Targeting Bacterial Signaling Cascades as a Novel Antibiotic Strategy

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Background and Veteran significance: Pathogens such as mycobacteria and Staphylococcus aureus, are not only intrinsically antibiotic resistant, but are rapidly acquiring multi-drug resistant traits. Two billion people are currently infected with Mycobacterium tuberculosis, and an increasing proportion are resistant to clinical treatments. Methicillin resistant Staphylococcus aureus (MRSA) is a massive burden in healthcare, and vancomycin resistant strains are increasingly problematic. These resistant infections are endemic to developing countries and areas with a regular military presence, causing an increased burden for the Veterans Administration healthcare system. Furthermore, MRSA infections are a major concern in VA hospitals and are a significant cost in time, resources, and lives. New effective antibiotics are needed, and development against novel targets is needed to treat infections caused by these resistant organisms. Novel antibiotic targets: Protein kinases and phosphatases are critical in transducing cellular and environmental signals to trigger growth and division or to respond to stress and environmental changes. Human phosphorylation signaling is well studied, but bacterial signaling is less known and no antibiotics targeting these pathways exist. Penicillin-binding And Serine/Threonine Associated (PASTA) kinases are unique transmembrane kinases present in Actinobacteria and Firmicutes. PASTA kinases are necessary for virulence, making them attractive drug targets. Genetic knockouts in MRSA and pharmacological inhibition in MRSA or tuberculosis increase β-lactam susceptibility. Furthermore, genetic deletion of the cognate S/T phosphatase in MRSA increases this synergy and decreases in vivo virulence. Inhibiting both kinases and phosphatases to enhance β-lactam synergy is a novel approach to antibiotic development. Goals of this award: The goals of this award are to 1) provide time and resources to facilitate Dr. Wlodarchak’s transition from a mentored scientist to an independent translational investigator in the VA system and to 2) develop lead compounds against novel antibiotic targets in MRSA and tuberculosis. The central hypothesis tested here is that a coordinated attack on several nodes of the serine/threonine phosphorylation signaling cascade will be an effective pharmacological strategy with low likelihood of resistance development. This hypothesis will be tested by characterizing hits from a biochemical phosphatase screen, developing MRSA kinase inhibitors, and performing the first global transcriptomic and phosphoproteomic screen on MRSA under pharmacologic stress on this pathway. Expected outcomes and impacts: Upon completion of this award, it is expected that Dr. Wlodarchak will transition to a fully independent VA investigator with several well-characterized lead compounds against MRSA and tuberculosis and leads on other potential targets in this pathway. This research will provide preliminary data for a competitive VA Merit award for a translational drug development project. This will have a positive impact on not only Dr. Wlodarchak’s career but also on Veterans’ health by providing mechanistic insight on how phosphorylation signaling controls virulence and by providing tangible compounds that may become clinical drugs. This will help provide options to stifle the current antibiotic resistance crisis and thus improve patient survival and quality of life.