Fosfomycin is a broad-spectrum antibiotic that is effective against both Gram-positive and Gram-negative bacteria via inhibition of cell wall biosynthesis. A limitation to fosfomycin efficacy has evolved in the form of fosfomycin-modifying enzymes. FosB is a Mn2+-dependent bacillithiol-S-transferase found in high-profile pathogens like methicillin-resistant Staphylococcus aureus (MRSA). FosB knockout strains of MRSA demonstrate significantly increased susceptibility to fosfomycin, identifying suppression of the enzyme as a potential therapeutic strategy. Our goal is to identify and develop inhibitors of FosB that restore fosfomycin efficacy against MRSA. We use the Schrödinger program suite to dock compounds from virtual libraries into currently available high-resolution crystal structures of the FosB enzymes. Using this approach, we have identified over 140 potential compounds. Preliminary FosB kinetic assays using 31P-NMR have identified inhibitors: phosphonoformate (PPF) and (1-hydroxy-2-methylpropyl)phosphonic acid (1H2M). Absorbance-based kinetic assays have allowed us to investigate inhibitor potency with PPF. Our kinetic analysis with respect to increasing fosfomycin concentration has revealed a previously unknown cooperative binding mechanism in FosB. In addition to the kinetic assays, we have successfully obtained a 1.95 Å crystal structure of FosB with PPF bound, a significant step forward in our structure-based drug design workflow. Moreover, we have synthesized 1H2M and are currently working to complete kinetic analyses and obtain a crystal structure. Initial results indicate 1H2M is a more potent FosB inhibitor than PPF. We are working to design and synthesize derivatives of 1H2M for further development toward enhancing inhibitory potency.

Structure-based design of phosphonoformate analogues as inhibitors of FosB from Staphylococcus aureus

ACS Spring 2022

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