Novel Inhibitors Targeting Urease Promote Fungicidal and Antivirulence Effects in Cryptococcus.

Cryptococcosis is a life-threatening fungal disease caused by the Cryptococcus neoformans and Cryptococcus gattii species complexes, frequently associated with cryptococcal meningitis and high mortality. Urease is an important virulence factor that promotes intracellular survival and blood-brain barrier translocation, representing an antifungal target absent in humans. Here, a library of new synthetic urease inhibitors was evaluated against Cryptococcus growth and enzymatic activity. Four lead compounds AF19 (Schiff's base), AF36 (benzoylselenourea), AF55, and AF57 (Biginelli's adducts) displayed fungicidal activity and urease inhibition at micro- to nanomolar concentrations. Furthermore, using kinetics analysis and molecular dynamic simulations approaches revealed distinct inhibition modes: AF19 and AF57 acted as competitive nickel-coordinating inhibitors; AF36 exhibited noncompetitive inhibition via mobile-flap modulation; and AF55 behaved as a mixed-type inhibitor engaging both catalytic and allosteric sites. All leads reduced capsule thickness and increased membrane permeability, while AF55 and AF57 additionally suppressed melanin production. Together, the urease inhibitors AF55 and AF57 emerge as promising anticryptococcal candidates, combining a potent urease targeting, fungicidal activity, multivirulence inhibition, and favorable predicted pharmacokinetic and toxicological properties.