Activities of the Microtubule-stabilizing Agents Epothilones A and B with Purified Tubulin and in Cells Resistant to Paclitaxel (Taxol®)
24 Jan 1997-Journal of Biological Chemistry (American Society for Biochemistry and Molecular Biology)-Vol. 272, Iss: 4, pp 2534-2541
TL;DR: The epothilones are competitive inhibitors of the binding of [3H]paclitaxel to tubulin polymers, and there were different proportions of various mitotic aberrations following treatment with different drugs.
About: This article is published in Journal of Biological Chemistry.The article was published on 1997-01-24 and is currently open access. It has received 520 citations till now. The article focuses on the topics: Epothilones & Epothilone.
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Abstract: Microbes have made a phenomenal contribution to the health and well-being of people throughout the world. In addition to producing many primary metabolites, such as amino acids, vitamins and nucleotides, they are capable of making secondary metabolites, which constitute half of the pharmaceuticals on the market today and provide agriculture with many essential products. This review centers on these beneficial secondary metabolites, the discovery of which goes back 80 years to the time when penicillin was discovered by Alexander Fleming.
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> …Throughout our evolution, the importance of natural products for medicine and health has been enormous
Owing …
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TL;DR: Natural products continue to provide greater structural diversity than standard combinatorial chemistry and so they offer major opportunities for finding novel low molecular weight lead structures that are active against a wide range of assay targets.
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TL;DR: The unification of taxane, epothilone, and sarcodictyin chemistries in a single pharmacophore provides a framework to study drug-tubulin interactions that should assist in the rational design of agents targeting tubulin.
Abstract: The epothilones are naturally occurring antimitotic drugs that share with the taxanes a similar mechanism of action without apparent structural similarity. Although photoaffinity labeling and electron crystallographic studies have identified the taxane-binding site on β-tubulin, similar data are not available for epothilones. To identify tubulin residues important for epothilone binding, we have isolated two epothilone-resistant human ovarian carcinoma sublines derived in a single-step selection with epothilone A or B. These epothilone-resistant sublines exhibit impaired epothilone- and taxane-driven tubulin polymerization caused by acquired β-tubulin mutations (β274Thr→Ile and β282Arg→Gln) located in the atomic model of αβ-tubulin near the taxane-binding site. Using molecular modeling, we investigated the conformational behavior of epothilone, which led to the identification of a common pharmacophore shared by taxanes and epothilones. Although two binding modes for the epothilones were predicted, one mode was identified as the preferred epothilone conformation as indicated by the activity of a potent pyridine-epothilone analogue. In addition, the structure–activity relationships of multiple taxanes and epothilones in the tubulin mutant cells can be fully explained by the model presented here, verifying its predictive value. Finally, these pharmacophore and activity data from mutant cells were used to model the tubulin binding of sarcodictyins, a distinct class of microtubule stabilizers, which in contrast to taxanes and the epothilones interact preferentially with the mutant tubulins. The unification of taxane, epothilone, and sarcodictyin chemistries in a single pharmacophore provides a framework to study drug–tubulin interactions that should assist in the rational design of agents targeting tubulin.
464 citations
References
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TL;DR: The SRB assay provides a sensitive measure of drug-induced cytotoxicity, is useful in quantitating clonogenicity, and is well suited to high-volume, automated drug screening.
Abstract: We have developed a rapid, sensitive, and inexpensive method for measuring the cellular protein content of adherent and suspension cultures in 96-well microtiter plates. The method is suitable for ordinary laboratory purposes and for very large-scale applications, such as the National Cancer Institute's disease-oriented in vitro anticancer-drug discovery screen, which requires the use of several million culture wells per year. Cultures fixed with trichloroacetic acid were stained for 30 minutes with 0.4% (wt/vol) sulforhodamine B (SRB) dissolved in 1% acetic acid. Unbound dye was removed by four washes with 1% acetic acid, and protein-bound dye was extracted with 10 mM unbuffered Tris base [tris (hydroxymethyl)aminomethane] for determination of optical density in a computer-interfaced, 96-well microtiter plate reader. The SRB assay results were linear with the number of cells and with values for cellular protein measured by both the Lowry and Bradford assays at densities ranging from sparse subconfluence to multilayered supraconfluence. The signal-to-noise ratio at 564 nm was approximately 1.5 with 1,000 cells per well. The sensitivity of the SRB assay compared favorably with sensitivities of several fluorescence assays and was superior to those of both the Lowry and Bradford assays and to those of 20 other visible dyes. The SRB assay provides a colorimetric end point that is nondestructive, indefinitely stable, and visible to the naked eye. It provides a sensitive measure of drug-induced cytotoxicity, is useful in quantitating clonogenicity, and is well suited to high-volume, automated drug screening. SRB fluoresces strongly with laser excitation at 488 nm and can be measured quantitatively at the single-cell level by static fluorescence cytometry.
9,019 citations
TL;DR: It is reported here that taxol acts as a promoter of calf brain microtubule assembly in vitro, in contrast to plant products such as colchicine and podophyllotoxin, which inhibit assembly.
Abstract: TAXOL (Fig. 1) was isolated from the plant Taxus brevifolia (western yew) by Wani et al., who reported that the molecule has antitumour activity in several experimental systems1. In our laboratory we have found that taxol, a low molecular weight neutral compound, completely inhibits division of exponentially growing HeLa cells at low concentrations of drug (0.25 µM) that have no significant effects on DNA, RNA or protein synthesis during a 4-h incubation with the cells. HeLa cells incubated with taxol for 20 h are blocked in late G2 and/or M (ref. 2). We report here that taxol acts as a promoter of calf brain microtubule assembly in vitro, in contrast to plant products such as colchicine and podophyllotoxin, which inhibit assembly. Taxol decreases the lag time for microtubule assembly and shifts the equilibrium for assembly in favour of the microtubule, thereby decreasing the critical concentration of tubulin required for assembly. Microtubules polymerised in the presence of taxol are resistant to depolymerisation by cold (4 °C) and CaCl2 (4 mM).
3,430 citations
TL;DR: It is reported here that microtubules in vitro coexist in growing and shrinking populations which interconvert rather infrequently and this dynamic instability is a general property of micro Tubules and may be fundamental in explaining cellular microtubule organization.
Abstract: We report here that microtubules in vitro coexist in growing and shrinking populations which interconvert rather infrequently. This dynamic instability is a general property of microtubules and may be fundamental in explaining cellular microtubule organization.
3,108 citations
Journal Article•
1,385 citations
Journal Article•
TL;DR: Epothilones represent a novel structural class of compounds, the first to be described since the original discovery ofTaxol, which not only mimic the biological effects of taxol but also appear to bind to the same microtubule-binding site as taxol.
Abstract: Tubulin polymerization into microtubules is a dynamic process, with the equilibrium between growth and shrinkage being essential for many cellular processes. The antineoplastic agent taxol hyperstabilizes polymerized microtubules, leading to mitotic arrest and cytotoxicity in proliferating cells. Using a sensitive filtration-calorimetric assay to detect microtubule nucleating activity, we have identified epothilones A and B as compounds that possess all the biological effects of taxol both in vitro and in cultured cells. The epothilones are equipotent and exhibit kinetics similar to taxol in inducing tubulin polymerization into microtubules in vitro (filtration, light scattering, sedimentation, and electron microscopy) and in producing enhanced microtubule stability and bundling in cultured cells. Furthermore, these 16-membered macrolides are competitive inhibitors of [3H]taxol binding, exhibiting a 50% inhibitory concentration almost identical to that of taxol in displacement competition assays. Epothilones also cause cell cycle arrest at the G2-M transition leading to cytotoxicity, similar to taxol. In contrast to taxol, epothilones retain a much greater toxicity against P-glycoprotein-expressing multiple drug resistant cells. Epothilones, therefore, represent a novel structural class of compounds, the first to be described since the original discovery of taxol, which not only mimic the biological effects of taxol but also appear to bind to the same microtubule-binding site as taxol.
1,188 citations