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PubMed2000

Isolation and characterization of the epothilone biosynthetic gene cluster from Sorangium cellulosum.

The epothilone biosynthetic gene cluster was isolated from Sorangium cellulosum strain SMP44. The gene cluster contains seven genes and spans approx. 56kb. The genes encoding the PKS, epoA, epoC, epoD, epoE, and epoF, are divided into nine modules. The EpoB protein is a non-ribosomal peptide synthetase (NRPS) that catalyzes formation of the thiazole found in the epothilones. EpoK is a P450 enzyme responsible for the epoxidation of epothilones C and D to epothilones A and B, respectively. EpoK was expressed in Escherichia coli, and the purified protein was shown to convert epothilone D to epothilone B in vitro.

Amino Acid Sequence↗

PubMed2000

Chemical synthesis and biological properties of pyridine epothilones.

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.

Antineoplastic Agents↗

PubMed2002

Enzymatic assembly of epothilones: the EpoC subunit and reconstitution of the EpoA-ACP/B/C polyketide and nonribosomal peptide interfaces.

The biosynthesis of epothilones, a family of hybrid polyketide (PK)/nonribosomal peptide (NRP) antitumor agents, provides an ideal system to study a hybrid PK/NRP natural product with significant biomedical value. Here the third enzyme involved in epothilone production, the five domain 195 kDa polyketide synthase (PKS) EpoC protein, has been expressed and purified from Escherichia coli. EpoC was combined with the first two enzymes of the epothilone biosynthesis pathway, the acyl carrier protein (ACP) domain of EpoA and EpoB, to reconstitute the early steps in epothilone biosynthesis. The acyltransferase (AT) domain of EpoC transfers the methylmalonyl moiety from methylmalonyl-CoA to the holo HS-acyl carrier protein (ACP) in an autoacylation reaction. The ketosynthase (KS) domain of EpoC decarboxylates the methylmalonyl-S-EpoC acyl enzyme to generate the carbon nucleophile that reacts with methylthiazolylcarboxyl-S-EpoB. The resulting condensation product can be reduced in the presence of NADPH by the ketoreductase (KR) domain of EpoC and then dehydrated by the dehydratase (DH) domain to produce the methylthiazolylmethylacrylyl-S-EpoC acyl enzyme intermediate that serves as the acyl donor for subsequent elongation of the epothilone chain. The acetyl-CoA donor can be replaced with propionyl-CoA, isobutyryl-CoA, and benzoyl-CoA and the acyl chains accepted by both EpoB and EpoC subunits to produce ethyl-, isopropyl-, and phenylthiazolylmethylacrylyl-S-EpoC acyl enzyme intermediates, suggesting that future combinatorial biosynthetic variations in epothilone assembly may be feasible. These results demonstrate in vitro reconstitution of both the PKS/NRPS interface (EpoA-ACP/B) and the NRPS/PKS interface (EpoB/C) in the assembly line for this antitumor natural product.

Acrylates↗

PubMed2004

EpoK, a cytochrome P450 involved in biosynthesis of the anticancer agents epothilones A and B. Substrate-mediated rescue of a P450 enzyme.

The epothilones are a new class of highly promising anticancer agents with a mode of action akin to that of paclitaxel but with distinct advantages over that drug. The principal natural compounds are epothilones A and B, which have an epoxide in the macrocyclic lactone ring, and C and D, which have a double bond instead of the epoxide group. The epoxidation of epothilones C and D to A and B, respectively, is mediated by EpoK, a cytochrome P450 enzyme encoded in the epothilone gene cluster. Here we report high-yield expression of EpoK, characterization of the protein, demonstration that the natural substrate can prevent-and even reverse-denaturation of the protein, identification of ligands and surrogate substrates, development of a high-throughput fluorescence activity assay based on the H(2)O(2)-dependent oxidation of 7-ethoxy-4-trifluoromethylcoumarin, and identification of effective inhibitors of the enzyme. These results will facilitate improvements in the yields of epothilones C and D and the engineering of EpoK to prepare novel epothilone analogues. Furthermore, the finding that the denatured enzyme is rescued by the substrate offers a potential paradigm for control of the P450 catalytic function.

Antineoplastic Agents↗

PubMed2005

Epothilone D affects cell cycle and microtubular pattern in plant cells.

Epothilones, macrocyclic lactones from culture filtrates of the myxobacterium Sorangium cellulosum, are known as taxol-like microtubular drugs in human medicine. To date, nothing is known about the effect of epothilones on microtubules (MTs) in plant cells and/or on the plant cell cycle. As shown in this report, the treatment of tomato cell suspension cultures with epothilone D produced a continuous increase in the mitotic index. Dose-response curves revealed that epothilone D alters the mitotic index at concentrations as low as 1.5 microM. Mitotic arrest was already visible after only 2 h of treatment, and 55% of the cells were arrested after 24 h. As shown by immunocytological methods, abnormal spindles are formed during metaphase, which leads to a random distribution of chromosomes in the whole cell and prevents the formation of a metaphase plate. The process of chromosome decondensation does not seem to be affected, because micronuclei form at the same place with the distributed chromosomes. This suggests that epothilone D influences the stability of plant MTs mainly during metaphase of the mitotic cycle. In metaphase, the effects of epothilone D seem to be irreversible, because cells with an abnormal spindle could not be recovered after removal of the drug.

Cell Cycle↗

PubMed2001

Chemical synthesis and biological evaluation of cis- and trans-12,13-cyclopropyl and 12,13-cyclobutyl epothilones and related pyridine side chain analogues.

The design, chemical synthesis, and biological evaluation of a series of cyclopropyl and cyclobutyl epothilone analogues (3-12, Figure 1) are described. The synthetic strategies toward these epothilones involved a Nozaki-Hiyama-Kishi coupling to form the C15-C16 carbon-carbon bond, an aldol reaction to construct the C6-C7 carbon-carbon bond, and a Yamaguchi macrolactonization to complete the required skeletal framework. Biological studies with the synthesized compounds led to the identification of epothilone analogues 3, 4, 7, 8, 9, and 11 as potent tubulin polymerization promoters and cytotoxic agents with (12R,13S,15S)-cyclopropyl 5-methylpyridine epothilone A (11) as the most powerful compound whose potencies (e.g. IC(50) = 0.6 nM against the 1A9 ovarian carcinoma cell line) approach those of epothilone B. These investigations led to a number of important structure-activity relationships, including the conclusion that neither the epoxide nor the stereochemistry at C12 are essential, while the stereochemistry at both C13 and C15 are crucial for biological activity. These studies also confirmed the importance of both the cyclopropyl and 5-methylpyridine moieties in conferring potent and potentially clinically useful biological properties to the epothilone scaffold.

Antineoplastic Agents↗

PubMedDate not supplied

Assessment of fed-batch, semicontinuous, and continuous epothilone D production processes.

Epothilone D is a member of a class of potent antineoplastic natural products produced by myxobacteria. Previously, we have described a fed-batch epothilone D production process in which an adsorber resin is incorporated into the bioreactor setup to capture and stabilize the product in situ, preventing its degradation within the bioreactor. The capture of epothilone D by these relatively large resin beads enables the development of continuous and semicontinuous culturing systems incorporating bead retention mechanisms to completely retain the product within the bioreactor, increasing the epothilone D product titer by almost 3-fold in both cases over a baseline fed-batch system. These product retention strategies, described here for production of the epothilones, are generally applicable to any system using adsorber resins as a method to capture product during a microbial cultivation.

Epothilones↗

PubMed2001

On the interactivity of complex synthesis and tumor pharmacology in the drug discovery process: total synthesis and comparative in vivo evaluations of the 15-aza epothilones.

The total syntheses of 12,13,15-desoxy-15(S)-aza-epothilone B (aza-dEpoB; dEpoB-lactam) and 12,13,15-desoxy-15(R)-aza-epothilone B (15-epi-aza-dEpoB; 15-epi-dEpoB-lactam) have been accomplished via a highly convergent strategy. We have also successfully oxidized 12,13,15-desoxy-15(S)-aza-epothilone B to aza-epothilone B (aza-EpoB; EpoB-lactam). Aza-epothilone B has been advanced to phase I clinical trials by the Bristol-Myers Squibb group. Our synthesis is efficient and was amenable to the production of significant quantities of these lactams. Using our fully synthetically derived lactams, in vitro and in vivo studies were conducted in comparison with advanced clinical candidates, 12,13-desoxyepothilone B and 12,13-desoxyepothilone F, also derived by total synthesis.

Animals↗

PubMed2002

A new cytotoxic epothilone from modified polyketide synthases heterologously expressed in Myxococcus xanthus.

A new epothilone, 10,11-didehydroepothilone D (5), was isolated from a strain of the heterologous host Myxococcus xanthus genetically engineered to produce epothilone D (4). The structure of 5 was determined from NMR and MS data. The epothilone polyketide synthase was further modified in a recombinant M. xanthus strain to produce 5 as the major epothilone-related metabolite. The cytotoxicity of 5 against a panel of tumor cell lines, including several with multidrug resistance, and its effect on tubulin polymerization were comparable to epothilone D (4).

Antineoplastic Agents↗

PubMed2005

The epothilones and related analogues-a review of their syntheses and anti-cancer activities.

The macrocylic polyketide class of compounds known as the epothilones has generated substantial interest over the last few years in the areas of chemistry, biology, and medicine due to their interesting structure and, more importantly, their activity against numerous cancer cell lines, including drug-resistant, especially Taxol-resistant, cancer cell lines. To date, numerous total syntheses have been published, hundreds of epothilone analogues have been synthesized, and detailed structure activity relationship studies have been conducted. The purpose of this review is to give a brief summary of the latest advances made concerning the epothilones. Recent total or partial syntheses will be presented along with the syntheses of new epothilone analogues and their corresponding biological data. In addition, we will look at the current state of research into an economically viable method for the biosynthesis of the epothilones and related analogues.

Animals↗

PubMed2005

Activity of epothilones.

Epothilones represent a novel class of anticancer drugs which inhibit the cell cycle and strongly influence cell division. The biological activity of epothilones is associated with their capacity to bind to the protein tubulin of microtubules and to disturb the dynamic equilibrium between microtubule assembling and disassembling. Consequently, in dividing cells, it leads to mitotic arrest and to apoptotic cell death. A number of epothilones demonstrate a potent inhibitory effect on cell proliferation in human tumor cell lines, as well as antitumor activity in experimental animals bearing human tumor xenografts. This review will focus on the recent preclinical studies of the in vitro and in vivo activities of four epothilones, EPO-906, BMS-247550, KOS-862 and BMS-310705. Several epothilones are undergoing clinical evaluation and are promising candidates for advanced cancer therapy.

Animals↗

PubMed1995

Epothilones, a new class of microtubule-stabilizing agents with a taxol-like mechanism of action.

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.

Animals↗

PubMed2002

Optimizing the heterologous production of epothilone D in Myxococcus xanthus.

The heterologous production of epothilone D in Myxococcus xanthus was improved by 140-fold from an initial titer of 0.16 mg/L with the incorporation of an adsorber resin, the identification of a suitable carbon source, and the implementation of a fed-batch process. To reduce the degradation of epothilone D in the basal medium, XAD-16 (20 g/L) was added to stabilize the secreted product. This greatly facilitated its recovery and enhanced the yield by three-fold. The potential of using oils as a carbon source for cell growth and product formation was also evaluated. From a screen of various oils, methyl oleate was shown to have the greatest impact. At the optimal concentration of 7 mL/L in a batch process, the maximum cell density was increased from 0.4 g dry cell weight (DCW)/L to 2 g DCW/L. Product yield, however, depended on the presence of trace elements in the production medium. With an exogenous supplement of trace metals to the basal medium, the peak epothilone D titer was enhanced eight-fold. This finding demonstrates the significant role of metal ions in cell metabolism and in epothilone biosynthesis. To further increase the product yield, a continuous fed-batch process was used to promote a higher cell density and to maintain an extended production period. The optimized fed-batch cultures consistently yielded a cell density of 7 g DCW/L and an average production titer of 23 mg/L.

Drug Stability↗

PubMed2003

Nutrient regulation of epothilone biosynthesis in heterologous and native production strains.

Fermentation media with different initial concentrations of ammonium and phosphate salts were used to study the inhibitory effects of those ions on growth and production of epothilone in Sorangium cellulosum and Myxococcus xanthus. The native epothilone producer, S. cellulosum was more sensitive to ammonium and phosphate than the heterologous producer, M. xanthus. An ammonium concentration of 12 mM reduced epothilone titers by 90% in S. cellulosum but by only 40% in M. xanthus. When 5 mM phosphate was added to the medium, production in both strains was 60% lower. Higher phosphate concentrations had little additional effect on M. xanthus titers, but epothilone production with 17 mM extra-cellular phosphate in S. cellulosum was 95% lower than in the control condition. The effect of iron supplementation to the fermentation medium was also investigated. Both strains showed best production with 20 microM iron added to the medium.

Antineoplastic Agents↗

PubMed2001

Epothilone biosynthesis: assembly of the methylthiazolylcarboxy starter unit on the EpoB subunit.

BACKGROUND: Polyketides (PKs) and non-ribosomal peptides (NRPs) are therapeutically important natural products biosynthesized by multimodular protein assembly lines, termed the PK synthases (PKSs) and NRP synthetases (NRPSs), via a similar thiotemplate-mediated mechanism. The potential for productive interaction between these two parallel enzymatic systems has recently been demonstrated, with the discovery that PK/NRP hybrid natural products can be of great therapeutic importance. One newly discovered PK/NRP product, epothilone D from Sorangium cellulosum, has shown great potential as an anti-tumor agent. RESULTS: The chain-initiating methylthiazole ring of epothilone has been generated in vitro as an acyl-S-enzyme intermediate, using five domains from two modules of the polymodular epothilone synthetase. The acyl carrier protein (ACP) domain, excised from the EpoA gene, was expressed in Escherichia coli, purified as an apo protein, and then post-translationally primed with acetyl-CoA using the phosphopantetheinyl transferase enzyme Sfp. The four-domain 150-kDa EpoB subunit (cyclization-adenylation-oxidase-peptidyl carrier protein domains: Cy-A-Ox-PCP) was also expressed and purified in soluble form from E. coli. Post-translational modification with Sfp and CoASH introduced the HS-pantP prosthetic group to the apo-PCP, enabling subsequent loading with L-cysteine to generate the Cys-S-PCP acyl enzyme intermediate. When acetyl-S-ACP (EpoA) and cysteinyl-S-EpoB were mixed, the Cy domain of EpoB catalyzed acetyl transfer from EpoA to the amino group of the Cys-S-EpoB, generating a transient N-Ac-Cys-S-EpoB intermediate that is cyclized and dehydrated to the five-membered ring methylthiazolinyl-S-EpoB. Finally, the FMN-containing Ox domain of EpoB oxidized the dihydro heterocyclic thiazolinyl ring to the heteroaromatic oxidation state, the methylthiazolylcarboxy-S-EpoB. When other acyl-CoAs were substituted for acetyl-CoA in the Sfp-based priming of the apo-CP domain, additional alkylthiazolylcarboxy-S-EpoB acyl enzymes were produced. CONCLUSIONS: These experiments establish chain transfer across a PKS and NRPS interface. Transfer of the acetyl group from the ACP domain of EpoA to EpoB reconstitutes the start of the epothilone synthetase assembly line, and installs and converts a cysteine group into a methyl-substituted heterocycle during this natural product chain growth.

Acyl Coenzyme A↗

PubMed2004

Elucidating the mechanism of cis double bond formation in epothilone biosynthesis.

The epothilones, originally isolated from the myxobacterium Sorangium cellulosum, are macrocyclic compounds that are synthesized by a modular polyketide synthase, an enzyme complex composed of six large, multifunctional proteins. The penultimate intermediates in epothilone production, and the products of the PKS-catalyzed reactions, are epothilones D and C, which contain a 12,13-cis-double bond. The 12 and 13 positions of epothilones are generated during the fourth elongation step that is governed by module 4. Module 4 does not contain a dehydratase (DH) domain, which is required for dehydration to create the double bond. A DH domain, present in module 5 and presumed to act in the fifth elongation step at the 10 and 11 positions, was proposed to act as well to generate the 12,13-cis-double bond. Inactivation of the DH domain in module 5 resulted in the production of 10,11-dehydro-13-hydroxyepothilone D as the major product, confirming that DH5 is required for 12,13 dehydration. A mechanistic model based on domain skipping and modular stuttering is presented to explain the basis for the iterative DH5 activity observed.

Enzyme Activation↗

PubMed2004

Precursor-directed biosynthesis of epothilone in Escherichia coli.

Engineered biosynthetic pathways provide a powerful method for generating complex molecules. Precursor-directed biosynthesis, which combines chemical synthesis and enzymatic transformations, allows non-native starting materials to be incorporated into biosynthetic pathways. Using this approach, we achieved the production of the anticancer agent epothilone C in Escherichia coli. An E. coli strain was engineered to express the last three modules of the epothilone biosynthetic pathway (epoD-M6, epoE, and epoF) and the substrate required to complement the biosynthetic enzymes was obtained by chemical synthesis. Under high-density cell culture conditions, the E. coli strain processed exogenously fed synthetic substrate into epothilone C at levels comparable to the native host (1 mg/L) and at higher levels than other heterologous hosts. Importantly, this precursor-directed approach will allow chemical modifications to be introduced into the polyketide backbone and may ultimately provide access to epothilone analogues with improved pharmacological properties in quantities sufficient for clinical development.

Antibiotics, Antineoplastic↗

PubMed2003

Conformation-activity relationships in polyketide natural products. Towards the biologically active conformation of epothilone.

The conformation-activity relationships for the biologically active polyketide, epothilone, have been determined. Computer-based molecular modeling and high field NMR techniques have provided the solution preferences for epothilones and. For the C1-C8 polypropionate region, two conformational families, conformers 1 and 2, have been identified as having significant populations in polar and non-polar solvents. In the C11-C15 region, additional flexibility was observed and two local conformations have been identified as important, conformers 3 and 4. Epothilone analogues with altered conformational profiles have been designed and synthesized. Conformational analysis and the results of biological assays have been correlated to provide increased understanding of the biologically active conformation for the epothilone class of natural product. Conformation-activity relationships have been shown to be an important complement to structure-activity data.

Drug Design↗