Discovery of a small-molecule inhibitor of {beta}-1,6-glucan synthesis

Abstract

It is possible that antifungal drugs with novel modes of action will provide favorable options to treat fungal infections. In the course of our screening for antifungal compounds acting on the cell wall, a pyridobenzimidazole derivative with unique activities, named D75-4590, was discovered. During treatment of Saccharomyces cerevisiae with D75-4590, (i) incorporation of [(14)C]glucose into the beta-1,6-glucan component was selectively reduced, (ii) proteins released from the cell had lost the beta-1,6-glucan moiety, and (iii) cells tended to clump, resulting in impaired cell growth. Genetic analysis of a D75-4590-resistant mutant of S. cerevisiae indicated that its primary target was Kre6p, which is considered to be one of the beta-1,6-glucan synthases. These results strongly suggest that D75-4590 is a specific inhibitor of beta-1,6-glucan synthesis. D75-4590 showed potent activities against various Candida species. It inhibited hyphal elongation of C. albicans as well. KRE6 is conserved in various fungi, but no homologue has been found in mammalian cells. These lines of evidence indicate that D75-4590 is a promising lead compound for novel antifungal drugs. To our knowledge, this is the first report of a beta-1,6-glucan inhibitor.

FIG. 1.

Chemical structure of D75-4590.

FIG. 2.

The principle of the screening system. Reporter proteins are fixed onto the cell wall (wall-type arming yeast) or onto the cell membrane (membrane-type arming yeast) by means of genetic engineering. Both yeasts are treated with the test compound. If the compound acts on the process of protein fixation to the cell wall (e.g., β-1,6-glucan synthesis), reporter proteins will be released from the wall-type arming yeast only. If the compounds act on the process of protein fixation to cell membranes, reporter proteins will be released from both yeasts. If the compounds do not act in protein fixation, no reporter proteins will be released from either yeast.

FIG. 3.

Effect of D75-4590 on growing cells of C. glabrata IFO0622. Cells were incubated in RPMI medium with or without D75-4590. The viable cells were counted at intervals using SDA plates. The concentrations of D75-4590 are shown.

FIG. 4.

Multibudding phenotype of yeasts treated with D75-4590. Growing cells of S. cerevisiae YPH500, C. glabrata IFO0622, and C. albicans ATCC 24433 were treated with D75-4590. Microscopic observation was carried out after 18 h. a, S. cerevisiae, without drug; b, S. cerevisiae, 2 μg/ml of drug; c, C. glabrata, without drug; d, C. glabrata, 2 μg/ml of drug; e, C. albicans, without drug; f, C. albicans, 2 μg/ml of drug.

FIG. 5.

Inhibitory effect of D75-4590 against hyphal elongation of C. albicans ATCC 24433. Cells were cultured in HFM-7 with or without D75-4590. Microscopic observations were carried out after 6 h of treatment with D75-4590 at concentrations of (a) 0 μg/ml, (b) 1 μg/ml, (c) 4 μg/ml, and (d) 16 μg/ml and after 24 h of treatment at concentrations of (e) 0 μg/ml, (f) 1 μg/ml, (g) 4 μg/ml, and (h) 16 μg/ml.

FIG. 6.

Effects of D75-4590 and aculeacin A on the incorporation of radioactive precursors into macromolecules in growing cells of S. cerevisiae AY-10 and C. glabrata IFO0622. Growing cells in RPMI medium were treated with or without drug in the presence of radioactive glucose, leucine, uridine, or acetic acid. β-1,3-Glucan, β-1,6-glucan, chitin, mannan, RNA, protein, and sterol fractions were prepared by the methods described in Materials and Methods. The percent changes of the incorporated radioactivities by drug treatment for 1 h (upper panels) or 3 h (lower panels) at each concentration tested are displayed. (a) S. cerevisiae AY-10 treated with aculeacin A. (b) S. cerevisiae AY-10 treated with D75-4590. (c) C. glabrata IFO0622 treated with D75-4590.

FIG. 7.

Western analysis of proteins secreted from S. cerevisiae AY-10 treated with D75-4590. Cells were treated with D75-4590 for 6 h, and the extracellular medium was concentrated and used for Western blot analysis with anti-pustulan (β-1,6-glucan) antiserum. Concentrations of D75-4590 added were as follows: 1, 20 μg/ml; 2, 10 μg/ml; 3, 5 μg/ml; 4, 2.5 μg/ml; 5, 1.25 μg/ml; 6, 0.625 μg/ml; 7, 0.313 μg/ml; and 8, 0 μg/ml. M, marker.

FIG. 8.

Comparison of partial sequences of Kre6p homologues from various fungal species. The amino acid residues are shown in single-letter amino acid code. The region near the position of the residue (indicated by an arrow) that, when mutated, confers D75-4590 resistance in S. cerevisiae is displayed in the alignment. Abbreviations: SC, S. cerevisiae; CG, C. glabrata; CA, C. albicans; CK, C. krusei; CP, C. parapsilosis; AF, A. fumigatus; AT, A. terreus; AC, A. clavatus.