Discovery of a Small-Molecule Inhibitor of β-1,6-Glucan Synthesis

Fungal infections have increased in frequency over the past several decades due to a growing number of immunocompromised patients (8, 9, 35). Present therapeutic options are limited, however, to three classes of compounds: polyenes, azoles, and recently introduced candins (3, 9, 48). The utility of polyenes is limited by their nephrotoxicity (35, 48). Although azoles are safer and most commonly used, the broad usage of these drugs has probably allowed the increase of less-susceptible species of Candida, such as C. glabrata (2, 17). In addition, drug-drug interactions and teratogenicity limit their clinical usage. Candins have solved some of these problems; however, they have not completely satisfied unmet medical needs mainly due to their poor oral absorption and limited spectrum (48). In addition, incidents of resistance to these drugs have also emerged (14, 28). Therefore, development of an orally active antifungal drug with a novel mode of action is desirable. The fungal cell wall is an attractive target for antifungal agents because it is an essential, fungal-specific organelle that is absent from human cells. The cell wall of Saccharomyces cerevisiae is basically composed of β-1,3-glucan, β-1,6-glucan, chitin, and highly mannosylated glycoproteins, which are interconnected (5, 20). Many fungal-specific enzymes, such as Fks1p, Kre6p, and Chs1p, are involved in the synthesis of β-1,3-glucan, β-1,6-glucan, and chitin, respectively (5). In addition to the synthases of cell wall components, several enzymes have been shown to be involved in the interconnection of these components (5). A number of β-1,3-glucan synthase inhibitors, such as echinocandins, papulacandins, and enfumafungin, have already been reported (21, 34, 44). Several chitin synthase inhibitors have been reported as well (13, 42). In contrast, an inhibitor of β-1,6-glucan synthase or the enzymes involved in the interconnection of cell wall components has not been reported. Genetic analyses of S. cerevisiae and C. albicans have provided us with valuable information regarding β-1,6-glucan synthesis (16, 27, 30, 39). Many proteins encoded by KRE genes, such as KRE6, KRE9, and KRE1, are involved in the biosynthesis in a sequential manner (5). However, no precise functions, either catalytic or regulatory, have been definitively assigned to any KRE gene products. Lack of enzymatic information hampers the discovery of their inhibitors. Structural and biochemical analyses of the yeast cell wall, however, have provided a way to obtain inhibitors. Most cell wall proteins are glycosylphosphatidylinositol (GPI) dependent and are attached to β-1,3-glucan and/or chitin via β-1,6-glucan (19, 22, 23). Recent progress in genetic technology allowed us to attach heterologous protein to the cell wall by constructing a gene of interest fused to a secretion signal and GPI attachment signal (46, 47). Using this technology, we have developed a cell-based assay system for screening various inhibitors of cell wall components, including β-1,6-glucan (A. Kitamura, K. Someya, and R. Nakajima, U.S. patent application 20040091949 [PCT/JP01/03630]). In the course of screening for antifungal compounds using this system, we discovered the compound D75-4590, which has unique activities. To gain a better insight into this compound, we studied the nature of its antifungal activities and its mechanism of action.