Abstract
BACKGROUND: Clostridioides difficile is one of only three bacteria categorized as an ‘urgent’ drug-resistant threat by the CDC. This pathogen is responsible for nearly 500,000 hospital-acquired infections and 29,000 deaths per year at a cost of nearly $4.8 billion. There is a critical need for the identification of novel, anti-difficile agents with narrow-spectrum activity that can spare the human microbiome. We previously reported the essentiality of the FAS-II enzyme, enoyl-ACP reductase (FabK) in C. difficile, and the narrow-spectrum activity of a series of phenylimidazole inhibitors. We present here experimental and virtual compound screening studies that identified novel FabK inhibitors with sub-micromolar activity and follow-up SAR and structural studies. METHODS: Using a novel luminescence assay, 20K diverse compounds from the St. Jude drug-like and lead-like libraries were screened. In parallel, a ligand-based virtual screen was performed against 2.4 million lead- and drug-like compounds from commercial libraries. Hit compounds were confirmed using an orthogonal fluorescence-intensity assay and SAR studies were performed by testing commercially available hit analogs. The most potent inhibitor was advanced into co-crystallography structural studies. RESULTS: The compound screening campaigns resulted in the identification of several confirmed hit compounds with low to sub-micromolar activity and novel scaffolds relative to the known phenylimidazole inhibitors. Importantly, the first confirmed nanomolar inhibitor of C. difficile FabK was identified and validated with an IC(50) of 0.35 μM. SAR studies of the hits along with a high-resolution co-crystal structure have allowed new insights into the key binding determinants and structural requirements for activity as well as the structural requirements for species specificity. CONCLUSION: The C. difficile FabK enzyme offers a promising narrow-spectrum drug target that can potentially spare the human microbiome. The known activity of the phenylimidazole inhibitor series supports the druggability of this target. The newly discovered inhibitor class presented here further demonstrates the potential of FabK inhibition and will facilitate the development of clinically relevant, narrow-spectrum anti-difficile agents. DISCLOSURES: All Authors: No reported disclosures