Combating antimicrobial resistance : novel targets and anti-infectives development

Abstract

Antimicrobial resistance (AMR) is projected to cost 100 trillion USD and 10 million lives yearly by 2050 with the rise of superbugs, overuse of antimicrobials, and a lack of new antibiotics in the pipeline. To combat AMR, new antibiotics are sorely needed. This thesis reports a structural microbiology approach of three targets across the bacterial defense lines that will support the modern drug development efforts against them. The first target is the virulence factor regulator (Vfr) for which a drug repurposing campaign has found that Auranofin gold drug is a potential inhibitor. Vfr is an attractive drug target for its role of driving pathogenicity in Pseudomonas aeruginosa. Four crystal structures were solved to reveal the molecular details of protein alone and when bound with various gold analogues. This work supports the model of a biphasic cAMP concentration dependence activity and how two of the gold binding sites can affect the DNA and the secondary cAMP binding. The second target is the indirect aminoacylation pathway found in most bacteria. It was reported to drive an adaptive mistranslation mechanism for AMR in mycobacteria. The key enzymes involved are the heterotrimeric glutaminyl-amidotransferase (GatCAB), and a non-discriminating tRNA synthetase (ND-RS), which are believed to form a mega assembly with its substrate tRNAs, called transamidosome (Tdt), for ensuring translational fidelity. It remains unclear whether the Tdt forms in mycobacteria and there were no direct and high throughput assays compatible for drug screening to target this pathway. In my work, I show that homologous pull-down of GatB only resulted in GatCAB without the ND-RS or tRNA. A key significance of this finding could be that there are other regulatory mechanisms at play for ensuring translational fidelity in the indirect aminoacylation pathway. I have also developed a novel mass spectrometry-based assay for direct analysis of the pathway and drug screening. The third and last target is the efflux pump A (efpA) in mycobacteria where a recent chemical-genetics screening strategy has uncovered potent lead compounds against it. Efflux pumps are key mediators of expelling toxic drugs out of the bacteria, but this drug target space remains undeveloped. I determined the efpA structure at 6-7 Å using cryoEM method. Further improvement of the cryoEM sample preparation is needed to obtain atomic model of efpA. Nonetheless, this work paves the way for the rational design of novel inhibitors and shed new light on efflux pump structure-function relationships. Overall, the work spans across novel targets and infectives that are aligned with the innovations required to combat AMR.