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

Norseeda, Krissada; Pavel, Fahad Bin Aziz; Rutherford, Jacob; Meer, Humna N.; Dureja, Chetna; Hurdle, Julian G.; Hevener, Kirk E.; Sun, Dianqing

doi:10.1016/j.bmc.2023.117330

Cited by 3 publications

(18 citation statements)

References 37 publications

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“…The test panel had reported IC 50 s of 0.1 to >10 μM against Cd FabK and MICs of 1.6 to >100 μg/mL against C. difficile cells . However, against F.…”

Section: Results and Discussionmentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

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

Rutherford,

Avad,

Dureja

et al. 2024

ACS Infect. Dis.

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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 IC 50 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 narrowspectrum antimicrobial agents.

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“…The test panel had reported IC 50 s of 0.1 to >10 μM against Cd FabK and MICs of 1.6 to >100 μg/mL against C. difficile cells . However, against F.…”

Section: Results and Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Rutherford,

Avad,

Dureja

et al. 2024

ACS Infect. Dis.

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“…Compound 296 was synthesized as previously described (29, 35) and tested against CDI-associated ribotypes ( Table 1 ), including strains that were resistant to metronidazole, vancomycin and fidaxomicin. 296 had MIC 50 and MIC 90 values of 0.5 µg/ml and 2 µg/ml, respectively in BHI broth, which were comparable to vancomycin (MIC 50 and MIC 90 values of 0.5 µg/ml and 1 µg/ml).…”

Section: Resultsmentioning

confidence: 99%

“…A desirable property for anti- C. difficile antibiotics is that they are non-absorbed, achieving high local concentrations at the site of infection in the large intestine. Previously, in silico analysis of phenylimidazole Cd FabK inhibitors in QikProp in Schrödinger/Maestro predicted they are low absorption molecules (35). To experimentally test this for 296, we used the Caco2 permeability assay, which also suggested it was non-absorbed with an apparent permeability (Papp) coefficient for apical to basolateral permeability of 21.65 ± 13.57 nm/s, when compared to non-absorbed and absorbed controls vinblastine 5.48 ± 1.66 nm/s and carbamazepine 136.12 ± 50.72 nm/s, respectively.…”

Section: Resultsmentioning

confidence: 99%

“…We must emphasize that 296 is not a clinical candidate, but it was only deployed as an experimental probe. Nonetheless, from a drug development standpoint, the 296-related class of phenylimidazole Cd FabK inhibitors also possess lipophilic characteristics to effectively bind to the hydrophobic catalytic pocket of the enzyme, and this physicochemical property may contribute to poor absorption from the intestinal tract (35). Taken together, the studies conducted herein provide experimental evidence supporting that Cd FabK is druggable in an acute CDI setting and that a first-generation inhibitor (296) spares the microbiome and maintains colonization resistance.…”

Section: Discussionmentioning

confidence: 99%

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In vivoevaluation ofClostridioides difficileenoyl-ACP reductase II (FabK) Inhibition by phenylimidazole unveils a promising narrow-spectrum antimicrobial strategy

Dureja,

Rutherford,

Pavel

et al. 2023

Preprint

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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 MIC90of 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.

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

Dureja,

Rutherford,

Pavel

et al. 2024

Antimicrob Agents Chemother

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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.

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

Cited by 3 publications

References 37 publications

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

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

In vivoevaluation ofClostridioides difficileenoyl-ACP reductase II (FabK) Inhibition by phenylimidazole unveils a promising narrow-spectrum antimicrobial strategy

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

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