HWY-289

Virulence ofCandida albicansis attributable to its unique dimorphic transition from nonpathogenic yeast cells to pathogenic hyphal cells. We previously discovered a novel antifungal agent, known as HWY‐289. To characterize the mechanism underlying HWY‐289 antifungal activity, we performed 2‐DE to identify proteins that were differentially expressed during yeast‐to‐hyphal transition and in response to HWY‐289. Twenty‐four differentially expressed protein spots were identified in HWY‐289‐treated yeast. Most differentially expressed proteins were involved in carbohydrate‐derived energy metabolism, cellular detoxification, and antioxidant defenses. Two proteins were involved in cell cycle regulation and DNA processing, and both were downregulated by HWY‐289, suggesting that this agent might promote cell death by weakening cellular defense systems. HWY‐289 inhibited yeast‐to‐hyphal transition in a dose‐dependent manner. 2‐DE analysis of hyphae uncovered several proteins that were induced during yeast‐to‐hyphal transition. Of these, aconitase and phosphatidylinositol transfer protein were downregulated by HWY‐289, suggesting that they mediate the antifungal effects of HWY‐289. Finally, RT‐PCR analysis revealed that HWY‐289 induced expression of three RAS‐related genes (CcCST20,CaHST7, andCaCPH1) in yeast cells, but suppressed their expression in hyphae. Thus, the antifungal action of HWY‐289 may be attributable to its ability to disrupt prohyphal RAS signaling.

To determine whether Candida albicans acyl CoA:sterol acyltransferase (ASAT) can be a potential target enzyme for the protoberberine derivative (HWY-289), we have isolated a gene encoding Ca-ASAT and examined inhibitory effects of HWY-289 on the overexpressed Ca-ASAT. HWY-289 specifically inhibits Ca-ASAT in a non-competitive manner in vitro (IC(50) [9.2microM], K(i) [5.15microM]). The cloned CaARE2 gene (1830 nucleotides [nt]) encodes active Ca-ASAT protein that exhibits a calculated molecular mass of 71.3kDa. The amino acid sequence of CaAre2p is 33.4% and 35.1% identical to those of Saccharomyces cerevisiae ScAre1p and ScAre2p homologues, respectively. Recombinant and endogenous Ca-ASAT displayed identical patterns of inhibition upon exposure to HWY-289 and a preference for cholesterol and oleoyl-CoA as substrates. Northern blot analysis showed that CaARE2 was activated by HWY-289, but not by CI-976 (a human acyl-coenzyme A:cholesterol acyltransferase inhibitor), in a dose-dependent manner (up to 5mg/L), suggesting different selectivities of action between HWY-289 and CI-976 on Ca-ASAT activity.