IDENTIFICATION OF A NEW ANTIFUNGAL TARGET SITE THROUGH A DUAL BIOCHEMICAL AND MOLECULAR-GENETICS APPROACH

The target site of the antifungal compound LY214352 [8-chloro-4-(2-chl oro-4-fluorophenoxy) quinoline] has been identified through a dual bio chemical and molecular-genetics approach. In the molecular-genetics ap proach, a cosmid library was prepared from an Aspergillus nidulans mut ant that was resistant to LY214352 because of a dominant mutation in a single gene. A single cosmid (6A6-6) that could transform an LY214352 -sensitive strain of A. nidulans to LY214352-resistance was isolated f rom the library by sib-selection. Restriction fragments from cosmid 6A 6-6 containing the functional resistance gene were identified by trans formation, and sequenced. The LY214352-resistance gene coded for a pro tein of 520 amino acids that had a 34% identity and a 57% similarity i n a 333 amino-acid overlap to E. coli dihydroorotate dehydrogenase (DH O-DH). The results of a series of biochemical mechanism-of-action stud ies initiated simultaneously with molecular-genetic experiments also s uggested that DHO-DH was the target of LY214352. Assays measuring the inhibition of DHO-DH activity by LY214352 in a wild-type strain (I-50 = 40 ng/ml) and a highly resistant mutant (I-50 > 100 mu g/ml) conclus ively demonstrated that DHO-DH is the target site of LY214352 in A. ni dulans. Several mutations in the DHO-DH (pyrE) gene that resulted in r esistance to LY214352 were identified.