Development of Microbiome-Sparing Antibacterials for Clostridioides difficile Infection - PROJECT SUMMARY/ABSTRACT Hypervirulent, drug-resistant strains of Clostridioides difficile now greatly contribute to the overall morbidity and mortality of C. difficile infection (CDI), resulting in relapse rates of up to 35%, nearly 30,000 deaths per year, and a national burden over $4.5 billion annually. As such, there is an urgent need for the identification of novel therapeutics for the treatment of recurrent CDI (rCDI). We have characterized the bacterial enzyme FabK, enoyl-acyl carrier protein (ACP) reductase II, as an essential, druggable, and narrow-spectrum target for CDI. FabK is a key enzyme in the bacterial fatty acid synthesis pathway (FAS II). Inhibition of enzymes in this pathway has been shown to result in a strong antibacterial effect, as with the FabI (enoyl-ACP reductase I) inhibitor isoniazid, and other FabI inhibitors currently in clinical trials. We have shown in mouse studies that CdFabK inhibition is efficacious in CDI and results in low disruption to the normal flora of the lower bowel. Further, our lead inhibitor series is compartmentalized to the gut with low oral absorption. Here, our objectives are (i) to expand the SAR of our lead CdFabK inhibitor series while improving potency, (ii) to identify and characterize novel chemotypes of CdFabK inhibitors, (iii) to demonstrate an in vivo narrow spectrum anti- difficile effect with low disruption of microbiota. To achieve these objectives, we have organized a skilled team with expertise in C. difficile microbiology, protein biochemistry, structural biology, synthetic chemistry, and in vivo PK and efficacy to pursue the following specific aims: 1. Optimize PTIM and chemical related lead series to improve antimicrobial potency. To accomplish this aim, we will employ structure-guided lead optimization guided by enzymology, microbiology, and biophysical testing to advance 2nd generation compounds with improved potency and new chemical features. 2. Identify novel inhibitor chemotypes and expand our knowledge of inhibitor selectivity for CdFabK. This aim addresses the need to expand the chemical landscape of FabK inhibitors to further understand the structural requirements for FabK inhibition and selectivity. To accomplish this aim, we will employ a unique cell-based high-throughput screen, followed by hit validation, lead characterization, and synthetic lead optimization. 3. Biological evaluation of lead compounds to obtain efficacious drug candidates. In this aim, we will explore the pharmacological efficacy of CdFabK inhibitors against rCDI, using in vitro antimicrobial, pharmacokinetic and animal model systems to prioritize best-in-class molecules. We will also begin to define mechanistically the underlying protective effects of CdFabK inhibitors in the microbiome environment. We expect these studies to yield fundamental insights regarding the inhibition of the FAS-II pathway and CdFabK for treatment of CDI and will advance our understanding of the microbiological effects of gut flora FAS-II inhibition. These results will further advance CdFabK inhibitors as a new strategy for primary CDI treatment and prevention of recurrence.
