Targeting SleC and CspB in the Inhibition of Spore Germination in Clostridioides difficile

Clostridioides difficile, a Gram-positive, spore-forming anaerobic bacterium, is a major healthcare threat. Its spores colonize the gut following dysbiosis caused by broad-spectrum antibiotics, remaining dormant until host's bile acid triggers germination into vegetative cells that produce toxins, leading to diarrhea, colitis, and potentially death. Current antibiotics to treat C. difficile infection target vegetative cells but not spore germination, a pivotal step in infection development. This study unveils 1,2,4-oxadiazoles as a novel class of spore germination inhibitors and delineates the structure-activity relationship. Screening of 120 oxadiazoles revealed compound 110 (IC₅₀ = 14 ± 1 μM or 6.3 ± 0.4 μg/mL). Compound 110 targets mature SleC (K_d = 12 ± 1.0 μM) and CspB (K_d = 8.0 ± 1.0 μM) on spores, inhibiting their enzymatic activities, thus preventing spore germination. To our knowledge, compound 110 is the first reported spore germination inhibitor targeting SleC/CspB, offering a promising avenue for C. difficile therapies.