To address the nature of intrinsic resistance in S. aureus, one focus has been to study if MRSA strains can become sensitive to β lactams. This has been demonstrated to be the case based on key experiments where central coordination of cell wall synthesis has been perturbed at the points of teichoic acid synthesis and separation of daughter cells. These findings reveal intrinsic bacterial factors that are required for MRSA strains to be β lactam resistant and thus can serve as targets for novel therapeutics that sensitizes resistant strains. Such a candidate has potentially been identified in extracts of Actinomyces and future analysis will pinpoint active compound. Another approach is to revitalize β lactams in resistant strains is to use combinations β lactamase inhibitors such as clavulanic acids. To understand the mode of action the structure of MRSA PBP2a was determined bound to amoxicillin, both with and without clavulanic acid, offering valuable insights into β lactam interactions with this resistance associated protein.
New drug candidates and targets have been sought through pathway and structural analysis involving firstly examination of genes that affect cell wall porosity. To this end transposon mutagenesis combined with FACS based sorting revealed two genes representing new potential targets for enhancing antibiotic susceptibility. To assess the potential of these hits, work is in progress to screen inhibitors both virtually and experimentally. Inhibitors of cell wall synthesis has been investigated by virtual compound screening guided by AlphaFold structural predictions of the L,D transpeptidases and from a successful crystal structure of the L,D-transpeptidase from Clostridioides difficile. Further this work identified isatin as a promising hit. A synthetic route for generating multiple isatin based variants was successfully developed, demonstrating that the isatin scaffold is highly amenable to chemical modification. For screening purposes, a novel data analysis pipeline has been developed to identify promising natural product candidates based on mass spectrometry dataset. This approach has led to several new molecular hits, including a purified compound tentatively identified as a novel cyclic pentapeptide with a long fatty acid chain.
To examine the potential of repurposing of drugs, patented ticagrelor derived compounds devoid of antiplatelet activity were engineered and tested for potency against MRSA and other Gram-positive bacteria. All derivatives retained a multi target antimicrobial mode of action involving membrane disruption and inhibition of peptidoglycan biosynthesis. MRSA strains evolved for ticagrelor resistance showed increased resistance to most derivatives, and notable differences were observed in how the transcriptional regulator Spx contributes to resistance across the compound series. Further 39 novel ticagrelor-derived compounds analogues designed to retain strong antibacterial activity while eliminating unwanted antiplatelet effects and been synthesized. Mode of action studies have shown that three representative compounds act similarly to ticagrelor by targeting the bacterial cell envelope, binding to membrane lipids, and disrupting membrane potential.
As another alternative approach to target antibiotic resistant cells a high throughput screening system has been developed to test combinations of phages and antibacterial compounds and against a library of 103 compounds, resulting in the discovery of several promising phage compound combinations. Also, novel phage encoded, antimicrobial endolysins have been examined and structurally determined and activity studies are ongoing.
