Identification of Saccharomyces cerevisiae Tub1 alpha-tubulin as a potential target for NKH-7, a cytotoxic 1-naphthol derivative compound.

In a screening for small-molecule compounds that alleviate the deleterious effects of external CaCl(2) on zds1 Delta strain yeast, we found 2-((1-(hydroxymethyl) cyclohexyl) methyl) naphthalen-1-ol (NKH-7) to be an active compound. NKH-7 also inhibited cell growth at higher concentrations. To identify its target in growth inhibition, we isolated NKH-7-resistant mutants and selected those mutants that exhibited dominant or semi-dominant resistance specifically to NKH-7. By gene cloning, a TUB1 mutant gene encoding alpha-tubulin with a Ser248Pro mutation was identified. Deletion of the TUB3 gene, a minor gene encoding alpha-tubulin, led to supersensitivity to NKH-7. Cellular tubulin-containing arrays as visualized by green fluorescent protein (GFP)-labeled alpha-tubulin diminished rapidly on exposure to the inhibitor. The mutation was situated proximal to the alpha-beta interface of alpha-tubulin in microtubule protofilaments, suggesting the possibility that NKH-7 affects the hydrolysis of GTP bound to beta-tubulin. A functional connection perhaps exists between the tubulin inhibition and Ca(2+)-dependent cell-cycle regulation.

Inhibition of Ca2+-signal-dependent growth regulation by radicicol in budding yeast.

Compelled activation of Ca(2+) signaling by exposure of zds1Delta strain Saccharomyces cerevisiae cells to external CaCl(2) leads to characteristic physiological consequences such as growth inhibition in the G(2) phase and polarized bud growth. Screening of microbial metabolites for activity alleviating the deleterious physiological effects of external CaCl(2) identified the Hsp90 inhibitor radicicol as an inhibitor of Ca(2+)-signal-dependent cell-cycle regulation in yeast. Radicicol alleviated analogous physiological effects due to the expression of a constitutively active form of calcineurin or overexpression of Swe1, the negative regulatory kinase of the Cdc28-Clb complex. Western blot analysis indicated that radicicol inhibited Ca(2+)-induced accumulation of Swe1 and Cln2.

YCM1008A, a novel Ca2+-signaling inhibitor, produced by Fusarium sp. YCM1008.

In the course of screening for drugs that suppress the Ca(2+)-mediated growth inhibition in a yeast mutant, we found that the metabolite of Fusarium sp. strain YCM1008 inhibited Ca(2+)-signaling. A novel pyrano-pyridone, YCM1008A was isolated from the fermentation broth using HLB column chromatography followed by HPLC, and the structure was elucidated by spectral analysis. YCM1008A suppressed Ca(2+)-induced growth inhibition of the Saccharomyces cerevisiae (Deltazds1Deltasyr1) mutant.