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E Hamel

Publications and source records attributed to E Hamel.

At least 19 recordsLinked to original sources
PubMed1997

Activities of the microtubule-stabilizing agents epothilones A and B with purified tubulin and in cells resistant to paclitaxel (Taxol(R)).

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.

Drug Resistance, Multiple↗
PubMed1997

Inhibition of angiogenesis and breast cancer in mice by the microtubule inhibitors 2-methoxyestradiol and taxol.

2-Methoxyestradiol (2-ME), an endogenous estrogen metabolite which disrupts microtubule function, has been shown to inhibit proliferating cells in vitro and suppress certain murine tumors in vivo. In vitro screening has determined that breast cancer cell lines are most sensitive to inhibition by 2-ME. Additionally, 2-ME has been shown to inhibit angiogenesis in vitro. We tested whether 2-ME suppresses cytokine-induced angiogenesis in vivo and inhibits growth of a human breast carcinoma in severe combined immunodeficient mice. A model of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF)-induced corneal neovascularization in C57BL/6 mice was used to evaluate the antiangiogenic effects of 2-ME and other microtubule inhibitors such as Taxol, vincristine, and colchicine. 2-ME (150 mg/kg p.o., n = 20) inhibited bFGF and VEGF-induced neovascularization by 39% and 54%, respectively. Taxol (6 mg/kg i.p., n = 17) inhibited bFGF and VEGF-induced neovascularization by 45% and 37%, respectively. Vincristine (0.2 mg/kg i.p., n = 8) and colchicine (0.25 mg/kg i.p., n = 8) had no effect. Treatment with 2-ME (75 mg/kg p.o., n = 9) for 1 month suppressed the growth of a human breast carcinoma in mice by 60% without toxicity. Recognition of the antiangiogenic and antitumor properties of 2-ME and Taxol may be crucial in planning clinical applications to angiogenesis-dependent diseases.

2-Methoxyestradiol↗
PubMed1996

Characterization of the interaction of cryptophycin 1 with tubulin: binding in the Vinca domain, competitive inhibition of dolastatin 10 binding, and an unusual aggregation reaction.

The antimitotic depsipeptide cryptophycin 1 (CP1) was compared to the antimitotic peptide dolastatin 10 (D10) as an antiproliferative agent and in its interactions with purified tubulin. The potent activity of CP1 as an inhibitor of cell growth was confirmed. The agent had an IC50 of 20 pM against L1210 murine leukemia cells versus 0.5 nM for D10. Both drugs were comparable as inhibitors of the glutamate-induced assembly of purified tubulin, with D10 being slightly more potent. CP1, like D10, was a noncompetitive inhibitor of the binding of [3H]vinblastine to tubulin (apparent Ki, 3.9 microM); and the depsipeptide was a competitive inhibitor of the binding of [3H]D10 to tubulin (apparent Ki, 2.1 microM). CP1 was less potent than D10 as an inhibitor of nucleotide exchange on tubulin, but the two drugs were equivalent in stabilizing the colchicine binding activity of tubulin. CP1, like D10, caused the formation of extensive structured aggregates of tubulin when present in stoichiometric amounts relative to the protein. Whereas at lower concentrations the drugs were equivalent in causing formation of small oligomers detected by gel permeation high-performance liquid chromatography, there were notable differences in the aggregation reactions induced by the two drugs. The electron micrographic appearance of the D10-induced aggregate differed substantially from that of the CP1-induced aggregate. With D10, but not CP1, aggregate morphology was greatly altered in the presence of microtubule-associated proteins. Finally, although CP1 caused the formation of massive aggregates, as did D10, there was little turbidity change with the depsipeptide as opposed to the peptide.

Antifungal Agents↗
PubMed1996

Ipsilateral alterations in tryptophan hydroxylase activity in rat brain after hypothalamic 5,7-di-hydroxytryptamine lesion.

The in vivo relationship between the amounts of tryptophan hydroxylase (TPH) protein and its intrinsic synthetic activity, measured by quantifying the amounts of alpha-[3H]methyl-5-hydroxytryptamine (alpha-[3H]M5-HT), is reported in cell body and terminal areas of intact and disturbed serotonergic neurons following a unilateral 5,7-dihydroxytryptamine (5,7-DHT) lesion of the dorsolateral hypothalamus. Five days after the lesion, the relationships between TPH and its synthetic product 5-HT were evaluated on adjacent brain sections in serotonergic cells bodies of the dorsal raphe nucleus (DRN) and nerve fibres of the medial forebrain bundle (MFB). On the side contralateral to the lesion, TPH and alpha-[3H]M5-HT levels in the intact hemi-DRN exhibited a caudo-rostral distribution and were positively and significantly correlated (p < or = 0.001); the calculated TPH-specific activity was 0.76 nCi of alpha-[3H]M5-HT formed per U TPH. In the MFB, quantitative measurements of TPH and alpha-[3H]M5-HT showed no correlation between enzyme and product and no specific activity for TPH could be determined. On the side ipsilateral to the lesion, the density of TPH-immunoreactive fibers was drastically decreased in the dorsolateral hypothalamus where a significant reduction in TPH content (45.5% of control side, P < 0.001) was found. In the overall ipsilateral hemi-DRN, TPH and alpha-[3H]M5-HT levels, their correlation as well as TPH-specific activity were unaltered by the lesion but a significant increase in alpha-[3H]M5-HT and TPH contents was observed in the lateral wings of the DRN. The lesion also induced a significant increase in alpha-[3H]M5-HT and TPH levels (136% and 93.8%, P < 0.001, respectively) in the ipsilateral MFB, which resulted in a positive and significant correlation between these two markers and yielded a TPH-specific activity of 1.0 nCi of alpha-[3H]M5-HT formed per U TPH. TPH topological area was also significantly increased in the lateral aspect of the ipsilateral MFB 5 days post lesion. These results show that 5-HT synthesis in the intact DRN is proportional to and dependent on TPH activity while in the MFB, 5-HT accumulation appears unrelated to TPH content which is most likely in an inactive enzymatic form. Moreover, the data show that a local disruption of serotonergic terminals in the dorsolateral hypothalamus does not affect 5-HT synthesis in the overall ipsilateral DRN neurons but results in local activation of TPH within the serotonergic projection neurons and the ipsilateral MFB, as evidenced by active de novo synthesis of 5-HT. Altogether the results point to circumscribed activation of compensatory mechanisms in 5-HT synthesis after selective destruction of serotonergic terminals.

5,7-Dihydroxytryptamine↗
PubMed1996

Identification of cysteine 354 of beta-tubulin as part of the binding site for the A ring of colchicine.

The colchicine analog 3-chloroacetyl-3-demthylthio-colchicine (3CTC) is a competitive inhibitor of colchicine binding to tubulin, binds to tubulin at 37 degrees C, but not at 0 degree C, and covalently reacts with beta-tubulin at 37 degree C, but not at 0 degree C, in a reaction inhibited by colchicine site drugs. The approximate intramolecular distance between the oxygen at position C-3 in 3CTC and the chlorine atom of the 3-chloroacetyl group is 3 A. using decylagarose chromatography, we purified beta-tubulin that had reacted with 3-(chloromethyl-[14C] Carbonyl)-3- demethylthiocolchicine ([14C]3CTC). This beta-tubulin that had reacted with 3-(chloromethyl-[14C]carbonyl)- 3-demethythiocolchicine ([14C]3CTC). This beta-tubulin was digested with formic acid, cyanogen bromide, endoproteinase Glu-C, or endoproteinase Lys-C, and the radio-labeled peptide(s) were isolated. The sequences of these peptides indicated that as much as 90% of the covalent reaction between the [14C]3CTC and beta-tubulin occurred at cysteine 354. This finding indicates that the C-3 oxygen atom of colchicinoids is within 3 A of the sulfur atom of the Cys-354 residue, suggests that the colchicine A ring lies between Cys-354 and Cys-239, based on the known 9 A distance between these residues, and may indicate that the tropolone C ring lies between the peptide region containing Cys-239 and the amino-terminal beta-tubulin sequence, based on the labeling pattern observed following direct photoactivation of tubulin-bound colchicine.

Amino Acid Sequence↗
PubMed1996

Interactions of 2-methoxyestradiol, an endogenous mammalian metabolite, with unpolymerized tubulin and with tubulin polymers.

2-Methoxyestradiol (2ME) is an endogenous mammalian catabolite of estradiol with antimitotic activity. Although it is a competitive inhibitor of the binding of colchicine to tubulin, it has unusual effects on glutamate-induced tubulin polymerization. Polymer that was little changed in morphology assembled at a reduced rate and was relatively cold stable. We have now examined interactions of [4-3H]-2ME with unpolymerized tubulin and polymer. The [3H]2ME binds avidly to tubulin even on ice, and it is readily displaced by other colchicine site drugs. An association rate constant on ice of 1.9 x 10(2) M-1s-1 was obtained. Scatchard analysis indicated a single class of binding site and an association equilibrium constant of 5.7 x 10(5) M-1. These values lead to a calculated dissociation rate constant of 3.3 x 10(-4) s-1. In glutamate-induced tubulin assembly, a reaction that requires GTP and leads to the formation of sheets of parallel protofilaments, increasing amounts of [3H]2ME were incorporated into polymer, reaching near-stoichiometry with tubulin at 100 microM 2ME. Equivalent binding of [3H]2ME occurred when the drug was added to preformed polymer, but binding of [3H]2ME to polymer was not readily inhibited by colchicine site drugs. Significant amounts of [3H]2ME were also incorporated into microtubule polymer formed with microtubule-associated proteins, glycerol, or 4-morpholineethanesulfonate buffer, but the stoichiometry was substantially lower than that in the sheet polymer induced by either glutamate or 1,4-piperazineethanesulfonate buffer. The structural differences between the microtubule and sheet polymers leading to these differences in apparent affinity for 2ME are unknown, but presumably interaction of the estrogen metabolite with cellular microtubules has functional significance related to the antimitotic properties of the compound.

2-Methoxyestradiol↗
PubMed1996

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.

Animals↗
PubMed1996

Discodermolide, a cytotoxic marine agent that stabilizes microtubules more potently than taxol.

Computer-assisted structure analysis indicated (+)-discodermolide, a polyhydroxylated alkatetraene lactone marine natural product, was an antimitotic compound, and we confirmed this prediction. Previous work had shown an accumulation of discodermolide-treated cells in the G2/M portion of the cell cycle, and we have now found that discodermolide arrests Burkitt lymphoma cells in mitosis. Discodermolide-treated breast carcinoma cells displayed spectacular rearrangement of the microtubule cytoskeleton, including extensive microtubule bundling. Microtubule rearrangement that occurred with 10 nM discodermolide required 1 microM taxol. Discodermolide had equally impressive effects on tubulin assembly in vitro. Near-total polymerization occurred at 0 degree C with tubulin plus microtubule-associated proteins (MAPs) under conditions in which taxol at an identical concentration was inactive. Without MAPs and/or without GTP, tubulin assembly was also more vigorous with discodermolide than with taxol under every reaction condition examined. Discodermolide-induced polymer differed from taxol-induced polymer in that it was completely stable at 0 degree C in the presence of high concentrations of Ca2+. In a quantitative assay designed to select for agents more effective than taxol in inducing assembly, discodermolide had an EC50 value of 3.2 microM versus 23 microM for taxol.

Alkanes↗
PubMed1996

Angiotensin II-induced fluid phase endocytosis in human cerebromicrovascular endothelial cells is regulated by the inositol-phosphate signaling pathway.

The involvement of the early signaling messengers, inositol tris-phosphate (IP3), intracellular calcium, [Ca2+]i, and protein kinase C (PKC), in angiotensin II (AII)-induced fluid phase endocytosis was investigated in human brain capillary and microvascular endothelial cells (HCEC). ALL (0.01-10 microM) stimulated the uptake of Lucifer yellow CH, an inert dye used as a marker for fluid phase endocytosis, in HCEC by 50-230%. AII also triggered a fast accumulation of IP3 and a rapid increase in [Ca2+]i in cells loaded with the Ca(2+)-responsive fluorescent dye fura-2. The prompt AII-induced [Ca2+]i spike was not affected by incubating HCEC in Ca(2+)-free medium containing 2 mM EGTA or by pretreating the cultures with the Ca2+ channel blockers, methoxyverapamil (D600; 50 microM), nickel (1 mM), or lanthanum (1 mM), suggesting that the activation of AII receptors on HCEC triggers the release of Ca2+ from intracellular stores. The AII-triggered increases in IP3, [Ca2+]i, and Lucifer yellow uptake were inhibited by the nonselective AII receptor antagonist, Sar1, Val5, Ala8-AII (SVA-AII), and by the phospholipase C (PLC) inhibitors, neomycin and U-73122. By contrast, the protein kinase C (PKC) inhibitors, staurosporine and calphostin C, failed to affect any of these AII-induced events. This study demonstrates that increased fluid phase endocytotosis induced by AII in human brain capillary endothelium, an event thought to be linked to the observed increases in blood-brain barrier permeability in acute hypertension, is likely dependent on PLC-mediated changes in [Ca2+]i and independent of PKC.

Angiotensin II↗
PubMed1996

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

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 microM) 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 microM 2-debenzoyl-2-meta-azidobenzoylpaclitaxel and 11 microM 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).

Animals↗
PubMed1996

Computational and molecular modeling evaluation of the structural basis for tubulin polymerization inhibition by colchicine site agents.

The computer-automated structure evaluation programs MultiCASE and CASE were used to perform a quantitative structure-activity relationship study on tubulin polymerization inhibitors. A learning set of 536 chemicals (202 active. 27 marginal, and 307 inactive), built using IC50 values for inhibition of tubulin polymerization or mitosis from this and previous studies, was used for artificial intelligence self-teaching. The algorithms successfully predicted the activity of agents in the learning set with > 90% accuracy. Seventeen MultiCASE and twelve CASE (mostly included in the MultiCASE set) biophores (substructures significantly correlated with activity) were identified with a probability > 0.95. Here we present the biophores of podophyllotoxins, colchicinoids, and certain combretastatins, each examined for structure-activity relationships. For the podophyllotoxins and colchicinoids in the learning set, the correlations between observed and predicted potencies were > 0.85. The algorithms recognized the importance of several known site, electronic, and steric effects in the two classes. A predictive QSAR (R2 = 0.98) was developed for combretastain A-2 and dihydrocombretastatin analogues. The MultiCASE/CASE analyzes were used in combination with molecular models to study relative orientations of colchicine, podophyllotoxin, combretastatin A-4, and steganacin at the colchicine site. This resulted in a new hypothesis, consistent with extensive published experimental data, in which the C-ring and part of the B-ring of colchicine overlap with the A- and B-rings of podophyllotoxin. Consequently, the trimethoxyphenyl rings of colchicine and podophyllotoxin occupied different regions of space, each pointing out from a hydrophobic 'core' occupied by the overlapping biophores. The molecular model of the highly potent combretastatin A-4 could fit into the model binding site in at least three different ways. The developed QSARs were used to identify the potent microtubule stabilizer discodermolide. Its identification, in concert with recently reported findings, suggest potential overlap in the colchicine and paclitaxel binding sites on tubulin.

Antineoplastic Agents, Phytogenic↗
PubMed1996

Serotonin in the regulation of brain microcirculation.

Manipulation of brainstem serotonin (5-HT) raphe neurons induces significant alterations in local cerebral metabolism and perfusion. The vascular consequences of intracerebrally released 5-HT point to a major vasoconstrictor role, resulting in cerebral blood flow (CBF) decreases in several brain regions such as the neocortex. However, vasodilatations, as well as changes in blood-brain barrier (BBB) permeability, which are blocked by 5-HT receptor antagonists also can be observed. A lack of relationship between the changes in flow and metabolism indicates uncoupling between the two variables and is suggestive of a direct neurogenic control by brain intrinsic 5-HT neurons on the microvascular bed. In line with these functional data are the close associations that exist between 5-HT neurons and the microarterioles, capillaries and perivascular astrocytes of various regions but more intimately and/or more frequently so in those where CBF is altered significantly following manipulation of 5-HT neurons. The ability of the microvascular bed to respond directly to intracerebrally released 5-HT is underscored by the expression of distinct 5-HT receptors in the various cellular compartments of the microvascular bed. Thus, it appears that while some 5-HT-mediated microvascular functions involve directly the blood vessel wall, others would be relayed through the perivascular astrocyte. The strategic localization of perivascular astrocytes and the different 5-HT receptors that they harbor strongly emphasize their putative pivotal role in transmitting information between 5-HT neurons and microvessels. It is concluded that the cerebral circulation has full capacity to adequately and locally adapt brain perfusion to changes in central 5-HT neurotransmission either directly or indirectly via the neuronal-astrocytic-vascular tripartite functional unit. Dysfunctions in these neurovascular interactions might result in perfusion deficits and might be involved in specific pathological conditions.

Animals↗
PubMed1996

Differential expression of sumatriptan-sensitive 5-hydroxytryptamine receptors in human trigeminal ganglia and cerebral blood vessels.

The efficacy of sumatriptan in migraine relief has been attributed to its interaction with 5-hydroxytryptamine1D (5-HT1D) receptors in cerebral blood vessels and/or on nerve endings of the trigeminovascular system in the dura mater. Using the high sensitivity of polymerase chain reaction (PCR) amplification, we investigated the expression of the sumatriptan-sensitive 5-HT receptors, namely, the 5-HT1D alpha, 5-HT1D beta, and 5-HT1F subtypes in human trigeminal ganglia (10 experiments) and cerebral blood vessels (seven experiments) obtained postmortem. Messages for the 5-HT1D alpha and 5-HT1D beta receptors were expressed in all except one of the 10 trigeminal ganglia studied. Expression of the 5-HT1F receptor was detected by gel electrophoresis of the PCR products in six ganglia and by Southern blot hybridization in two additional cases. In human brain vessels, message for the 5-HT1D beta receptor was present in all samples, whereas specific PCR products corresponding to the 5-HT1D alpha receptor could hardly be detected in only two preparations. PCR products indicative of the 5-HT1F receptor message were detected by gel electrophoresis in three brain vessel preparations and confirmed in the other four by Southern blot hybridization. Restriction mapping and sequence analysis of all PCR products identified the expected human 5-HT receptor DNA sequences. The data confirm that the 5-HT1D beta receptor is the dominant species in human cerebral blood vessels and further show that this receptor and the 5-HT1F are expressed in both neural and vascular tissues. In contrast, the data point to a preferential expression of 5-HT1D alpha receptors in neural versus vascular tissues and strongly reemphasize the need for selective 5-HT1D alpha agonists in the identification of the target tissue(s) for antimigraine drugs. Moreover, the data stress the importance to better understand the role of 5-HT1F receptors in cerebrovascular functions and dural inflammation and further raise interest regarding their possible involvement in migraine therapy.

Adult↗
PubMed1995

Interactions of tubulin with guanine nucleotides that have paclitaxel-like effects on tubulin assembly: 2',3'-dideoxyguanosine 5'-[alpha,beta-methylene]triphosphate, guanosine 5'-[alpha,beta-methylene]triphosphate, and 2',3'-dideoxyguanosine 5'-triphosphate.

Despite reduced affinity for the exchangeable nucleotide binding site of tubulin relative to GTP, 2',3'-dideoxyguanosine 5'-triphosphate (ddGTP) and guanosine 5'-[alpha, beta-methylene]triphosphate [pp(CH2)pG] are highly active in promoting tubulin assembly. Like the antimitotic drug paclitaxel, which interacts with the same part of the beta-tubulin molecule as exchangeable-site GTP, both analogs enhance nucleation reactions and promote formation hyperstable polymers. These observations led us to synthesize the doubly modified analog 2',3'-dideoxyguanosine 5'-[alpha, beta-methylene]triphosphate [pp(CH2)pddG]. We compared the effects of pp(CH2)pddG to those of ddGTP, pp(CH2)pG, and the three-cognate diphosphates in their interactions with tubulin. We found that pp(CH2)pddG was as active as ddGTP and pp(CH2)pG in supporting formation of polymer of increased stability, but that its affinity for the exchangeable site was lower than that of both singly modified analogs [relative affinities for the exchangeable site for pp(CH2)pddG:ddGTP:pp(CH2)-pG:GTP were 1:2.8:10:273]. There were significant differences in interactions of each of the three analogs with tubulin, and the behavior of pp(CH2)pddG was intermediate between that of ddGTP and that of pp(CH2)pG. Most importantly, under the reaction conditions studied, with heat-treated microtubule-associated proteins (MAPs) ddGTP-induced polymer consisted of short microtubules, while polymer formed with both pp(CH2)pddG and pp(CH2)pG consisted of short sheets. On the other hand, assembly without MAPs had a fivefold lower critical concentration for tubulin with ddGTP and pp(CH2)pddG (0.5 mg/ml) than with pp(CH2)pG (2.5 mg/ml). De novo assembly, which occurs readily with 2',3'-dideoxyguanosine 5'-diphosphate, was not observed with either alpha, beta-methylenediphosphate GDP analog.

Antineoplastic Agents↗
PubMed1995

The spongistatins, potently cytotoxic inhibitors of tubulin polymerization, bind in a distinct region of the vinca domain.

The highly cytotoxic, sponge-derived, antimitotic macrolide polyether spongistatin 1 has been previously shown to inhibit microtubule assembly, the binding of vinblastine and GTP to tubulin, and displacement of GDP bound in the exchangeable site of tubulin. We have now examined in detail inhibition by spongistatin 1 of both [3H]vinblastine and [3H]dolastatin 10 binding to tubulin. We found spongistatin 1 to be a noncompetitive inhibitor of the binding of both radiolabeled drugs to tubulin, in contrast to competitive patterns obtained with vincristine versus [3H]vinblastine and with a chiral isomer of dolastatin 10 versus [3H]dolastatin 10. Since dolastatin 10 is itself a noncompetitive inhibitor of vinca alkaloid binding to tubulin, this implies at least three distinct binding sites for the structurally complex and diverse natural products that interfere with each others binding to tubulin and with nucleotide exchange. Spongistatin 1, in contrast to both vinca alkaloids and peptide antimitotic agents like dolastatin 10, does not induce formation of a GTP-independent, morphologically distinctive polymer ("aggregate"). We also examined eight compounds closely related structurally to spongistatin 1 (spongistatins 2-9). The most distinctive in their properties were spongistatins 6 and 8. These two compounds, despite activity comparable to spongistatin 1 as inhibitors of tubulin polymerization and [3H]vinblastine binding, had much reduced activity as inhibitors of nucleotide exchange and [3H]dolastatin 10 binding. Spongistatins 1 and 6 were compared for effects on dolastatin 10-induced aggregate formation in conjunction with effects on [3H]dolastatin 10 binding. Spongistatin 6 was about 4-fold less active than spongistatin 1 as an inhibitor of aggregation and over 20-fold less active as an inhibitor of dolastatin 10 binding.

Animals↗
PubMed1995

Synthesis, antitubulin and antimitotic activity, and cytotoxicity of analogs of 2-methoxyestradiol, an endogenous mammalian metabolite of estradiol that inhibits tubulin polymerization by binding to the colchicine binding site.

In order to define the structural parameters associated with the antitubulin activity and cytotoxicity of 2-methoxyestradiol, a mammalian metabolite of estradiol, an array of analogs was synthesized and evaluated. The potencies of the new congeners as inhibitors of tubulin polymerization and colchicine binding were determined using tubulin purified from bovine brain, and the cytotoxicities of the new compounds were studied in a variety of cancer cell cultures. Maximum antitubulin activity was observed in estradiols having unbranched chain substituents at the 2-position with three non-hydrogen atoms. 2-Ethoxyestradiol and 2-((E)-1-propenyl)-estradiol were substantially more potent than 2-methoxyestradiol itself. The tubulin polymerization inhibitors in this series displayed significantly higher cytotoxicities in the MDA-MB-435 breast cancer cell line than in the other cell lines studied. The potencies of the analogs as cytotoxic and antimitotic agents in cancer cell cultures correlated with their potencies as inhibitors of tubulin polymerization, supporting the hypothesis that inhibition of tubulin polymerization is the mechanism of the cytotoxic action of 2-methoxyestradiol and its congeners. Several of the more potent analogs were tested in an estrogen receptor binding assay, and their affinities relative to estradiol were found to be very low.

2-Methoxyestradiol↗
PubMed1995

Antineoplastic agents. 291. Isolation and synthesis of combretastatins A-4, A-5, and A-6(1a)

The antineoplastic constituents of Combretum caffrum (Eckl. and Zeyh) Kuntze (Combretaceae family), a species indigenous to South Africa, have been investigated. Subsequently we isolated a series of closely related bibenzyls, stilbenes, and phenanthrenes from C. caffrum. Some of the stilbenes proved to be potent antimitotic agents which inhibited both tubulin polymerization and the binding of colchicine to tubulin. Combretastatin A-4 has been shown to be the most potent cancer cell growth inhibitor of the series. Presently this cis-stilbene is the most effective inhibitor of colchicine binding to tubulin and the simplest natural product yet described with such potent antitubulin effects. Combretastatin A-4, A-5, and A-6 were also found to inhibit growth of Neisseria gonorrhoeae. Details of the isolation and syntheses of combretastatins A-4 (2a), A-5 (2c), and A-6 (3a) have been described.

Anti-Bacterial Agents↗