A new screening method for pathway-specific antifungal compounds.

Recently there has been a dramatic rise in the number of immunocompromised patients within our population, resulting from infection with HIV or from aggressive medical practices. These patients are especially susceptible to infections by opportunistic fungi leading to mycoses1). In the course of our antifungal screening program, we have devised an efficient screening method for selective toxicity for fungi. We have taken advantage of recent advances in genomics research and technical advances in highthroughput screening (HTS). This paper describes a screening strategy to detect compounds that inhibit any of the steps in the biosynthetic pathway of fungi. This screening method is rapid, robust, sensitive, and suitable for HTS. We sought homologous proteins between fungi and mammals, which were essential in fungi. It is appropriate to choose target proteins that have important roles for cell division or biosynthesis of essential cell components. Some antifungal medicines, fluconazole (FCZ), itraconazole (ICZ) and miconazole (MCZ), are notable because they show low toxicity towards humans. They have selective inhibitory activities against biosynthetic enzyme of ergosterol for fungi and cholesterol for humans2). We also chose target proteins that participate in biosynthetic pathway of ergosterol. Biosynthesis of cholesterol in humans is the same as that of ergosterol in fungi until the middle of their pathways; biosynthetic enzymes are homologous in each step3). Lanosterol 14-alpha demethylase (DM) is a typical enzyme for biosynthesis of ergosterol. In this paper we describe the screening method for selective inhibitors of fungal DM using the engineered strains of yeast. We prepared three types of strains for this screening. One expressed DM of Candida albicans (S1) and others expressed that each of human (S2) and rat (S3). If there are compounds that have specific inhibitory activity for the fungal enzyme, S1 will not survive in the presence of these compounds but S2 and S3 will survive. So we can easily screen specific activity of compounds by monitoring difference of growth among S1, S2 and S3.