The African tree Combretum caffrum (Combretacae) has been found to contain a powerful inhibitor of tubulin polymerization (IC50 2-3 microM), the growth of murine lymphocytic leukemia (L 1210 and P 388 with ED50 approximately 0.003 microM and human colon cancer cell lines [(e.g. LoVo (ED50 = 0.005 microgram/ml), HT 29 (ED50 0.02 microgram/ml, Colo 205 (ED50 = 0.07 microgram/ml), DLD-1 (ED50 = 0.005 microgram/ml) and HCT-15 (ED50 = 0.0009 microgram/ml] designated combretastatin A-4 (1c). The structure assigned by spectral techniques was confirmed by synthesis.

Epothilones A and B, natural products with minimal structural analogy to taxoids, have effects similar to those of paclitaxel (Taxol(R)) in cultured cells and on microtubule protein, but differ from paclitaxel in retaining activity in multidrug-resistant cells. We examined interactions of the epothilones with purified tubulin and additional cell lines, including a paclitaxel-resistant ovarian carcinoma line with an altered beta-tubulin. The epothilones, like paclitaxel, induced tubulin to form microtubules at low temperatures and without GTP and/or microtubule-associated proteins. The epothilones are competitive inhibitors of the binding of [3H]paclitaxel to tubulin polymers. The apparent Ki values for epothilones A and B were 1.4 and 0.7 microM by Hanes analysis and 0.6 and 0.4 microM by Dixon analysis. In the paclitaxel-sensitive human cell lines we examined, epothilone B had greater antiproliferative activity than epothilone A or paclitaxel, while epothilone A was usually less active than paclitaxel. A multidrug-resistant colon carcinoma line and the paclitaxel-resistant ovarian line retained sensitivity to the epothilones. With Potorous tridactylis kidney epithelial (PtK2) cells examined by indirect immunofluorescence, microtubule bundles appeared more rapidly following epothilone B treatment, and there were different proportions of various mitotic aberrations following treatment with different drugs.

Combretastatin A-4 (CS-A4), 3,4,5-trimethoxy-3'-hydroxy-4'-methoxy-(Z)-stilbene, and combretastatin A-2 (CS-A2), 3,4-(methylenedioxy)-5-methoxy-3'-hydroxy-4'-methoxy-(Z)-stilbene, are structurally simple natural products isolated from the South African tree Combretum caffrum. They inhibit mitosis and microtubule assembly and are competitive inhibitors of the binding of colchicine to tubulin [Lin et al. (1988) Mol. Pharmacol. 34, 200-208]. In contrast to colchicine, drug effects on tubulin were not enhanced by preincubating CS-A4 or CS-A2 with the protein. The mechanism of their binding to tubulin was examined indirectly by evaluating their effects on the binding of radiolabeled colchicine to the protein. These studies demonstrated rapid binding of both compounds to tubulin even at 0 degrees C (binding was complete at the earliest times examined), in contrast to the relatively slow and temperature-dependent binding of colchicine. Although the binding of the C. caffrum compounds to tubulin was quite tight, permitting ready isolation of near-stoichiometric amounts of drug-tubulin complex even in the absence of free drug, both CS-A4 and CS-A2 dissociated rapidly from tubulin in the presence of high concentrations of radiolabeled colchicine. Apparent rate constants for drug dissociation from tubulin at 37 degrees C were 3.2 x 10(-3) s-1 for CS-A4, 4.8 x 10(-3) s-1 for CS-A2, and 2.9 x 10(-5) s-1 for colchicine (half-lives of 3.6, 2.4, and 405 min, respectively). Thus, the effectiveness of the C. caffrum compounds as antimitotic agents appears to derive primarily from the rapidity of their binding to tubulin.(ABSTRACT TRUNCATED AT 250 WORDS)

A metabolite of estradiol, 2-methoxyestradiol (2ME), inhibits angiogenesis In the chicken embryo chorioallantoic membrane assay. Since 2ME causes mitotic perturbalions, we ea ined Its interactions with tubulin. In our standard 1.0 M glutamate system (plus 1.0 mM MgCl2 at 3rC), superstoichiometric concentrations (relative to tubulin) of 2ME inhibited the nucleation and propagation phases of tubulin assembly but did not affect the reaction extent. Although polymer formed In the presence of 2ME was more cold-stable than control polymer, morphology was little changed. Under suboptimal reaction conditions (0.8 M glutamate/no MgCl2 at 26WC), substoichiometric 2ME totally inhibited polymerization. No other estrogenic compound was as effective as 2ME as an inhibitor of polymerization or of the binding of colchicine to tubulin. Inhibition ofcolchicine binding was competitive (KM,22 aM). Thus, a m an metabolite of estradiol binds to the colchicine site of tubulin and, depending on reaction conditions, either inhibits assembly or seems to be incorporated into a polymer with altered stability properties.There is growing evidence that estrogenic compounds affect cell division and act directly on microtubules by interacting with tubulin. The most extensively studied of these compounds is the synthetic analog diethylstilbestrol (DES; Fig. 1, compound 1) and related agents (1-3). These drugs cause disturbances in mitosis, inhibit microtubule assembly at relatively high concentrations, and inhibit the binding of colchicine to tubulin. Estradiol (compound 2), the major estrogenic hormone of human beings, also causes disturbances ofmitosis in cultured cells (1,4,5). These perturbations include aneuploidy, multinucleation, and mitotic arrest, and estradiol has been reported to inhibit the polymerization of rat brain tubulin (6). Seegers et al. (5) found that 2-methoxyestradiol (2ME; compound 3), a metabolite of both estradiol (7) and the oral contraceptive agent 17-ethynylestradiol (compound 4), was more potent than estradiol in producing mitotic perturbations and proposed that it was 2ME rather than estradiol that caused the observed disturbances. Although 2-methoxyestrogens are extremely weak in binding to cytosol estrogen receptors (8), 2ME is found in blood and urine after sequential hepatic hydroxylation and methylation ofestradiol (7).We recently found that 2ME inhibited angiogenesis in the chicken embryo chorioallantoic membrane assay of Crum et al. (9). Disks of2ME (100 Mg) produced large avascular zones 48 hr after implantation on a 6-day embryo, similar to in vitro results of others (10). In attempting to define the mechanistic basis, we observed that 2ME inhibited microtubule assembly. This finding led us to study the interactions of 2ME with purified tubulin.MATERIALS AND METHODS Materials. Purified bovine brain tubulin and H2-CSA4 were prepared as described (11,12). Monosodium glutamate (2.0 M) was adjusted to pH 6.6 with HCl. 2ME, 2-fluoroestradiol, 2-methoxy-17-ethynylestradiol, 2-methoxyestradiol 3-0-meth...