Design of β-lactam Prodrugs to Selectively Suppress β-lactam Resistant, Pathogenic Bacteria in Microbial Mixtures

Abstract

The emergence of extended-spectrum-β-lactamases (ESBLs) poses a significant threat to conventional β-lactam antibiotics. Current treatment strategies often fail against ESBL-producing bacteria, necessitating alternative approaches to combat resistance. A fragment-based drug design (FBDD) platform was developed, validated, and used to design next-generation β-lactam prodrugs capable of selectively suppressing resistant strains. Unlike conventional β-lactam antibiotics, which target penicillin-binding proteins (PBPs), ideal β-lactam prodrugs would exhibit inverted reactivity to avoid PBPs recognition and still require enzymatic activation by ESBLs. By hijacking ESBL activity, these prodrugs transform a key resistance mechanism into a vulnerability, selectively eliminating ESBL-producing bacteria while sparing non-resistant strains. This targeted strategy not only combats resistance but also aims to protect commensal bacteria, preserving the microbiome and reducing collateral damage associated with broad-spectrum antibiotics.From a catalog of chemically diverse β-lactam fragment and prodrug pairs, the R1 functional group of the β-lactam core was investigated for its role in modulating penicillin-binding-protein (PBP) recognition and maintaining recognition and reactivity with ESBLs. The findings demonstrate that the activity of a β-lactam fragment is predictive for prodrug activity. Structural analyses by time-course 1H NMR confirm β-lactam hydrolysis and prodrug activation by ESBLs in vitro and in cells. Additionally, prodrug activation rate and ESBL product inhibition is shown to be a determinant of growth suppression of ESBL-producing bacteria. In a mixed microbial setting, the prodrug AcephPT selectively suppresses the proliferation of ESBL-producing clinical isolate, while sustaining and recovering the growth of a non-producer, the first β-lactam prodrug of its kind to demonstrate this activity. These findings establish a foundation and platform for design of β-lactam prodrugs that selectively exploit ESBL activity to counteract β-lactam resistance while promoting antimicrobial stewardship.