In vivo evaluation of Clostridioides difficile enoyl-ACP reductase II (FabK) inhibition by phenylimidazole unveils a promising narrow-spectrum antimicrobial strategy

Abstract

Clostridioides difficile infection (CDI) is a leading cause of hospital-acquired diarrhea, which often stems from disruption of the gut microbiota by broad-spectrum antibiotics. The increasing prevalence of antibiotic-resistant C. difficile strains, combined with disappointing clinical trial results for recent antibiotic candidates, underscores the urgent need for novel CDI antibiotics. To this end, we investigated C. difficile enoyl ACP reductase ( Cd FabK), a crucial enzyme in de novo fatty acid synthesis, as a drug target for microbiome-sparing antibiotics. To test this concept, we evaluated the efficacy and in vivo spectrum of activity of the phenylimidazole analog 296, which is validated to inhibit intracellular Cd FabK. Against major CDI-associated ribotypes 296 had an Minimum inhibitory concentration (MIC 90 ) of 2 µg/mL, which was comparable to vancomycin (1 µg/mL), a standard of care antibiotic. In addition, 296 achieved high colonic concentrations and displayed dosed-dependent efficacy in mice with colitis CDI. Mice that were given 296 retained colonization resistance to C. difficile and had microbiomes that resembled the untreated mice. Conversely, both vancomycin and fidaxomicin induced significant changes to mice microbiomes, in a manner consistent with prior reports. Cd FabK, therefore, represents a potential target for microbiome-sparing CDI antibiotics, with phenylimidazoles providing a good chemical starting point for designing such agents.

Evaluation of Fusobacterium nucleatum Enoyl-ACP Reductase (FabK) as a Narrow-Spectrum Drug Target

“…Studies of a lead phenylimidazole, designated as 296, inhibited Clostridioides difficile FabK in cellular and enzymatic assays and was efficacious in treating C. difficile infection (CDI) in mice. 26 , 35 , 36 Importantly, 296 did not appear to significantly disrupt the gut microbiome of mice, in contrast to the standard-of-care drugs for CDI, vancomycin, and fidaxomicin. 35 The FabK proteins from F. nucleatum ( Fn FabK) and C. difficile ( Cd FabK) are closely related, with 56% overall identity and 74% overall similarity, and their active sites differ by only a single residue at amino acid position 46; Figure S1 .…”

“… 26 , 35 , 36 Importantly, 296 did not appear to significantly disrupt the gut microbiome of mice, in contrast to the standard-of-care drugs for CDI, vancomycin, and fidaxomicin. 35 The FabK proteins from F. nucleatum ( Fn FabK) and C. difficile ( Cd FabK) are closely related, with 56% overall identity and 74% overall similarity, and their active sites differ by only a single residue at amino acid position 46; Figure S1 . This prompted us to investigate whether Fn FabK could be a target.…”

“…Studies of a lead phenylimidazole, designated as 296, inhibited Clostridioides difficile FabK in cellular and enzymatic assays and was efficacious in treating C. difficile infection (CDI) in mice. ,, Importantly, 296 did not appear to significantly disrupt the gut microbiome of mice, in contrast to the standard-of-care drugs for CDI, vancomycin, and fidaxomicin . The FabK proteins from F.…”