STRUCTURES OF CANDIDA ALBICANS DIHYDROFOLATE REDUCTASE: HOLOENZYME AND TERNARY INHIBITOR COMPLEXES Marc Whitlow, Andrew J. Howard, David Stewart, Karl D. Hardman (Genex Corp., Gaithersburg, MD 20877); Lee F. Kuyper, David P. Baccanari, Mary Fling, Joseph H. Chan, Robert L. Tansik (Glaxo Wellcome Inc., Research Triangle Park, NC, 27709, USA).

Crystal structures of Candida albicans dihydrofolate reductase (DHFR) were determined as part of an effort to develop selective inhibitors for the treatment of systemic fungal infections. The incidence of systemic fungal infections has risen considerably in recent years: C. albicans is now the fifth leading cause of microbial infection in the hospital setting, and opportunistic fungal infections present a major problem to AIDS patients. The success of selective DHFR inhibitors as antibacterials and antimalarials prompted us to explore the development of compounds for the selective inhibition of C. albicans DHFR (versus human DHFR). The enzyme was cloned, expressed, and crystallized, and its structure was determined as the holoenzyme and in ternary complex with a number of inhibitors. A potent but nonselective inhibitor (a 2,4-diaminopyrrolo[3,2-d]quinazoline) bound to the enzyme as expected. However, a selective class of inhibitor (2,4-diamino-5-arylthioquinazolines) showed unusual binding properties. Two of the most selective inhibitors bound to the enzyme with the 5-arylthio moiety positioned in the site that is normally occupied by the nicotinamide ring of NADPH. The diphospho bridge and adenosine portion of NADPH was clearly bound to those complexes in typical fashion, but the density for the nicotinamide ring was not visible. That portion of the cofactor was disordered but appeared to extended into solvent, being displaced from its normal binding site by the inhibitor. The width of the binding cleft of C. albicans DHFR was significantly larger (1.3-2.0 Å) than the corresponding dimensions of human DHFR and may be a feature that is exploited by the 5-arylthioquinazoline inhibitors. C. albicans DHFR crystallized in space group P2₁ with two molecules in the asymmetric unit. Final structures had R-factors of 0.155-0.199 at resolution values of 1.6-1.85 Å.