Targeting SleC and CspB in the Inhibition of Spore Germination in <i>Clostridioides difficile</i>

<i>Clostridioides difficile</i>, 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 <i>C. difficile</i> 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 <b>110</b> (IC₅₀ = 14 ± 1 μM or 6.3 ± 0.4 μg/mL). Compound <b>110</b> targets mature SleC (<i>K</i>_d = 12 ± 1.0 μM) and CspB (<i>K</i>_d = 8.0 ± 1.0 μM) on spores, inhibiting their enzymatic activities, thus preventing spore germination. To our knowledge, compound <b>110</b> is the first reported spore germination inhibitor targeting SleC/CspB, offering a promising avenue for <i>C. difficile</i> therapies.