Mutations in the β-tubulin binding site for peloruside A confer resistance by targeting a cleft significant in side chain binding

• Published in: Cell cycle (Georgetown, Tex.) Vol. 10; no. 19; pp. 3387 - 3396
• Main Authors: Begaye, Adrian, Trostel, Shana, Zhao, Zhiming, Taylor, Richard E., Schriemer, David C., Sackett, Dan L.
• Format: Journal Article
• Language: English
• Published: United States Taylor & Francis 01.10.2011; Landes Bioscience
• Subjects: Amino Acid Substitution; Binding; Binding Sites; Biology; Bioscience; Bridged Bicyclo Compounds, Heterocyclic - metabolism; Calcium; Cancer; Cell; Cell Line, Tumor; Cycle; Deuterium Exchange Measurement; Drug Resistance, Neoplasm - genetics; G2 Phase Cell Cycle Checkpoints; Humans; Lactones - metabolism; Landes; Microtubules - metabolism; Mutation; Organogenesis; Protein Structure, Tertiary; Proteins; Tubulin - genetics; Tubulin - metabolism
• ISSN: 1538-4101; 1551-4005
• DOI: 10.4161/cc.10.19.17706

Peloruside A is a microtubule-stabilizing macrolide that binds to beta tubulin at a site distinct from the taxol site. The site was previously identified by H-D exchange mapping and molecular docking as a region close to the outer surface of the microtubule and confined in a cavity surrounded by a continuous loop of protein folded so as to center on Y340. We have isolated a series of peloruside A-resistant lines of the human ovarian carcinoma cell line A2780(1A9) to better characterize this binding site and the consequences of altering residues in it. Four resistant lines (Pel A-D) are described with single-base mutations in class I β-tubulin that result in the following substitutions: R306H, Y340S, N337D, and A296S in various combinations. The mutations are localized to peptides previously identified by Hydrogen-Deuterium exchange mapping, and center on a cleft in which the drug side chain appears to dock. The Pel lines are 10-15-fold resistant to peloruside A and show cross resistance to laulimalide but not to any other microtubule stabilizers. They show no cross-sensitivity to any microtubule destabilizers, nor to two drugs with targets unrelated to microtubules. Peloruside A induces G2/M arrest in the Pel cell lines at concentrations 10-15 times that required in the parental line. The cells show notable changes in morphology compared to the parental line.