Chii M. Lin

Isolation and structure of the strong cell growth and tubulin inhibitor combretastatin A-4

The African treeCombretum caffrum (Combretaceae) has been found to contain a powerful inhibitor of tubulin polymerization (IC502–3 μM), the growth of murine lymphocytic leukemia (L 1210 and P 388 with ED50≈0.003 μM and human colon cancer cell lines [(e.g. LoVo (ED50=0.005 μg/ml), HT29 (ED50 0.02 μg/ml, Colo 205 (ED50=0.07 μg/ml), DLD-1 (ED50=0.005 μg/ml) and HCT-15 (ED50=0.0009 μg/ml)] designated combretastatin A-4 (1c). The structure assigned by spectral techniques was confirmed by synthesis.

Glutamate-induced polymerization of tubulin: Characteristics of the reaction and application to the large-scale purification of tubulin

Abstract A three-stage method is presented for the large-scale purification of calf brain tubulin, exploiting the ability of high concentrations of glutamate to stabilize tubulin and, in the presence of GTP, to induce its polymerization. The purified tubulin contained 1.9 mol of guanine nucleotide per mole of protein and was essentially free of nucleoside diphosphate kinase and ATPase activities. The glutamate-induced polymerization reaction required GTP, while ATP, CTP, and UTP were completely ineffective. Polymerization was temperature dependent and cold reversible and was inhibited by colchicine, GDP, and Ca 2+ , but no absolute requirement for Mg 2+ could be demonstrated. The purified tubulin had only a minimal GTPase activity unless glutamate was present, and the hydrolytic reaction varied with the glutamate concentration. At higher glutamate levels the onset of hydrolysis was closely linked to the onset of polymerization with a rapid burst of GTP hydrolysis ending as polymerization approached its plateau, followed by a slower, linear rate of hydrolysis. Initially the molar ratio of GTP hydrolyzed to tubulin polymerized was about 1:1. At lower glutamate concentrations GTP hydrolysis did not appear to be linked to tubulin polymerization.

Antitumor 2,3-dihydro-2-(aryl)-4(1H)-quinazolinone derivatives: Interactions with tubulin

A series of derivatives of 2,3-dihydro-2-(aryl)-4(1H)-quinazolinone (DHQZ) with known antitumor activity was re-evaluated in the National Cancer Institute cancer cell line screen. Analysis by the COMPARE algorithm suggested that their cytotoxicity derived from interactions with tubulin. Significant inhibition of tubulin assembly and of the binding of radiolabeled colchicine to tubulin was demonstrated with several of the compounds, particularly NSC 145669, 175635, and 175636. The DHQZ derivatives are structurally analogous to a number of antimitotic agents, flavonols and derivatives of 2-styrylquinazolin-4(3H)-one and of 2-phenyl-4-quinolone. Structure-activity analogies between these agents, the combretastatins, and the colchicinoids were analyzed and summarized.

Centaureidin, a cytotoxic flavone from Polymnia fruticosa, inhibits tubulin polymerization

Abstract The characteristics pattern of differential cytotoxicity of a crude extract of the tropical plant Polymnia fruticosa was found to be similar to those of known tubulin-interactive compounds. Fractionation of the extract led to centraureidin as the major cytotoxic principle. Centaureidin inhibited tubulin polymerization, inhibited the binding of [3H]-colchicine to tubulin, and induced mitotic figure formation in whole cells at cytotoxic concentrations. This is the first known example of a flavone with antimitotic activity.

Maytansine inhibits nucleotide binding at the exchangeable site of tubulin

The antineoplastic drug maytansine inhibits the binding of exogenously added radiolabeled GDP and GTP to tubulin (50% inhibition at 9-10 microM drug at 0 degrees). Vinblastine was 1/10-th as inhibitory. Neither maytansine nor vinblastine displaced GDP from tubulin, and both drugs virtually eliminated dissociation of radiolabeled GDP from the exchangeable site. Maytansine also inhibits binding of nucleotides to a vacant exchangeable site. Maytansine thus prevents nucleotide exit and entry at the exchangeable site because of a direct physical obstruction or a conformational change in the tubulin molecule.

Stabilization of the colchicine-binding activity of tubulin by organic acids

A number of carboxylic acids and organic phosphates were found to be highly effective in stabilizing the colchicine-binding activity of calf brain tubulin. The most active were glutamate, glutarate, delta-aminovalerate, glucose 1-phosphate, glucose 6-phosphate, fructose 1,6-(bis)phosphate, creatine phosphate and 6-phosphogluconate Maximum effects occurred at high concentrations. Combinations of agents were also examined, and the most effective mixture for stabilizing tubulin found thus far was the combination of 1.0 M glutamate, 100 mM glucose 1-phosphate, 1 mM GTP and 0.5 mg/ml of albumin. No loss of activity occurred over 48 h at 37 degrees C with tubulin was present at a concentration of 100 microgram/ml.

A convenient tubulin-based quantitative assay for paclitaxel (Taxol) derivatives more effective in inducing assembly than the parent compound

Abstract A room temperature biochemical assay, based on centrifugal removal of tubulin polymer, was developed to permit ready detection of paclitaxel analogs more active than the parent compound and to permit reliable quantification of differences in activity relative to paclitaxel in terms of drug concentration. The assay was validated by comparing paclitaxel to two compounds (docetaxel and 2-debenzoyl-2-meta-azidobenzoylpaclitaxel) known to be more active under multiple reaction conditions. The assay was designed to yield a relatively high EC50 (23 μM) for paclitaxel. This was possible because paclitaxel only weakly induced tubulin assembly at room temperature in 0.4 M glutamate without exogenous GTP. Under these same reaction conditions 50% assembly occurred with 4.7  μM 2-debenzoyl-2-meta-azidobenzoylpaclitaxel and 11 μM docetaxel. These biochemical EC50 values were in agreement with the relative cytotoxicity of the three compounds for human Burkitt lymphoma CA46 cells (IC50 values for paclitaxel, docetaxel, and 2-debenzoyl-2-meta-azidobenzoylpaclitaxel were 40, 10, and 3 nM, respectively).

Synthesis and biological evaluation of paclitaxel analogs modified in ring C

Abstract Both 7-deoxy-7α-azidopaclitaxel (6) and 7-deoxy-Δ6,7-paclitaxel (4) can be prepared from paclitaxel-7-0-triflate (2b). Oxidation of 7-deoxy-Δ6,7-paclitaxel with dioxirane yields the epoxide 7, while oxidation with osmium tetroxide yields 6α-hydroxy-7-epipaclitaxel (9), and acylation of this gives the 6α-acyloxy-7-epipaclitaxel derivatives 11a-d. No compound was as effective at promoting tubulin assembly as paclitaxel, but most stabilized polymer as well as or better than paclitaxel. Compounds 4, 6, 7, 9, and 11d differed little from paclitaxel in their cytotoxicity for human Burkitt lymphoma CA46 cells.

Differential effects of magnesium on tubulin-nucleotide interactions

Magnesium-depleted 2-(N-morpholino)ethanesulfonate (Mes), glutamate, tubulin and microtubule-associated proteins were prepared and used to study the effects of exogenously added MgCl2 on tubulin-nucleotide interactions in 0.1 M Mes with microtubule-associated proteins and in 1.0 M glutamate. Endogenous levels of Mg2+ in the systems studied were approximately stoichiometric with the tubulin concentrations and largely derived from the tubulin. We examined the effects of added Mg2+ on tubulin polymerization, GDP inhibition of polymerization, binding of GDP and GTP to tubulin, and GTP hydrolysis. Exogenously added Mg2+ had markedly different effects on these reactions. The order of their sensitivity for a requirement for added Mg2+ was as follows: GTP binding greater than GTP hydrolysis greater than polymerization greater than GDP binding. Inhibition of polymerization by GDP varied inversely with the Mg2+ concentration and was greatest in the absence of the cation. These results indicate that GDP and GDP-Mg2+ interact with similar affinity at the exchangeable site, while GTP-Mg2+ has a higher affinity for tubulin than does free GTP. Nevertheless, under appropriate conditions, free GTP can interact sufficiently well with tubulin to permit both nucleation and elongation reactions.

Effects of pH on tubulin-nucleotide interactions.

Significant GTP-independent, temperature-dependent turbidity development occurs with purified tubulin stored in the absence of unbound nucleotide, and this can be minimized with a higher reaction pH. Since microtubule assembly is optimal at lower pH values, we examined pH effects on tubulin-nucleotide interactions. While the lowest concentration of GTP required for assembly changed little, GDP was more inhibitory at higher pH values. The amounts of exogenous GTP bound to tubulin at all pH values were similar, but the amounts of exogenous GDP bound and endogenous GDP (i.e., GDP originally bound in the exchangeable site) retained by tubulin rose as reaction pH increased. Endogenous GDP was more efficiently displaced by exogenous GTP than GDP at all pH values, but displacement by GTP was 10-15% greater at pH 6 than at pH 7. Dissociation constants for GDP and GTP were about 1.0 microM at pH 6 and 0.02 microM at pH 7. A small increase in the affinity of GDP relative to that of GTP occurs at pH 7 as compared to pH 6, together with a 50-fold absolute increase in the affinity of both nucleotides for tubulin at pH 7. The time courses of microtubule assembly and GTP hydrolysis were compared at pH 6 and pH 7. At pH 6, the two reactions were simultaneous in onset and initially stoichiometric. At pH 7, although the reactions began simultaneously, hydrolysis seemed to lag substantially behind assembly. Unhydrolyzed radiolabeled GTP was not incorporated into microtubules, however, indicating that GTP hydrolysis is actually closely coupled to assembly. The apparent lag in hydrolysis probably results from a methodological artifact rather than incorporation of GTP into the microtubule with delayed hydrolysis.