Microtubule Interactions with Chemically Diverse Stabilizing Agents: Thermodynamics of Binding to the Paclitaxel Site Predicts Cytotoxicity (original) (raw)
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The Journal of biological chemistry, 1997
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 ac...
Chemistry & Biology, 2004
gicas their good activity against ovarian, metastatic breast, head and neck, and lung cancer [4], paclitaxel has two Consejo Superior de Investigaciones Científicas Ramiro de Maeztu 9 factors that hamper its applicability. First, its low aqueous solubility, and second, the development of pleiotro-28040 Madrid Spain pic drug resistance mediated both by the overexpression of the P-glycoprotein [5, 6] and the presence of mutations in -tubulin [7, 8]. The discovery in recent years of several natural sub-Atlanta, Georgia 30322 3 Department of Chemistry and stances with a paclitaxel-like mechanism of action (epothilone, discodermolide, laulimalide, eleutherobin, The Skaggs Institute for Chemical Biology The Scripps Research Institute peloruside, dictyostatin-1, taccalonolide, and jatrophane polyesters; [9-16]) opened new possibilities in 10550 North Torrey Pines Road La Jolla, California 92037 the field. Of these compounds, the first one recognized as having a paclitaxel-like activity was an already known natural compound called epothilone [9], a secondary metabolite from the soil myxobacterium Sorangium cel-9500 Gilman Drive La Jolla, California 92093 lulosum [17-19]. Epothilones are the most promising of this group of new paclitaxel-like compounds because they offer several advantages. First of all, while most of the other compounds are isolated from marine organ-Summary isms in limited amounts, epothilone B can be obtained in kilogram amounts by fermentation [20]. Second, it The interactions of epothilone analogs with the paclihas higher solubility in water than paclitaxel [19]. Third, taxel binding site of microtubules were studied. The
Pharmacophore Models of Paclitaxel- and Epothilone-Based Microtubule Stabilizing Agents
Bulletin of the Korean Chemical Society, 2013
Microtubules play an important role in intracellular transport, mobility, and particularly mitosis. Paclitaxel (Taxol TM) and paclitaxel-like compounds have been shown to be anti-tumor agents useful for various human tumors. Paclitaxel-like compounds operate by stabilizing microtubules through interface binding at the interface between two β-tubulin monomers in adjacent protofilaments. In this paper we present the elucidation of the structural features of paclitaxel and paclitaxel-like compounds (e.g., epothilones) with microtubule stabilizing activities, and relate their activities to spatial and chemical features of the molecules. CATALYST program was used to generate three-dimensional quantitative structure activity relationships (3D-QSARs) resulting in 3D pharmacophore models of epothilone-and paclitaxel-derivatives. Pharmacophore models were generated from diverse conformers of these compounds resulting in a high correlation between experimental and predicted biological activities (r = 0.83 and 0.91 for epothilone and paclitaxel derivatives, respectively). On the basis of biological activities of the training sets, five-and four-feature pharmacophore hypotheses were generated in the epothilone and paclitaxel series. The validation of generated hypotheses was achieved by using twelve epothilones and ten paclitaxels, respectively, which are not in the training sets. The clustering (grouping) and merging techniques were used in order to supplement spatial restrictions of each of hypothesis and to develop more comprehensive models. This approach may be of use in developing novel inhibitor candidates as well as contributing a better understanding of structural characters of many compounds useful as anticancer agents targeting microtubules.
Mechanism of Action of Antitumor Drugs that Interact with Microtubules and Tubulin
Current Medicinal Chemistry-Anti-Cancer Agents, 2012
Microtubules, major structural components in cells, are the target of a large and diverse group of natural product anticancer drugs. Given the success of this class of drugs in cancer treatment, it can be argued that microtubules represent the single best cancer target identified to date. Microtubules are highly dynamic assemblies of the protein tubulin. They readily polymerize and depolymerize in cells, and they undergo two interesting kinds of dynamics called dynamic instability and treadmilling. These dynamic behaviors are crucial to mitosis, the process of chromosomal division to form new cells. Microtubule dynamics are highly regulated during the cell cycle by endogenous cellular regulators. In addition, many antitumor drugs and natural compounds alter the polymerization dynamics of microtubules, blocking mitosis, and consequently, inducing cell death by apoptosis. These drugs include several that inhibit microtubule polymerization at high drug concentrations, namely, the Vinca alkaloids, cryptophycins, halichondrins, estramustine, and colchicine. Another group of these compounds stimulates microtubule polymerization and stabilizes microtubules at high concentrations. These include Taxol™, Taxotere™, eleutherobins, epothilones, laulimalide, sarcodictyins, and discodermolide. Importantly, considerable evidence indicates that, at lower concentrations, these drugs have a common mechanism of action; they suppress the dynamics of microtubules without appreciably changing the mass of microtubules in the cell. The drugs bind to diverse sites on tubulin and at different positions within the microtubule, and they have diverse effects on microtubule dynamics. However, by their common mechanism of suppression microtubule dynamics, they all block mitosis at the metaphase/anaphase transition, and induce cell death. I. MICROTUBULES AS TARGETS FOR ANTI-CANCER DRUGS Microtubules are major dynamic structural components in cells. They are important in the development and maintenance of cell shape, in cell reproduction and division, in cell signaling, and in cellular movement [1]. Microtubules are the target of a diverse group of anticancer drugs, most of which are derived from natural products. Given the success of this class of drugs, the mitotic inhibitors, it can be argued that microtubules represent the single best cancer target identified to date [2] [3]. Microtubules are highly dynamic polymers of heterodimers of α and β tubulin, arranged parallel to a cylindrical axis to form tubes of 25 nm diameter that may be many µm long. Polymerization of microtubules occurs by a nucleation-elongation mechanism in which the formation of a short microtubule 'nucleus' is followed by elongation of the microtubule at its ends by the reversible, noncovalent addition of tubulin dimers. Microtubules are not simple equilibrium polymers. They exhibit complex polymerization dynamics that use energy provided by the hydrolysis of GTP, and these dynamics are crucial to their cellular functions. A large number of chemically diverse substances bind to
Biochemistry, 2002
Laulimalide is a cytotoxic natural product that stabilizes microtubules. The compound enhances tubulin assembly, and laulimalide is quantitatively comparable to paclitaxel in its effects on the reaction. Laulimalide is also active in P-glycoprotein overexpressing cells, while isolaulimalide, a congener without the drug's epoxide moiety, was reported to have negligible cytotoxic and biochemical activity [Mooberry et al. (1999) Cancer Res. 59, 653-660]. We report here that laulimalide binds at a site on tubulin polymer that is distinct from the taxoid site. We found that laulimalide, while as active as paclitaxel, epothilone A, and eleutherobin in promoting the assembly of cold-stable microtubules, was unable to inhibit the binding of radiolabeled paclitaxel or of 7-O-[N-(2,7-difluoro-4'-fluoresceincarbonyl)-L-alanyl]paclitaxel, a fluorescent paclitaxel derivative, to tubulin. Confirming this observation, we demonstrated that microtubules formed in the presence of both laulimalide and paclitaxel contained near-molar quantities, relative to tubulin, of both drugs. Laulimalide was active against cell lines resistant to paclitaxel or epothilones A and B on the basis of mutations in the M40 human -tubulin gene. We also report that a laulimalide analogue lacking the epoxide moiety, while less active than laulimalide in biochemical and cellular systems, is probably more active than isolaulimalide. Further exploration of the role of the epoxide in the interaction of laulimalide with tubulin is therefore justified.
Comparison of microtubules stabilized with the anticancer drugs cevipabulin and paclitaxel
Polymer Journal, 2020
Microtubules, one of the major components of the cytoskeleton, play important roles as pathways for neuronal transport of cellular traffic. Loss of structural stability of microtubules causes detrimental effects on neurons and may contribute to several neurodegenerative diseases, such as Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, etc. The triazolopyrimidine class compound cevipabulin is a synthetic microtubule-stabilizing agent that has recently emerged as a drug for the treatment of Alzheimer's disease. However, the mechanism of microtubule stabilization by cevipabulin has not yet been revealed. Here, we explored the effect of cevipabulin on stabilizing microtubules polymerized from purified tubulins in vitro. We observed the effects of the concentration of microtubule-stabilizing drugs, incubation time, and modification of the cevipabulin structure on the stabilization of microtubules in comparison to those of the most commonly used anticancer drug, paclitaxel. This study will provide insight into the action of cevipabulin in the treatment of neurodegenerative diseases.
Biochemistry, 1996
A fluorescent derivative of paclitaxel, 2-debenzoyl-2-(m-aminobenzoyl)paclitaxel (2-AB-PT), has been prepared. 2-AB-PT induces microtubule assembly in Vitro, but is about 3-fold less potent than paclitaxel itself. The absorption and emission characteristics of 2-AB-PT were analyzed as a function of solvent. It was found that both spectra were perturbed by specific solvent effects when the solvent contained a hydrogen bond donor. The absorption and fluorescence spectra of 2-AB-PT bound to microtubules could not be mimicked by a single solvent, but the absorption and emission maxima of the tubulin-bound species could be duplicated by a solvent mixture of DMSO and water. These results indicate that the fluorophore binding site on the microtubule is in an environment of intermediate polarity that is accessible to a hydrogen bond donor in the vicinity of the m-amino group. In addition, tubulin fluorescence is quenched in the 2-AB-PT/microtubule complex, and energy transfer from tubulin to 2-AB-PT is apparent.
Cancer Research, 2004
Discodermolide is a new microtubule-targeted antimitotic drug in Phase I clinical trials that, like paclitaxel, stabilizes microtubule dynamics and enhances microtubule polymer mass in vitro and in cells. Despite their apparently similar binding sites on microtubules, discodermolide acts synergistically with paclitaxel to inhibit proliferation of A549 human lung cancer cells (L. Martello et al., Clin. Cancer Res., 6: 1978 -1987. To understand their synergy, we examined the effects of the two drugs singly and in combination in A549 cells and found that, surprisingly, their antiproliferative synergy is related to their ability to synergistically inhibit microtubule dynamic instability and mitosis. The combination of discodermolide and paclitaxel at their antiproliferative IC 50 s (7 nM for discodermolide and 2 nM for paclitaxel) altered all of the parameters of dynamic instability synergistically except the time-based rescue frequency. For example, together the drugs inhibited overall microtubule dynamicity by 71%, but each drug individually inhibited dynamicity by only 24%, giving a combination index (CI) of 0.23. Discodermolide and paclitaxel also synergistically blocked cell cycle progression at G 2 -M (41, 9.6, and 16% for both drugs together, for discodermolide alone, and for paclitaxel alone, respectively; CI ؍ 0.59), and they synergistically enhanced apoptosis (CI ؍ 0.85). Microtubules are unique receptors for drugs. The results suggest that ligands that bind to large numbers of binding sites on an individual microtubule can interact in a poorly understood manner to synergistically suppress microtubule dynamic instability and inhibit both mitosis and cell proliferation, with important consequences for combination clinical therapy with microtubule-targeted drugs.
Biochemistry, 1995
Our finding that an analog of paclitaxel (Taxol) modified at position C-2 (2-debenzoyl-2-(m-azidobenzoy1)paclitaxel) was substantially more active than paclitaxel in promoting tubulin assembly [Chaudhary et al. (1994) J. Am. Chem. SOC. 116, 4097-40981 led us to perform an analysis of the modulating effects of microtubule-associated proteins, GTP, and temperature on assembly and polymer stability. The analog always showed superior activity to paclitaxel in inducing polymerization where it fails to occur without drug, probably indicating a greater ability than paclitaxel to "hypemucleate" assembly. In contrast, much smaller differences in effects on polymer stability were observed. The analysis was extended to a large series of derivatives modified at positions C-2, C-7, C-10, and C-3', including docetaxel, a clinically important analog of paclitaxel. While analog stabilization of polymer was frequently observed, neither qualitative nor quantitative analysis of this property reliably predicted whether a compound would have enhanced hypemucleation activity relative to that of paclitaxel. Stabilization was often observed at substoichiometric analog concentrations, while even superstoichiometric concentrations of most compounds failed to induce extensive tubulin polymerization at low temperatures or in the absence of microtubuleassociated proteins or GTP. Docetaxel was intermediate in activity between paclitaxel and 2-debenzoyl-2-(m-azidobenzoyl)paclitaxel in promoting assembly reactions. We conclude that the hypemucleation of tubulin assembly and polymer stabilization observed with paclitaxel represent two distinct properties of the drug. Our findings suggest that paclitaxel, docetaxel, and 2-debenzoyl-2-(m-azidobenzoyl)paclitaxel are able to interact with progressively smaller assemblages of tubulin at low temperatures or in the absence of microtubule-associated proteins or GTP. Antimitotic agents, by interfering with the microtubule system, inhibit cell growth and have potential roles in the treatment of neoplastic diseases. Most compounds in this class inhibit microtubule formation and may cause disassembly of existing microtubules. Exceptions are the taxoids paclitaxel and docetaxel [Taxol (1) and Taxotere (2); structures in Figure 11, which have great promise in cancer treatment (for reviews, see Kingston, 1991; Rowinsky & Donehower, 1992; Kingston et al., 1993; Nicolaou et al., 1994). Paclitaxel not only enhances microtubule assembly and stabilizes microtubules to disassembly induced by cold, calcium, and dilution (Schiff et al., 1979), but it also obviates many of the normal requirements for tubulin polymerization. Paclitaxel permits assembly reactions to occur at low temperatures and in the absence of microtubule-associated proteins (MAPs)' and GTP (
Dissecting Paclitaxel–Microtubule Association: Quantitative Assessment of the 2′-OH Group
Biochemistry, 2013
Paclitaxel (PTX) is a microtubule-stabilizing agent that is widely used in cancer chemotherapy. This structurally complex natural product acts by binding to β-tubulin in assembled microtubules. The 2′-hydroxyl group in the flexible side chain of PTX is an absolute requirement for activity, but its precise role in the drug−receptor interaction has not been specifically investigated. The contribution of the 2′-OH group to the affinity and tubulin-assembly efficacy of PTX has been evaluated through quantitative analysis of PTX derivatives possessing side chain deletions: 2′-deoxy-PTX, N-debenzoyl-2′-deoxy-PTX, and baccatin III. The affinity of 2′-deoxy-PTX for stabilized microtubules was more than 100-fold lower than that of PTX and only ∼3-fold greater than the microtubule affinity of baccatin III. No microtubule binding activity was detected for the analogue N-debenzoyl-2′-deoxy-PTX. The tubulin-assembly efficacy of each ligand was consistent with the microtubule binding affinity, as was the trend in cytotoxicities. Molecular dynamics simulations revealed that the 2′-OH group of PTX can form a persistent hydrogen bond with D26 within the microtubule binding site. The absence of this interaction between 2′-deoxy-PTX and the receptor can account for the difference in binding free energy. Computational analyses also provide a possible explanation for why N-debenzoyl-2′-deoxy-PTX is inactive, in spite of the fact that it is essentially a substituted baccatin III. We propose that the hydrogen bonding interaction between the 2′-OH group and D26 is the most important stabilizing interaction that PTX forms with tubulin in the region of the C-13 side chain. We further hypothesize that the substituents at the 3′-position function to orient the 2′-OH group for a productive hydrogen bonding interaction with the protein.