GS-164, A SMALL SYNTHETIC COMPOUND, STIMULATES TUBULIN POLYMERIZATIONBY A SIMILAR MECHANISM TO THAT OF TAXOL

Purpose: During our search for new microtubule effecters as anticancer agents, we have found that a small synthetic molecule designated GS-1 64 interferes with the assembly of porcine microtubule proteins and ha s cytotoxic activity against a wide range of human tumor cell lines. I n this study, we investigated mode of action of the compound in compar ison with Taxol and colcemid. Methods: To gain an insight into the mod e of action of GS-164, we used an in vitro microtubule polymerization assay and a flow-cytometric measurement technique. Microtubule organiz ation and the level of tubulin polymerization in HeLa cells were also examined by immunofluorescence microscopy and cytoskeletal protein ana lyses, respectively. Results: GS-164 stimulated assembly of microtubul e proteins in vitro in a concentration-dependent and a GTP-independent manner. Furthermore, as with Taxol, the microtubule polymerization in duced by GS-164 was antagonized by podophyllotoxin, a tubulin polymeri zation inhibitor, and microtubules formed by GS-164 were resistant to disassembly by calcium or low temperatures. GS-164 in the micromolar r ange arrested the cell cycle of HeLa cells in the mitotic phase leadin g to cell death. GS-164 also increased the amounts of cellular microtu bules in HeLa cells, resulting in the formation of microtubule bundles . Conclusion: These results indicate that GS-164 stimulates microtubul e assembly by a similar mechanism to that of Taxol. A comparative conf ormational analysis of GS-164 and Taxol suggested that the structure o f the former mimics the minimum essential sites of Taxol required to e xert the Taxol-like activities described above. Although the cytotoxic ity of GS-164 against human tumor cells was 1000-fold lower than that of Taxol and GS-164 was one-tenth as active as Taxol in vitro, these f indings pave the way for synthesizing clinically useful anticancer age nts using GS-164 as a lead compound.