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

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 (CdFabK), 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 CdFabK. Against major CDI-associated ribotypes 296 had an Minimum inhibitory concentration (MIC90) 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. CdFabK, therefore, represents a potential target for microbiome-sparing CDI antibiotics, with phenylimidazoles providing a good chemical starting point for designing such agents.

Synthesis and evaluation of phenylimidazole FabK inhibitors as new Anti-C. Difficile agents.

Previously, 1-((4-(4-bromophenyl)-1H-imidazol-2-yl)methyl)-3-(5-(pyridin-2-ylthio)thiazol-2-yl)urea bearing a p-bromine substitution was shown to possess selective inhibitory activity against the Clostridioides difficile enoyl-acyl carrier protein (ACP) reductase II enzyme, FabK. Inhibition of CdFabK by this compound translated to promising antibacterial activity in the low micromolar range. In these studies, we sought to expand our knowledge of the SAR of the phenylimidazole CdFabK inhibitor series while improving the potency of the compounds. Three main series of compounds were synthesized and evaluated based on: 1) pyridine head group modifications including the replacement with a benzothiazole moiety, 2) linker explorations, and 3) phenylimidazole tail group modifications. Overall, improvement in the CdFabK inhibition was achieved, while maintaining the whole cell antibacterial activity. Specifically, compounds 1-((4-(4-bromophenyl)-1H-imidazol-2-yl)methyl)-3-(5-((3-(trifluoromethyl)pyridin-2-yl)thio)thiazol-2-yl)urea, 1-((4-(4-bromophenyl)-1H-imidazol-2-yl)methyl)-3-(6-(trifluoromethyl)benzo[d]thiazol-2-yl)urea, and 1-((4-(4-bromophenyl)-1H-imidazol-2-yl)methyl)-3-(6-chlorobenzo[d]thiazol-2-yl)urea showed CdFabK inhibition (IC50 = 0.10 to 0.24 µM), a 5 to 10-fold improvement in biochemical activity relative to 1-((4-(4-bromophenyl)-1H-imidazol-2-yl)methyl)-3-(5-(pyridin-2-ylthio)thiazol-2-yl)urea, with anti-C. difficile activity ranging from 1.56 to 6.25 µg/mL. Detailed analysis of the expanded SAR, supported by computational analysis, is presented.

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

Fusobacterium nucleatum, a pathobiont inhabiting the oral cavity, contributes to opportunistic diseases, such as periodontal diseases and gastrointestinal cancers, which involve microbiota imbalance. Broad-spectrum antimicrobial agents, while effective against F. nucleatum infections, can exacerbate dysbiosis. This necessitates the discovery of more targeted narrow-spectrum antimicrobial agents. We therefore investigated the potential for the fusobacterial enoyl-ACP reductase II (ENR II) isoenzyme FnFabK (C4N14_ 04250) as a narrow-spectrum drug target. ENRs catalyze the rate-limiting step in the bacterial fatty acid synthesis pathway. Bioinformatics revealed that of the four distinct bacterial ENR isoforms, F. nucleatum specifically encodes FnFabK. Genetic studies revealed that fabK was indispensable for F. nucleatum growth, as the gene could not be deleted, and silencing of its mRNA inhibited growth under the test conditions. Remarkably, exogenous fatty acids failed to rescue growth inhibition caused by the silencing of fabK. Screening of synthetic phenylimidazole analogues of a known FabK inhibitor identified an inhibitor (i.e., 681) of FnFabK enzymatic activity and F. nucleatum growth, with an IC50 of 2.1 µM (1.0 µg/mL) and a MIC of 0.4 µg/mL, respectively. Exogenous fatty acids did not attenuate the activity of 681 against F. nucleatum. Furthermore, FnFabK was confirmed as the intracellular target of 681 based on the overexpression of FnFabK shifting MICs and 681-resistant mutants having amino acid substitutions in FnFabK or mutations in other genetic loci affecting fatty acid biosynthesis. 681 had minimal activity against a range of commensal flora, and it was less active against streptococci in physiologic fatty acids. Taken together, FnFabK is an essential enzyme that is amenable to drug targeting for the discovery and development of narrow-spectrum antimicrobial agents.

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

Clostridioides difficile infection (CDI) is a leading cause of hospital-acquired diarrhea, which often stem from disruption of the gut microbiota by broad-spectrum antibiotics. The increasing prevalence of antibiotic-resistant C. difficile strains, combined with disappointing clinical trials results for recent antibiotic candidates, underscore the urgent need for novel CDI antibiotics. To this end, we investigated C. difficile enoyl ACP reductase (CdFabK), 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 CdFabK. Against major CDI-associated ribotypes 296 had an MIC90 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. CdFabK therefore represents a potential target for microbiome-sparing CDI antibiotics, with phenylimidazoles providing a good chemical starting point for designing such agents.