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Differentiating between models of Epothilone binding to microtubules using tubulin mutagenesis, cytotoxicity, and molecular modeling
ChemMedChem (2012)
  • Ruth A. Entwistle, University of Kansas
  • Rania S. Rizk, University of Chicago
  • Daniel M. Cheng, University of Chicago
  • Gerald H. Lushington, University of Kansas
  • Richard H. Himes, University of Chicago
  • Mohan L. Gupta, Jr., University of Chicago
Microtubule stabilizers are powerful anti-mitotic compounds and represent a proven cancer
treatment strategy. Several classes of compounds in clinical use or trials, such as the taxanes and
epothilones, bind to the same region of β-tubulin. Determining how these molecules interact with
tubulin and stabilize microtubules is important both for understanding the mechanism of action
and enhancing chemotherapeutic potential, e.g. reducing side effects, increasing solubility, and
overcoming resistance. Structural studies using nonpolymerized tubulin or stabilized polymers
have produced different models of epothilone binding. Here, we used directed mutagenesis of the
binding site on Saccharomyces cerevisiae β-tubulin to analyze interactions between Epothilone B
and its biologically relevant substrate, dynamic microtubules. Five engineered amino acid changes
contributed to a 125-fold increase in Epothilone B cytotoxicity independent of inherent
microtubule stability. The mutagenesis of endogenous β-tubulin was done in otherwise isogenic
strains. This facilitated the correlation of amino acid substitutions with altered cytotoxicity using
molecular mechanics simulations. The results, which are based on the interaction between
Epothilone B and dynamic microtubules, most strongly support the binding mode determined by
NMR spectroscopy-based studies. This work establishes a system for discriminating between
potential binding modes and among various compounds and/or analogues using a sensitive
biological activity-based readout.
  • epothilone,
  • microtubule,
  • tubulin,
  • taxol binding site,
  • microtubule stabilizer,
  • drug design,
  • antitumor
Publication Date
September, 2012
Publisher Statement
Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Citation Information
Ruth A. Entwistle, Rania S. Rizk, Daniel M. Cheng, Gerald H. Lushington, et al.. "Differentiating between models of Epothilone binding to microtubules using tubulin mutagenesis, cytotoxicity, and molecular modeling" ChemMedChem Vol. 7 Iss. 9 (2012) p. 1580 - 1586
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