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Abstract
To compare TTI-237 (5-chloro-6-[2,6-difluoro-4-[3-(methylamino)propoxy]phenyl]-N-[(1S)-2,2,2-trifluoro-1-methylethyl]-[1, 2, 4]triazolo[1,5-a]pyrimidin-7-amine butanedioate) with paclitaxel and vincristine in order to better understand the properties of this new anti-microtubule agent.
Tubulin polymerization and depolymerization were followed by turbidimetric assays. Effects of compounds on the binding of [3H]guanosine triphosphate ([3H]GTP) to tubulin were studied by competition binding assays. Effects of compounds on the phosphorylation of a panel of intracellular proteins were determined by flow cytometry using phosphoprotein-specific antibodies.
At low molar ratios of TTI-237:tubulin heterodimer (about 1:30), TTI-237 enhanced depolymerization kinetics in response to low temperature, but stabilized the aggregates at higher ratios (about 1:4). Similarly, the aggregates induced in microtubule protein by TTI-237 were depolymerized by excess Ca++ at low TTI-237:tubulin-heterodimer molar ratios, but were stable at higher ratios. TTI-237 inhibited the exchange of [3H]GTP at the exchangeable nucleotide site of the tubulin heterodimer, and was similar to vincristine in its effects on the phosphorylation of eight intracellular proteins in HeLa cells.
TTI-237 has properties that distinguish it from typical vinca-site and taxoid-site ligands, and therefore it may exemplify a new class of microtubule-active compounds.