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

Abstract

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.

• Authors: Adrian Begaye, Shana Trostel, Zhiming Zhao, Richard E Taylor, David C Schriemer, Dan L Sackett
• Journal: Cell cycle (Georgetown, Tex.) (Cell Cycle) Vol. 10 Issue 19 Pg. 3387-96 (Oct 01 2011) ISSN: 1551-4005 [Electronic] United States
• PMID: 21926482 (Publication Type: Journal Article, Research Support, N.I.H., Intramural)
• Copyright: © 2011 Landes Bioscience

Chemical References

• Bridged Bicyclo Compounds, Heterocyclic
• Lactones
• Tubulin
• peloruside A

Topics

• Amino Acid Substitution
• Binding Sites
• Bridged Bicyclo Compounds, Heterocyclic (metabolism)
• Cell Line, Tumor
• Deuterium Exchange Measurement
• Drug Resistance, Neoplasm (genetics)
• G2 Phase Cell Cycle Checkpoints
• Humans
• Lactones (metabolism)
• Microtubules (metabolism)
• Mutation
• Protein Structure, Tertiary
• Tubulin (genetics, metabolism)