Tofacitinib Analogs as Oxacillin Potentiators against MRSA

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Project Summary Methicillin (oxacillin)-resistant Staphylococcus aureus (MRSA) infections are among the most frequently occurring and dangerous antibiotic-resistant public health threat. Clinical resistance to all front-line MRSA antibiotics (i.e. vancomycin, linezolid, daptomycin, and ceftaroline) has been observed, further exacerbating this threat and highlighting the urgent need for new strategies to overcome drug resistance in S. aureus. In recent years, there has been increased interest in targeting signal transduction systems for development as antimicrobials and anti-virulence agents. Targeting bacterial signal transduction is attractive because these systems are essential for many cellular processes in prokaryotes, and signal transduction pathways have been extensively studied in eukaryotic systems, as evidenced by the availability of chemical libraries containing small molecule probes with kinase inhibitor-like properties. With this strategy in mind, we screened a focused set of kinase inhibitors and identified tofacitinib, an antirheumatic Janus kinase inhibitor, as a potential chemistry starting point for the discovery of beta-lactam potentiators against MRSA. Several pyrrolopyridin-4- amine (P4A) analogs of tofacitinib were synthesized, which led to the discovery of potent analogs with the ability to re-sensitize oxacillin (OXA; a beta-lactam antibiotic) against MRSA. Preliminary pull-down proteomic experiments have identified PurM, SrrB, and PknB as putative targets, studies that have been complemented by gene knockout experiments. Therefore, the main scientific objective of this proposal is to confirm P4A engagement of putative targets using biophysical and biochemical techniques and to establish the therapeutic potential of tofacitinib analogs as anti-virulence agents and OXA potentiators against MRSA. In Aim 1, I will evaluate the binding of lead P4A compounds to putative targets, demonstrate inhibition of S. aureus kinase activity and determine the extent of potential off-target effects on mammalian kinases. Biophysical assays will be developed against the putative targets through a novel target engagement assay, which will be complemented with biochemical phosphorylation and ATP/ADP conversion assays. In Aim 2, I will evaluate the clinical potential of the P4A compounds through anti-biofilm studies, pharmacological ADME profiling, and in vivo efficacy against MRSA in a mouse model. The research plan will enhance my training in areas of target identification and validation, biophysical analysis, and anti-infective microbiology, benefiting from the expertise of my co-mentors, Drs. Richard Lee and Jason Rosch, and the state-of-the-art resources at St. Jude.