A common pharmacophore for cytotoxic natural products that stabilize microtubules.
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Taxol (paclitaxel), a complex diterpene obtained from the Pacific yew, Taxus brevifolia, is arguably the most important new drug in cancer chemotherapy. The mechanism of cytotoxic action for paclitaxel-i.e., the stabilization of microtubules leading to mitotic arrest-is now shared by four recently identified natural products, eleutherobin, epothilones A and B, and discodermolide. Their ability to competitively inhibit [3H]paclitaxel binding to microtubules strongly suggests the existence of a common binding site. Recently, we have developed nonaromatic analogues of paclitaxel that maintain high cytotoxicity and tubulin binding (e.g., nonataxel). We now propose a common pharmacophore that unites paclitaxel, nonataxel, the epothilones, eleutherobin, and discodermolide, and rationalizes the extensive structure-activity relationship data pertinent to these compounds. Insights from the common pharmacophore have enabled the development of a hybrid construct with demonstrated cytotoxic and tubulin-binding activity. (+info)
Cloning and heterologous expression of the epothilone gene cluster.
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The polyketide epothilone is a potential anticancer agent that stabilizes microtubules in a similar manner to Taxol. The gene cluster responsible for epothilone biosynthesis in the myxobacterium Sorangium cellulosum was cloned and completely sequenced. It encodes six multifunctional proteins composed of a loading module, one nonribosomal peptide synthetase module, eight polyketide synthase modules, and a P450 epoxidase that converts desoxyepothilone into epothilone. Concomitant expression of these genes in the actinomycete Streptomyces coelicolor produced epothilones A and B. Streptomyces coelicolor is more amenable to strain improvement and grows about 10-fold as rapidly as the natural producer, so this heterologous expression system portends a plentiful supply of this important agent. (+info)
The biosynthetic gene cluster for the microtubule-stabilizing agents epothilones A and B from Sorangium cellulosum So ce90.
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BACKGROUND: Epothilones are produced by the myxobacterium Sorangium cellulosum So ce90, and, like paclitaxel (Taxol((R))), they inhibit microtubule depolymerisation and arrest the cell cycle at the G2-M phase. They are effective against P-glycoprotein-expressing multiple-drug-resistant tumor cell lines and are more water soluble than paclitaxel. The total synthesis of epothilones has been achieved, but has not provided an economically viable alternative to fermentation. We set out to clone, sequence and analyze the gene cluster responsible for the biosynthesis of the epothilones in S. cellulosum So ce90. RESULTS: A cluster of 22 open reading frames spanning 68,750 base pairs of the S. cellulosum So ce90 genome has been sequenced and found to encode nine modules of a polyketide synthase (PKS), one module of a nonribosomal peptide synthetase (NRPS), a cytochrome P450, and two putative antibiotic transport proteins. Disruptions in the genes encoding the PKS abolished epothilone production. The first PKS module and the NRPS module are proposed to co-operate in forming the thiazole heterocycle of epothilone from an acetate and a cysteine by condensation, cyclodehydration and subsequent dehydrogenation. The remaining eight PKS modules are responsible for the elaboration of the rest of the epothilone carbon skeleton. CONCLUSIONS: The overall architecture of the gene cluster responsible for epothilone biosynthesis has been determined. The availability of the cluster should facilitate the generation of designer epothilones by combinatorial biosynthesis approaches, and the heterologous expression of epothilones in surrogate microbial hosts. (+info)
A common pharmacophore for epothilone and taxanes: molecular basis for drug resistance conferred by tubulin mutations in human cancer cells.
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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 beta-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 beta-tubulin mutations (beta274(Thr-->Ile) and beta282(Arg-->Gln)) located in the atomic model of alphabeta-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. (+info)
Taxol and discodermolide represent a synergistic drug combination in human carcinoma cell lines.
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Recently, three natural products have been identified, the epothilones, eleutherobin, and discodermolide, whose mechanism of action is similar to that of Taxol in that they stabilize microtubules and block cells in the mitotic phase of the cell cycle. In this report, we have compared and contrasted the effects of these new agents in Taxol-sensitive and -resistant cell lines. We also have taken advantage of a human lung carcinoma cell line, A549-T12, that was isolated as a Taxol-resistant cell line and found to require low concentrations of Taxol (2-6 nM) for normal cell division. This study then examined the ability of these new compounds to substitute for Taxol in sustaining the growth of A549-T12 cells. Immunofluorescence and flow cytometry have both indicated that the epothilones and eleutherobin, but not discodermolide, can substitute for Taxol in this Taxol-dependent cell line. In A549-T12 cells, the presence of Taxol significantly amplified the cytotoxicity of discodermolide, and this phenomenon was not observed in combinations of Taxol with either the epothilones or eleutherobin. Median effect analysis using the combination index method revealed a schedule-independent synergistic interaction between Taxol and discodermolide in four human carcinoma cell lines, an effect that was not observed between Taxol and epothilone B. Flow cytometry revealed that concurrent exposure of A549 cells to Taxol and discodermolide at doses that do not induce mitotic arrest caused an increase in the hypodiploid population, thereby indicating that a possible mechanism for the observed synergy is the potentiation of apoptosis. Our results suggest that Taxol and discodermolide may constitute a promising chemotherapeutic combination. (+info)
Chemical synthesis and biological properties of pyridine epothilones.
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BACKGROUND: Numerous analogs of the antitumor agents epothilones A and B have been synthesized in search of better pharmacological profiles. Insights into the structure-activity relationships within the epothilone family are still needed and more potent and selective analogs of these compounds are in demand, both as biological tools and as chemotherapeutic agents, especially against drug-resistant tumors. RESULTS: A series of pyridine epothilone B analogs were designed, synthesized and screened. The synthesized compounds exhibited varying degrees of tubulin polymerization and cytotoxicity properties against a number of human cancer cell lines depending on the location of the nitrogen atom and the methyl substituent within the pyridine nucleus. CONCLUSIONS: The biological screening results in this study established the importance of the nitrogen atom at the ortho position as well as the beneficial effect of a methyl substituent at the 4- or 5-position of the pyridine ring. Two pyridine epothilone B analogs (i.e. compounds 3 and 4) possessing higher potencies against drug-resistant tumor cells than epothilone B, the most powerful of the naturally occurring epothilones, were identified. (+info)
Studies on the biosynthesis of epothilones: the PKS and Epothilone C/D monooxygenase.
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Nonproducer mutants support the assumption that epothilones A and B are synthesized by the same polyketide synthase (PKS). The endproducts of the PKS, epothilones C and D, compete for the active site of a constitutively synthesized monooxygenase which is regulated by product inhibition. The postulated C-13 hydroxy-epothilones as direct precursors of epothilones C and D were not detected. (+info)
BMS-247550: a novel epothilone analog with a mode of action similar to paclitaxel but possessing superior antitumor efficacy.
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BMS-247550, a novel epothilone derivative, is being developed by Bristol-Myers Squibb Company (BMS) as an anticancer agent for the treatment of patients with malignant tumors. BMS-247550 is a semisynthetic analogue of the natural product epothilone B and has a mode of action analogous to that of paclitaxel (i.e., microtubule stabilization). In vitro, it is twice as potent as paclitaxel in inducing tubulin polymerization. Like paclitaxel, BMS-247550 is a highly potent cytotoxic agent capable of killing cancer cells at low nanomolar concentrations. Importantly, BMS-247550 retains its antineoplastic activity against human cancers that are naturally insensitive to paclitaxel or that have developed resistance to paclitaxel, both in vitro and in vivo. Tumors for which BMS-247550 demonstrated significant antitumor activity encompass both paclitaxel-sensitive and -refractory categories, i.e., (a) paclitaxel-resistant: HCT116/VM46 colorectal (multidrug resistant), Pat-21 breast and Pat-7 ovarian carcinoma (clinical isolates; mechanisms of resistance not fully known), and A2780Tax ovarian carcinoma (tubulin mutation); (b) paclitaxel-insensitive: Pat-26 human pancreatic carcinoma (clinical isolate) and M5076 murine fibrosarcoma; and (c) paclitaxel sensitive: A2780 ovarian, LS174T, and HCT116 human colon carcinoma. In addition, BMS-247550 is p.o. efficacious against preclinical human tumor xenografts grown in immunocompromised mice or rats. Schedule optimization studies indicate that BMS-247550 is efficacious when administered frequently (every 2 days x 5) or intermittently (every 4 days x 3 or every 8 days x 2). These efficacy data demonstrate that BMS-247550 has the potential to surpass Taxol in both clinical efficacy and ease of use (i.e., less frequent treatment schedule and/or oral administration). (+info)