Differentiating between models of epothilone binding to microtubules using tubulin mutagenesis, cytotoxicity, and molecular modeling.
Microtubule stabilizers are powerful antimitotic 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, for example, minimizing side effects, increasing solubility, and overcoming resistance. Structural studies using non-polymerized tubulin or stabilized polymers have produced different models of epothilone binding. In this study 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.
Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045-7534, USA.
Ruth A
RA
Entwistle
Rania S
RS
Rizk
Daniel M
DM
Cheng
Gerald H
GH
Lushington
Richard H
RH
Himes
Mohan L
ML
Gupta
D000595:Amino Acid Sequence; D001665:Binding Sites; D034261:Epothilones / Q000494:pharmacology*; D006801:Humans; D008870:Microtubules / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016296:Mutagenesis; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014404:Tubulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D050257:Tubulin Modulators / Q000494:pharmacology*
D034261:Epothilones; D029701:Saccharomyces cerevisiae Proteins; D014404:Tubulin; D050257:Tubulin Modulators; C093788:epothilone B