Differences in paclitaxel and docetaxel interactions with tubulin detected by mutagenesis of yeast tubulin.
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ABSTRACT:
SUBMITTER: Winefield RD
PROVIDER: S-EPMC3021245 | biostudies-literature | 2008 Dec
REPOSITORIES: biostudies-literature
Publications
Differences in paclitaxel and docetaxel interactions with tubulin detected by mutagenesis of yeast tubulin.
Winefield Robert D RD Entwistle Ruth A RA Foland Travis B TB Lushington Gerald H GH Himes Richard H RH
ChemMedChem 20081201 12
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Project description:We have successfully used mutagenesis to engineer Taxol (paclitaxel) binding activity in Saccharomyces cerevisiae tubulin. Taxol, a successful antitumor agent, acts by promoting tubulin assembly and stabilizing microtubules. Several structurally diverse antimitotic compounds, including the epothilones, compete with Taxol for binding to mammalian microtubules, suggesting that Taxol and these compounds share an overlapping binding site. However, Taxol has no effect on tubulin or microtubules from S. cerevisiae, whereas epothilone does. After considering data on Taxol binding to mammalian tubulin and recent modeling studies, we have hypothesized that differences in five key amino acids are responsible for the lack of Taxol binding to yeast tubulin. After changing these amino acids to those found in mammalian brain tubulin, we observed Taxol-related activity in yeast tubulin comparable to that in mammalian tubulin. Importantly, this experimental system can be used to reveal tubulin interactions with Taxol, the epothilones, and other Taxol-like compounds.
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Project description:BackgroundThe chemotherapeutic agent paclitaxel arrests cell division by binding to the hetero-dimeric protein tubulin. Subtle differences in tubulin sequences, across eukaryotes and among β-tubulin isotypes, can have profound impact on paclitaxel-tubulin binding. To capture the experimentally observed paclitaxel-resistance of human βIII tubulin isotype and yeast β-tubulin, within a common theoretical framework, we have performed structural principal component analyses of β-tubulin sequences across eukaryotes.ResultsThe paclitaxel-resistance of human βIII tubulin isotype and yeast β-tubulin uniquely mapped on to the lowest two principal components, defining the paclitaxel-binding site residues of β-tubulin. The molecular mechanisms behind paclitaxel-resistance, mediated through key residues, were identified from structural consequences of characteristic mutations that confer paclitaxel-resistance. Specifically, Ala277 in βIII isotype was shown to be crucial for paclitaxel-resistance.ConclusionsThe present analysis captures the origin of two apparently unrelated events, paclitaxel-insensitivity of yeast tubulin and human βIII tubulin isotype, through two common collective sequence vectors.
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Project description:IMPRINTS-CETSA data acquisition of MCF-7 cells treated with Vehicle (DMSO), Docetaxel or Paclitaxel using TMT 10 plex.
2024-10-23 | PXD056176 | Pride
Project description:IMPRINTS-CETSA data acquisition of MCF-7 cells treated with Vehicle (DMSO), Docetaxel or Paclitaxel using TMT 10 plex.
2024-10-23 | PXD056170 | Pride
Project description:Cancer treatment remains a pressing challenge, with paclitaxel playing a pivotal role in chemotherapy by disrupting mitotic spindle dynamics through microtubule stabilization. However, the molecular details of paclitaxel interaction with β-tubulin, its target, remain elusive, impeding efforts to overcome drug resistance and optimize efficacy. Here, we employ extensive molecular dynamics simulations to probe the binding modes of paclitaxel within tubulin protofilaments. Our simulations reveal a spectrum of paclitaxel binding poses, correlated with conformational changes in neighboring residues, proposing the ligand (un)binding route. These diverse binding modes exhibit varied interaction patterns and binding energies, elucidating the complex interplay between paclitaxel-tubulin interactions and the conformational dynamics of the M-loop. Furthermore, key residues influencing paclitaxel affinity and resistance are identified, enhancing our mechanistic understanding of the drug-binding mechanism. Finally, we uncover a novel high-affinity binding mode characterized by paclitaxel penetration into a subpocket formed by helices 1, 7, and loop B9-B10 of β-tubulin concerted with the rotational isomerization around a bond connecting the tetracyclic baccatin core with the N-benzoyl-β-phenylisoserine side chain, offering potential avenues for drug development. Our study advances the understanding of paclitaxel mode of action and informs strategies for rational drug design of antitumor agents.
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Project description:Members of the taxane class of chemotherapies, staples of cancer treatment since the 1990s, can induce chemotherapy-induced peripheral neuropathy (CIPN), a potentially irreversible outcome related to cumulative exposure. Switching between taxanes is often clinically necessary; however, different taxanes have different efficacies, toxicities, and dosing strategies, necessitating an evidence-based schema focused on toxicity. We performed a systematic review and meta-analysis of the literature on docetaxel and paclitaxel, extracting cumulative dose, rates of CIPN, and subject demographics, thereby establishing their dose-toxo-equivalence relationship through a Bayesian meta-analysis model, calculating doses of the two drugs that are expected to have comparable rates of CIPN, along with credible intervals. Our final model, based on 169 studies, produces credible interval widths that provide guidance within one treatment cycle. In practice, this model provides a framework under which oncologists can make treatment switching and dosing decisions, hopefully reducing patient risk of CIPN.
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Project description:Peripheral neuropathy is the dose-limiting toxicity of paclitaxel, a chemotherapeutic drug widely used to treat several solid tumors such as breast, lung, and ovary. The cytotoxic effect of paclitaxel is mediated through ?-tubulin binding in the cellular microtubules. In this study, we investigated the association between paclitaxel neurotoxicity risk and regulatory genetic variants in ?-tubulin genes.We measured variation in gene expression of three ?-tubulin isotypes (I, IVb, and IIa) in lymphocytes from 100 healthy volunteers, sequenced the promoter region to identify polymorphisms putatively influencing gene expression and assessed the transcription rate of the identified variants using luciferase assays. To determine whether the identified regulatory polymorphisms were associated with paclitaxel neurotoxicity, we genotyped them in 214 patients treated with paclitaxel. In addition, paclitaxel-induced cytotoxicity in lymphoblastoid cell lines was compared with ?-tubulin expression as measured by Affymetrix exon array.We found a 63-fold variation in ?-tubulin IIa gene (TUBB2A) mRNA content and three polymorphisms located at -101, -112, and -157 in TUBB2A promoter correlated with increased mRNA levels. The -101 and -112 variants, in total linkage disequilibrium, conferred TUBB2A increased transcription rate. Furthermore, these variants protected from paclitaxel-induced peripheral neuropathy [HR, 0.62; 95% confidence interval (CI), 0.42-0.93; P = 0.021, multivariable analysis]. In addition, an inverse correlation between TUBB2A and paclitaxel-induced apoptosis (P = 0.001) in lymphoblastoid cell lines further supported that higher TUBB2A gene expression conferred lower paclitaxel sensitivity.This is the first study showing that paclitaxel neuropathy risk is influenced by polymorphisms regulating the expression of a ?-tubulin gene.
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Project description:Cyclostreptin is the first microtubule-stabilizing agent whose mechanism of action was discovered to involve formation of a covalent bond with tubulin. Treatment of cells with cyclostreptin irreversibly stabilizes their microtubules because cyclostreptin forms a covalent bond to β-tubulin at either the T220 or the N228 residue, located at the microtubule pore or luminal taxoid binding site, respectively. Because of its unique mechanism of action, cyclostreptin overcomes P-glycoprotein-mediated multidrug resistance in tumor cells. We used a series of reactive cyclostreptin analogues, 6-chloroacetyl-cyclostreptin, 8-chloroacetyl-cyclostreptin, and [(14)C-acetyl]-8-acetyl-cyclostreptin, to characterize the cellular target of the compound and to map the binding site. The three analogues were cytotoxic and stabilized microtubules in both sensitive and multidrug resistant tumor cells. In both types of cells, we identified β-tubulin as the only or the predominantly labeled cellular protein, indicating that covalent binding to microtubules is sufficient to prevent drug efflux mediated by P-glycoprotein. 6-Chloroacetyl-cyclostreptin, 8-chloroacetyl-cyclostreptin, and 8-acetyl-cyclostreptin labeled both microtubules and unassembled tubulin at a single residue of the same tryptic peptide of β-tubulin as was labeled by cyclostreptin (219-LTTPTYGDLNHLVSATMSGVTTCLR-243), but labeling with the analogues occurred at different positions of the peptide. 8-Acetyl-cyclostreptin reacted with either T220 or N228, as did the natural product, while 8-chloroacetyl-cyclostreptin formed a cross-link to C241. Finally, 6-chloroacetyl-cyclostreptin reacted with any of the three residues, thus labeling the pathway for cyclostreptin-like compounds, leading from the pore where these compounds enter the microtubule to the luminal binding pocket.
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Project description:We have created 41 clustered charged-to-alanine scanning mutations of the mipA, gamma-tubulin, gene of Aspergillus nidulans and have created strains carrying these mutations by two-step gene replacement and by a new procedure, heterokaryon gene replacement. Most mutant alleles confer a wild-type phenotype, but others are lethal or conditionally lethal. The conditionally lethal alleles exhibit a variety of phenotypes under restrictive conditions. Most have robust but highly abnormal mitotic spindles and some have abnormal cytoplasmic microtubule arrays. Two alleles appear to have reduced amounts of gamma-tubulin at the spindle pole bodies and nucleation of spindle microtubule assembly may be partially inhibited. One allele inhibits germ tube formation. The cold sensitivity of two alleles is strongly suppressed by the antimicrotubule agents benomyl and nocodazole and a third allele is essentially dependent on these compounds for growth. Together our data indicate that gamma-tubulin probably carries out functions essential to mitosis and organization of cytoplasmic microtubules in addition to its well-documented role in microtubule nucleation. We have also placed our mutations on a model of the structure of gamma-tubulin and these data give a good initial indication of the functionally important regions of the molecule.
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