TARGETING THE FUNGAL PLASMA-MEMBRANE PROTON PUMP

The need for new mechanistic classes of broad spectrum antifungal agen ts has prompted development of the membrane sector and ectodomain of t he plasma membrane proton pumping ATPase as an antifungal target. The fungal proton pump is a highly abundant, essential enzyme in Saccharom yces cerevisiae. It belongs to the family of P-type ATPases, a class o f enzymes that includes the Na+,K+-ATPase and the gastric H+,K+-ATPase . These enzymes are cell surface therapeutic targets for the cardiac g lycosides and several anti-ulcer drugs, respectively. The effects of a cid-activated omeprazole show that extensive inhibition of the S. cere visiae ATPase is fungicidal. Fungal proton pumps possess elements with in their transmembrane loops that distinguish them from other P-type A TPases. These loops, such as the conformationally sensitive transmembr ane loop 1+2, can attenuate the activity of the enzyme. Expression in S. cerevisiae of fully functional chimeric ATPases that contain a fore ign target comprising transmembrane loops 1+2 and/or 3+4 from the fung al pathogen Candida albicans suggests that these loops operate as a do main. The chimera containing C. albicans transmembrane loops 1+2 and 3 +4 provides a prototype for mutational analysis of the target region a nd the screening of inhibitors directed against opportunistic fungal p athogens. Panels of mutants with modified ATPase regulation or with al tered cell surface cysteine residues are also described. Information a bout the ATPase membrane sector and ectodomain has been integrated int o a model of this region.