Tubulin polymerization in vitro

Analogs 38 were tested for their ability to promote in vitro tubulin polymerization, and their antiproliferative activity was assessed against the human epidermoid cancer cell lines KB-31 and KB-8511, which serve as representative examples of drug-sensitive and P-gp-overexpressing, multidrug-resistant human cancer cell lines, respectively (see, e.g.. Refs. 20 and 37). 

Noscapine (30) is a naturally occurring phthalideisoquino-line alkaloid obtained from opium that has weak anticancer properties. Subtle changes to the corresponding phenol (31) and aniline (32) resulted in a >50-fold increase in activity in an in vitro tubulin polymerization assay. Moreover, the phenol shows a modest 1.5-fold increase in oral bioavailability compared to noscapine (30). 

Fig. 6-17 Forward chemical-genetic screen compound activity from the initial cell-based for inhibitors of mitosis (data from Ref. 73). and in vitro tubulin polymerization assay.

Miura K, Inokawa M, Imura N (1984) Effects of methylmercury and some metal ions on microtubule networks in mouse glioma cells and in vitro tubulin polymerization. Toxicol Appl Pharmacol 73 218-231... 

A BMS research group found that 3 -r-butylaminocarbonyloxy paclitaxel analogs 14a and 14b (regioisomers of docetaxel and its 10-Ac derivative) were several times less active than paclitaxel in vitro, but 14b was equipotent to paclitaxel in vivo. They also prepared 3 -A-thiocarbamate and C3 -A-thiourea bearing analogs. Although C3 -A-thiocarbamate was found to be more potent than paclitaxel and docetaxel in both tubulin polymerization and cytotoxicity assays, thioureas are usually less active. ... 

Whole cell In vitro tubulin mitotic polymerization In vivo staining for arrest assay assay chromosomes and tubulin... 

Patupilone is naturally occurring epothilone B. It is twice as potent in promoting tubulin polymerization in vitro, compared with pac-litaxel and epothilone A [147 ]. Phase 1/11 studies have been conducted in patients with non-small cell lung cancer, renal cell cancer, and ovarian cancer. Patupilone crosses the blood-brain barrier and has activity in patients with non-small cell lung cancer and cerebral metastases [1 ]. 

Vinblastine, vincristine, and structurally related analogs inhibit microtubule polymerization by 50% at concentrations in the range 0.1-1 xM, and the process of tubulin addition to preformed microtubules, at steady state, is comparably sensitive to inhibition by these agents (5). As shown in Table I, the differences in values for inhibition of steady-state tubulin addition by vinblastine, vincristine, vindesine, and vinepidine were relatively small, but the pattern of activity in the tubulin addition system did not parallel that observed when the compounds were evaluated for effects on the proliferation of B16 melanoma cells in vitro. Vinepidine was more than twice as potent as vinblastine as an inhibitor of steady-state tubulin addition but nearly 10-fold less potent than vinblastine as an inhibitor of cell growth (i). 

The threshold concentration of monomer that must be exceeded for any observable polymer formation in a self-assembling system. In the context of Oosawa s condensation-equilibrium model for protein polymerization, the cooperativity of nucleation and the intrinsic thermodynamic instability of nuclei contribute to the sudden onset of polymer formation as the monomer concentration reaches and exceeds the critical concentration. Condensation-equilibrium processes that exhibit critical concentration behavior in vitro include F-actin formation from G-actin, microtubule self-assembly from tubulin, and fibril formation from amyloid P protein. Critical concentration behavior will also occur in indefinite isodesmic polymerization reactions that involve a stable template. One example is the elongation of microtubules from centrosomes, basal bodies, or axonemes. 

We can consider three situations (a) if [M]oo > [M] , there will be a net increase in polymerization (b) if [M] < [M]oo, there will be net depolymerization and (c) when [M] = [M] , the monomer and polymer are in equilibrium. The last case does not imply a static condition rather, monomer addition and loss can constantly occur, but the total polymer weight concentration will always remain unchanged. In head-to-tah polymerizations, hke those of actin and tubulin assembly in vitro, each of the two polymer ends can interact with monomer the critical concentration [M]o then equals (koff +... 

Although 2-methoxy-5-aryltropones lacking the B ring of colchicine are not true colchicinoids, they are discussed since some were found to be potent inhibitors of tubulin polymerization in vitro. (70). This has stimulated much interest in this type of compound as well as research to achieve their practical synthesis. 

Some daphniphyllum alkaloids, such as calydphyllines A (82) and B (83), exhibited moderate cytotoxicity against murine lymphoma L1210 cells in vitro [84]. Daphni-glaucin C showed inhibition of polymerization of tubulin at IC50 25mg/mL [82]. Recently, some daphniphyllum alkaloids such as daphmanidins E and F showed moderate vasorelaxant activity on rat aorta [80]. However, since the pharmacological activity of the daphniphyllum alkaloids is poorly studied, this area should be developed in future. 

Treatment of human cancer cells with low nM concentrations of Epo B leads to profound growth inhibition and cell death (Table 1-1). In line with the effects on tubulin polymerization in vitro, Epo B is a more potent antiproliferative agent than Epo A, which in turn is about equipotent with paclitaxel. As observed for paclitaxel, Epo B treatment produces aberrant mitotic spindles, results in cell cycle arrest in mitosis, and eventually leads to apoptotic cell death. It is often assumed that apoptosis is a direct consequence of G2/M arrest, which in turn would be a prerequisite for growth inhibition and cell death. However, as has been elegantly demonstrated by Chen et al. in a series of recent experiments, the situation is clearly more complex, such that low concentrations of Epo B (and paclitaxel... 

A protein that polymerizes to form microtubules. Tubulin is a target for anticancer therapy. See Feit, H Slusarek, L., and Shelanski, M.L., Heterogeneity of tubulin subunits, Proc. Natl. Acad. Sci. USA 68, 2028-2031, 1971 Fine, R.E., Heterogeneity of tubulin, Nat. New Biol. 233, 283-284, 1971 Rappaport, L., Leterrier, J.F., and Nunez, J., Non phosphorylation in vitro of 6 S tubulin from brain and thyroid tissue, FEBS Lett. 26, 239-352, 1972 Berry, R.W. and Shelanski, M.L., Interactions of tubulin with vinblastine and guanosine triphosphate, J. Mol. Biol. 71,71-80,1972 Hemminki, K., Relative turnover of tubulin subunits in rat brain, Biochim. Biophys. Acta 310, 285-288,1973 Timasheff, S.N., Frigon, R.P., and Lee,... 

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