Design of inhibitors of scytalone dehydratase: probing interactions with an asparagine carboxamide

Among the active-site residues of scytalone dehydratase, the side-chain carboxamide of asparagine 131 has the greatest potential for strong electrostatic interactions. Structure-based inhibitor design aimed at enhancing interactions with this residue led to the synthesis of a series of highly potent inhibitors that have a five- or six-membered ring containing a carbonyl functionality for hydrogen bonding. To achieve a good orientation for hydrogen bonding, the inhibitors incorporate a phenyl substituent that displaces a phenylalanine residue away from the five- or six-membered rings. Without the phenyl substituent, inhibitor binding potency is diminished by three orders of magnitude. Larger K(i) values of a site-directed mutant (Asn131Ala) of scytalone dehydratase in comparison to those of wild-type enzyme validate the design concept. The most potent inhibitor (K(i)=15 pM) contains a tetrahydrothiophenone that can form a single hydrogen bond with the asparagine carboxamide. Inhibitors with a butyrolactam that can form two hydrogen bonds with the asparagine carboxamide demonstrate excellent in vivo fungicidal activity.