Effect on Tubulin Polymerization

It was shown [100] that combretastatin A-4 (I), a potent inhibitor of tubulin polymerization (II), possessed growth inhibitory properties. 

Fourteen N-acetylated and nonacetylated 3,4,5-tri- or 2,5-dimethoxypyrazoline analogues with the same substituents as CA-4 was the most active compound in the series [102]. A cell-based assay indicated that compound I 

Conformationally restricted macrocyclic analogues of combretastatins have been evaluated as inhibitors of tubulin polymerization [103]. These compounds present a macrocyclic structure, in vhich the para positions of the aromatic moieties have been linked by a 5 - or 6-atoms chain, in order to produce a conformational restriction. This could contribute to detect the active conformation for these ligands. Such a conformational restriction and/or the steric hindrance made them less potent inhibitors than the model compound CA-4. 

An image-processing-based method to quantify the rate of extravasation of fluorescent contrast agents from tumor microvessels was developed, and the effect of combretastatin A-4-P 

Ophthalmic preparation containing combretastatin A-4 for treating diabetic retinopathy was patented [111]. The ophthalmic preparation was composed of combretastatin A-4 and other auxiliary materials acceptable for treating eye diseases. The authors claimed the preparation could be used as eye drop, ointment, and gel for treating diabetic retinopathy by inhibiting angiogenesis in a dose-dependent manner without affecting the development of retina vascular system. 

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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... 

The potency of the 3 -amino-4 -dimethylamino analogue against a panel of tumor cell lines (P-388, A-549, HT-29, MEL-28 or HeLa, HI 16, H-60) is even higher than that of Combretastatin A-4 or the related 3 -amino analogue. It is remarkable that this derivative, which is the most cytotoxic, has no effect on tubulin polymerization at concentrations below 30 )iM. This fact is in contrast to the usual behaviour of other combretastatin analogues, which always display a strong inhibitory effect on tubulin polymerization whenever they are cytotoxic in the submicromolar range. 

Grover S, Rimoldi JM, Molinero A A, Chaudhary AG, Kingston DGI, Hamel E (1995) Differential Effects of Paclitaxel (Taxol) Analogs Modified at Positions C-2, C-7, and C-3 on Tubulin Polymerization and Polymer Stabilization Identification of a Hyperactive Paclitaxel Derivative. Biochemistry 34 3927... 

In addition, the alkaloid colchicine (from Colchicum autumnale) blocks tubulin polymerization by binding to heterodimeric (3-tubulin between amino acids 239 and 254. Since it inhibits the MT-dependent migration of granulocytes into areas of inflammation and their MT-dependent release of proinflammatory agents, it is used to treat attacks of gout. Its antimitotic effect in the gastrointestinal system induces diarrhoea. Nocodazole competes for the binding site of colchicine and has similar effects on heterodimeric (3-tubulin. 

First we consider the acyl sector of the epothilones, which proved to be intolerant of modification. For example, inversion of stereochemistry at C3 (S toR), or reduction at C5 results in serious arrest of activity. Analogs with functionality at C3, C5, C6, C7 and C8 removed demonstrate both diminished tubulin-binding activity and cytotoxidty (structures not shown). Ddetion of the single methyl group at C8 has a highly pronounced deleterious effect on activity. Removal of the C9 methylene group resulting in a 15-membered macrolide, 87, results in a major loss of activity in tubulin polymerization/depolymerization assays. 

The cause of the cell cycle specificity of the bisindole alkaloids may be associated with the ability of these compounds to interact with the protein tubulin and thereby inhibit the polymerization (and depolymerization) of microtubules (16,17). In this respect the cellular pharmacology of vinca alkaloids is similar to that of other cytotoxic natural products such as colchicine or podophyllotoxin. On closer inspection, however, Wilson determined that the specific binding site on tubulin occupied by vinblastine or vincristine is chemically distinct from the site occupied by the other natural products (18). Subsequent experiments have determined that the maytansinoids, a class of ansa-macrocycles structurally distinct from the bisindoles, may bind to tubulin at an adjacent (or overlapping) site (19). A partial correlation of the antimitotic activity of these compounds with their tubulin binding properties has been made, but discrepancies in cellular uptake probably preclude any quantitative relationship of these effects (20). 

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). 

Vinflumine (Javlor ) is a second-generation Vinca alkaloid. It is more active than the nonfluorinated parent compound (vinorelbine) in several cancers (Figure 8.7). Vinflumine is currently in Phase III clinical trials as a chemotherapeutic agent against a variety of cancers (metastasic breast cancer, small cell lung cancer, and bladder cancer). This drug inhibits mitotic assembly, via inhibition of tubulin polymerization in microtubules, a major element of the cytoskeleton. Effects of fluorine substimtion on tubulin affinity or on metabolism are not responsible for the increased efficiency and decreased toxicity. The synthesis of vinflumine is reported in Chapter 4. ... 

Alterations in the cytoskeleton. The cytoskeleton depends on the intracellular Ca2+ concentration, which affects actin bundles, the interactions between actin and myosin and a-tubulin polymerization. The effect of increases in Ca2+ on the cytoskeletal attachments to the plasma membrane and the role of the cytoskeleton in cellular integrity have already been mentioned (see above). If the cytoskeleton is damaged or disrupted or its function altered by an increase in Ca2+, then blebs or protrusions appear on the plasma membrane (see below). As well as an increase in Ca2+, oxidation of, or reaction with sulfydryl groups, such as alkylation or arylation, for example, may disrupt the cytoskeleton, as thiols... 

Cyclic uptake and release of Ca2+ from the extracellular medium occur during mitosis in Physarum pofycephalum, and correlate with specific structural and kinetic events in the mitotic nuclei.442 The membrane system in the mitotic apparatus in Haemantkus endosperm cells functions in the localized release of Ca2+, so regulating the events of mitosis.443 It is known that calcium exerts effects on the stability of spindle microtubules. An alternative view is that free magnesium concentration acts as the fundamental regulator of the cell cycle.444 Tubulin polymerization depends on the presence of magnesium and the absence of calcium, and control of the Ca2+/Mg2+ ratio is relevant to spindle assembly. 

Most research has focused on the development of paclitaxel analogs or prodrugs with enhanced specificity MDR reversal and orally effective taxoids have also been developed recently. Meanwhile, scientists have gained insights into the mechanism of action of taxoids at molecular level, that is, binding sites on tubulin and dynamics of tubulin polymerization. It is worth pointing out that the SAR results derived from traditional medicinal chemistry have shown the essential... 

Since the discovery 117) of its action on tubulin 118) (the protein which, in the form of microtubules, constitutes the mitotic spindle), taxol has been of great utility to biologists. Numerous publications, not all of which need be cited here, describe the use of taxol for the isolation of tubulin from cellular preparations in which the concentration of this protein is too low to permit its polymerization, as in the pancreas 119) or the vegetal domain 120). Taxol has permitted not only the discovery of new microtubules in the Xenopus oocyte cortex 121) but also the study of the role of microtubules in certain cellular processes owing to its lack of destructive effects, in contrast to other known spindle poisons such as colchicine or vinblastine (722). Among other problems, taxol has helped in studies of the influence of the tubulin-microtubule equilibrium on the fluidity of platelet membranes 123) and of the function of the meiotic spindle in spermatocytes 124). 

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