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-chloro-4-fluorophenoxy) quinoline] has been identified through a dual biochemical and molecular-genetics approach. In the molecular-genetics approach, a cosmid library was prepared from an Aspergillus nidulans mutant 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 from the library by sib-selection. Restriction fragments from cosmid 6A6-6 containing the functional resistance gene were identified by transformation, and sequenced. The LY214352-resistance gene coded for a protein of 520 amino acids that had a 34% identity and a 57% similarity in a 333 amino-acid overlap to E. coli dihydroorotate dehydrogenase (DHO-DH). The results of a series of biochemical mechanism-of-action studies initiated simultaneously with molecular-genetic experiments also suggested that DHO-DH was the target of LY214352. Assays measuring the inhibition of DHO-DH activity by LY214352 in a wild-type strain (I50=40 ng/ml) and a highly resistant mutant (I50>100 microgram/ml) conclusively demonstrated that DHO-DH is the target site of LY214352 in A. nidulans. Several mutations in the DHO-DH (pyrE) gene that resulted in resistance to LY214352 were identified.
Gustafson
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G
Crop Disease Management Discovery Research, DowElanco, Indianapolis, IN 46268-1054, USA.
Davis
G
G
Waldron
C
C
Smith
A
A
Henry
M
M
D000935:Antifungal Agents / Q000737:chemistry / Q000494:pharmacology*; D001233:Aspergillus nidulans / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D003360:Cosmids; D004271:DNA, Fungal; D000090722:Dihydroorotate Dehydrogenase; D004352:Drug Resistance, Microbial / Q000235:genetics; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D011523:Protoplasts; D011804:Quinolines / Q000737:chemistry / Q000494:pharmacology*
D000935:Antifungal Agents; D004271:DNA, Fungal; D000090722:Dihydroorotate Dehydrogenase; D011804:Quinolines; C108506:8-chloro-4-(2-chloro-4-fluorophenoxy)quinoline; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors