Epothilone stability

Fig. 24 Laulimalide stabilizes microtubules, but destabilizes zinc sheets. This multipart figure illustrates the effect of addition of laulimalide upon the polymerization of tubulin. Tubulin polymers, as sheets or microtubules, are inherently unstable and disassemble rapidly at low temperatures. Addition of paclitaxel (or epothilone, not shown) protect sheets from disassembly, a Control zinc sheets stabilized with PTX after 20 minutes in a bath of ice and water, b The effect of laulimalide under the same ice bath conditions as a. After 20 minutes, sheets exposed to laulimalide have significantly disassembled and reformed into microtubules, c Microtubules formed at room temperature from addition of laulimalide to zinc-stabilized sheets and incubated overnight. Almost all sheets have converted to microtubules (enlarged microtubules shown inset). By comparison, paclitaxel and epothilone-stabilized sheets remain intact under the same conditions, d Magnified microtubules seen in b. Figure provided by Huilin Li...

Bollag et al. (88)at the Merck Research Laboratories discovered that the epothilones stabilize microtubule assembly and thus inhibit cell division by the same mechanism as that of paclitaxel (see above). This observation, together with their less complex chemical structure, increased water solubility, more rapid action in vitro, and effectiveness against multidrug-resistant tumor cells, has prompted... 

Epothilones A, B and E (4,5 and 6) (Fig. 2) are representative members of a new class of bacterially derived natural products which exhibit potent biological activity. Isolated by Hofle and coworkers [6] from a soil sample collected near the Zambesi river, the compounds have provided a great deal of excitement in the scientific community due to their potent cytotoxicity against a number of multiple drug-resistant tumor cell lines and because of the mechanism by which they exert this effect. Like Taxol [7], the epothilones promote the combination of a- and 3-tubulin subunits and stabilize the resulting microtubule structures. This mode of action inhibits the cell division process and is, therefore, an attractive strategy for cancer chemotherapy [7,8]. 

Chemical Synthesis and Biological Studies of the Epothilones - Microtubule Stabilizing Agents with Enhanced Activity Against Multidrug-Resistant Cell Lines and Tumors. 

Fig. 2.1 Structures of the naturally occurring mictrotubule stabilizing compounds paclitaxel, epothilones A and B, discodermolide, and eleutherobin.

Epothilone B (38) Polyketide macrol- actone Patupilone (Epothilone B, EPO-906) (38) Oncology Microtubulin stabilization Phase 111 (ovarian cancer) Novartis 913,914... 

Epothilone D (195) -do- 9,10-Didehydro-epothilone D (KOS-1584) (196) Oncology Tubulin stabilization Phase PII Kosan (Memorial Sloan-Kettering) 919... 

Rothermel J, Wartmann M, Chen T, Hohneker J. (2003) EPO906 (epothilone B) A promising novel microtnbnle stabilizer. Semin Oncol 30 51-55. 

Epothilones A-E (3, 17, 48-50, Fig. 12) were isolated by Hofle and Reichen-bach and co-workers from the myxobacterium Sorangium cellulosum [80, 81]. After initial selection for their antifungal activity, the epothilones were soon shown to possess significant cytotoxicity against mammalian cells, epothilone B being the most active one [82]. Their mode of action was shown to be microtubule stabilization in a fashion very similar to that of... 

Molnar 1, Schupp T, Ono M, et al. (2000) The biosynthetic gene cluster for the microtubule-stabilizing agents epothilones A and B from Sorangium ceUulosum So ce90. Chem. Biol. 7 97-109. 

Epothilones are naturally occurring cytotoxic macrolides, which were initially isolated from a mycobacterium. Their antitumor activity is similar to that of the clinically established taxoids (Taxol, Taxotere), by interrupting the dynamic mechanism of microtubule assembly/disassembly via microtubule stabilization. In contrast to taxoids, epothilones are remarkably efficient against multidrug resistant cells. 

Kowalski RJ, Giannakakous P et al (1997) Activities of the microtubule-stabilizing agents epothilones A and B with purified tubulin and in cells resistant to paclitaxel (Taxol). J Biol Chem 272 2534-2541... 

Vinblastine (6.73) is an antimitotic drug that prevents polymerization of tubulin (Figure 6.26). When incubated with tubulin, vinblastine complexes in a 1 1 ratio with tubulin proteins. By blocking polymerization, vinblastine prevents microtubule formation and therefore mitosis. In contrast, paclitaxel (Taxol, 6.74) and epothilone B (6.75) stabilize aggregated tubulin. As a result, in the presence of paclitaxel and epothilone B, cells form static bundles of microtubules that are nonfunctional. Vinblastine and paclitaxel are both approved for clinical use against cancer. Ixabepilone (6.76), an analogue of epothilone B (6.75), has been approved by the FDA for treatment of certain forms of breast cancer. The European Medicines Agency (EMEA) did not approve ixabepilone out of concern over severe side effects.27... 

The natural product eleutherobin (1) was isolated in 1994 by Fenical et al. from a marine soft coral from an Eleutherobia species and its structure was elucidated shortly afterwards (Figure 1) [1]. Eleutherobin is a diterpene glycoside that possesses remarkable cytotoxicity against a wide variety of cancer cells, which is likely to be based on binding to tubulin and stabilization of microtubules [2, 3]. Mitosis is interrupted and the cell division cycle is terminated. The mechanism of action of eleutherobin is comparable to that of highly potent cytostatic agents such as paclitaxel (Taxol), nonataxel, epothilones, and discodermolide. 

The fourth and fifth chapters review the efforts and achievements made in the characterization of the structure of the complexes of tubulin with microtubules stabilizing agents by NMR (Chapter 4) and EM (Chapter 5). Especially evident is the discrepancy of the results obtained for epothilones, where the two techniques deliver radically different structures of the bound drug. Both NMR and EM models are, however, able to explain a consistent set of SAR data. The authors of the two chapters discuss critically the advantages and limitations of each methodology. 

The study of the tubulin-bound conformation of paclitaxel has resulted in a number of protein-ligand models, partially or fully based on the electron diffraction structure of aP-tubulin in paclitaxel-stabilized Zn2+-induced sheets [5, 12], Obviously, the nature of the paclitaxel binding site and the paclitaxel conformation in the binding site have key implications for the design of new MSA. A deep knowledge of the bioactive conformation would also help to explain how compounds as structurally diverse as the epothilones [48], discodermolide [49], and eleutherobin [50] have very similar mechanisms of action. 

Peluroside A was the first microtubule stabilizing agent whose conformation has been determined bound to microtubules (those of Paclitaxel and Epothilone were determined in non-microtubular tubulin [5, 12, 38, 91]). In the bound state, the NMR data, assisted by molecular mechanics calculations and docking experiments, indicated that only one (that present in water, B) of the two major conformations existing in water solution is bound to microtubules (a-tubulin). A model of the binding mode to tubulin has also been proposed [27], involving the a-tubulin monomer, in contrast with paclitaxel, which binds to the p-monomcr. 

Fig. 11 Three-dimensional structures of epothilones determined in different environments (O red, S yellow, N dark blue). Top structures of free EpoA determined by X-ray crystallography from dichloromethane/petroleum ether (top left [9 8] (a)) and from methanol/water (top right [143](b)). Bottom structures of EpoA bound to tubulin determined by solution NMR in aqueous medium (bottom left [96]) and by electron crystallography from zinc-stabilized tubulin sheets (bottom right [26]).(a) The crystal structure data have been available from the author to interested research groups since October 1995.(b) H.-J. Hecht, G. Hofle, unpublished results CCDC 241333 and CCDC 241334 contain the crystallographic data of this structure. These data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retiieving.html (or from the Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK fax (+ 44) 1223-336-033 or deposit cede. cam. ac. uk)...

The framework for this discussion will be formed around three important classes of MT stabilizers shown in Scheme 1. Given the chemical similarity between epothilones and laulimalide, it is particularly surprising that, while taxanes and epothilones compete for the same binding site, laulimalide has been shown to be non-competitive with either. In fact, laulimalide may bind simultaneously with taxanes and produce a synergistic effect. This chapter provides details of experiments and analyses we have done to formulate and test hypotheses about these binding mechanisms. 

Fig. 1 Electron diffraction pattern of Zn-sheet stabilized with epothilone A. Reflections extend to the edge of the image at 2.5 A resolution...

We have found that nearly all of the compounds that stabilize MTs also stabilize the 2-D crystals. Compounds that have been used include paclitaxel, epothilone-A and B, discodermolide and eleutherobin. Laulimalide, however, disrupts the sheets and causes them to reform into MTs. Microtubule destabilizing compounds have also been found to disrupt the crystals. 

Fig. 23 Bridging biological space. The overlap of epothilone B (cyan carbons) and PTX (green carbons) models derived from EC reveal shared anchors between the exchangeable nucleotide site through H227 and the truncated B9-B10 loop of the beta tubulin site. Perhaps rigidifying this vector across the site is of greater importance to the MT stabilizing effect than picking up interactions within the deep hydrophobic pocket...

Chart 9 Schematic representation of the principal microtubule stabilizing agents (MSAA) paclitaxel (10) and docetaxel (11), epothilones A-D (12-15), discodermolide (16), eleutherobin (17) and sarcodictyin (18), laulimalide (19) and peloruside A (20)... 

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