA cytotoxin into mammalian cells. We conducted fragment-based screening using a differential scanning fluorimetry assay and identified 16 molecules that stabilize the protein suggesting that they bind Cagα. Several molecules affect binding of ADP and four of them inhibit the ATPase activity. Analysis of enzyme kinetics suggests that their mode of action is non-competitive, suggesting that they do not bind to the active site. Cross-linking suggests that the active molecules change protein conformation and gel filtration and transmission electron microscopy show that molecule 1G2 dissociates the Cagα hexamer. Addition of the molecule 1G2 inhibits the induction of interleukin-8 production in gastric cancer cells after co-incubation with H. pylori suggesting that it inhibits Cagα in vivo . Our results reveal a novel mechanism for the inhibition of the ATPase activity of VirB11-like proteins.

BMRB accession no. 26852 and PDB 5JBS.

The increasing frequency of antimicrobial resistance is a problem of global importance. Novel strategies are urgently needed to understand and inhibit antimicrobial resistance gene transmission that is mechanistically related to bacterial virulence functions. The conjugative transfer of plasmids by type IV secretion systems is a major contributor to antimicrobial resistance gene transfer. Here, we present a structure-based strategy to identify inhibitors of type IV secretion system-mediated bacterial conjugation. Using differential scanning fluorimetry we screened a fragment library and identified molecules that bind the essential TraE protein of the plasmid pKM101 conjugation machinery. Co-crystallization revealed that fragments bind two alternative sites of the protein and one of them is a novel inhibitor binding site. Based on the structural information on fragment binding we designed novel small molecules that have improved binding affinity. These molecules inhibit the dimerization of TraE, bind to both inhibitor binding sites on TraE and inhibit the conjugative transfer of plasmid pKM101. The strategy presented here is generally applicable for the structure-based design of inhibitors of antimicrobial resistance gene transfer and of bacterial virulence.

Methionine aminopeptidases (MetAPs) are essential enzymes that make them good drug targets in cancer and microbial infections. MetAPs remove the initiator methionine from newly synthesized peptides in every living cell. MetAPs are broadly divided into type I and type II classes. Both prokaryotes and eukaryotes contain type I MetAPs, while eukaryotes have additional type II MetAP enzyme. Although several inhibitors have been reported against type I enzymes, subclass specificity is scarce. Here, using the fine differences in the entrance of the active sites of MetAPs from Mycobacterium tuberculosis, Enterococcus faecalis, and human, three hotspots have been identified and pyridinylpyrimidine-based molecules were selected from a commercial source to target these hotspots. In the biochemical evaluation, many of the 38 compounds displayed differential behavior against these three enzymes. Crystal structures of four selected inhibitors in complex with human MetAP1b and molecular modeling studies provided the basis for the binding specificity.