Zhou, Jingyuan; (2021) Targeting Enzymes Involved in Antimicrobial Resistance (AMR) in Gram-negative Bacteria. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Antimicrobial resistance (AMR) emerged rapidly after the introduction of the penicillins, the first generation of β-lactam antibiotics, in 1946. Resistance to antibiotics of last resort has highlighted AMR in bacterial pathogens as a pressing therapeutic issue. Gram-negative bacteria manifest high-level resistance to most classes of antibiotics and are the leading cause of severe infectious disease globally. Therefore, reversing their resistant status is of our interest. Among many mechanisms discovered, the expression of drug-inactivating enzymes is the major cause that leads to Gram-negative bacterial AMR. We aim to probe the chemical biology of two proteins associated with drug resistance: Klebsiella pneumoniae carbapenemase (KPC-2) which hydrolyses β-lactam antibiotics and a bacterial glutathione transferase, glutathione transferase (GST-A) which plays roles in antibiotic conjugation and inactivation. Based on the known crystal structures of the proteins (KPC-2: PDB id 3RXX, GST-A: PDB id 1A0F), small molecules were designed and synthesised and tested as inhibitors of the purified enzymes. Promising inhibitors for KPC-2 have been developed with a scaffold containing a 1,4-disubstituted 1,2,3-triazole. In vitro tests indicated that the compounds have a clear SAR and the best inhibitors have nanomolar Ki values. Antibiotic susceptibility tests were used to validate boronic acid KPC-2 inhibitors as potentiators of β-lactam antibiotic activity in cellulo. The compounds showed the successful reversal of resistance to cefotaxime (CTX) and meropenem (MEM) in cellulo in KPC-2 producing Escherichia coli (over 512-fold more sensitive). A small library of glutathione (GSH) analogues was synthesised and tested against GST-A. Binding assays and enzyme kinetics studies suggested that the Gly moiety of GSH is less important than Glu in protein G-site binding, and π-stacking is a critical factor in GST-A H-site binding. We also used susceptibility tests to explore whether GST-A plays a role in antibiotic detoxification and may serve as a target to combat AMR. However, target validation work suggested that GST-A is not essential for E. coli survival and inhibiting the protein may not be a promising approach for drug discovery.

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