Discovery of beta-lactamase CMY-10 inhibitors for combination therapy against multi-drug resistant Enterobacteriaceae

beta-lactam antibiotics are the most widely used antimicrobial agents since the discovery of benzylpenicillin in the 1920s. Unfortunately, these life-saving antibiotics are vulnerable to inactivation by continuously evolving beta-lactamase enzymes that are primary resistance determinants in multi-drug resistant pathogens. The current study exploits the strategy of combination therapeutics and aims at identifying novel beta-lactamase inhibitors that can inactivate the beta-lactamase enzyme of the pathogen while allowing the beta-lactam antibiotic to act against its penicillin-binding protein target. Inhibitor discovery applied the Site-Identification by Ligand Competitive Saturation (SILCS) technology to map the functional group requirements of the beta-lactamase CMY-10 and generate pharmacophore models of active site. SILCS-MC, Ligand-grid Free Energy (LGFE) analysis and Machine-learning based random-forest (RF) scoring methods were then used to screen and filter a library of 700,000 compounds. From the computational screens 74 compounds were subjected to experimental validation in which beta-lactamase activity assay, in vitro susceptibility testing, and Scanning Electron Microscope (SEM) analysis were conducted to explore their antibacterial potential. Eleven compounds were identified as enhancers while 7 compounds were recognized as inhibitors of CMY-10. Of these, compound 11 showed promising activity in beta-lactamase activity assay, in vitro susceptibility testing against ATCC strains (E. coli, E. cloacae, E. agglomerans, E. alvei) and MDR clinical isolates (E. cloacae, E. alvei and E. agglomerans), with synergistic assay indicating its potential as a beta-lactam enhancer and beta-lactamase inhibitor. Structural similarity search against the active compound 11 yielded 28 more compounds. The majority of these compounds also exhibited beta-lactamase inhibition potential and antibacterial activity. The non-beta-lactam-based beta-lactamase inhibitors identified in the current study have the potential to be used in combination therapy with lactam-based antibiotics against MDR clinical isolates that have been found resistant against last-line antibiotics.

Automated summary

This study explores the use of combination therapy to identify novel beta-lactamase inhibitors that can inactivate the enzyme of pathogens, providing a new approach in combating multi-drug resistant pathogens. By utilizing computational screening and experimental validation, potential compounds were identified with beta-lactamase inhibition and antibacterial activity against MDR clinical isolates. These non-beta-lactam-based inhibitors have the potential to be used in combination therapy with beta-lactam antibiotics against resistant strains.