Integration of molecular modelling and in vitro studies to inhibit LexA proteolysis

IntroductionAs antibiotic resistance has become more prevalent, the social and economic impacts are increasingly pressing. Indeed, bacteria have developed the SOS response which facilitates the evolution of resistance under genotoxic stress. The transcriptional repressor, LexA, plays a key role in this response. Mutation of LexA to a non-cleavable form that prevents the induction of the SOS response sensitizes bacteria to antibiotics. Achieving the same inhibition of proteolysis with small molecules also increases antibiotic susceptibility and reduces drug resistance acquisition. The availability of multiple LexA crystal structures, and the unique Ser-119 and Lys-156 catalytic dyad in the protein enables the rational design of inhibitors. MethodsWe pursued a binary approach to inhibit proteolysis; we first investigated beta-turn mimetics, and in the second approach we tested covalent warheads targeting the Ser-119 residue. We found that the cleavage site region (CSR) of the LexA protein is a classical Type II beta-turn, and that published 1,2,3-triazole compounds mimic the beta-turn. Generic covalent molecule libraries and a beta-turn mimetic library were docked to the LexA C-terminal domain using molecular modelling methods in FlexX and CovDock respectively. The 133 highest-scoring molecules were screened for their ability to inhibit LexA cleavage under alkaline conditions. The top molecules were then tested using a RecA-mediated cleavage assay. ResultsThe beta-turn library screen did not produce any hit compounds that inhibited RecA-mediated cleavage. The covalent screen discovered an electrophilic serine warhead that can inhibit LexA proteolysis, reacting with Ser-119 via a nitrile moiety. DiscussionThis research presents a starting point for hit-to-lead optimisation, which could lead to inhibition of the SOS response and prevent the acquisition of antibiotic resistance.

Automated summary

This study focuses on the issue of antibiotic resistance and explores the structure and function of LexA protein to find small molecule inhibitors that can inhibit its degradation. The research reveals that a specific covalent inhibitor can react with the Ser-119 residue in LexA protein, thus inhibiting the SOS response. This study provides a starting point for future research on preventing antibiotic resistance.