title: 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.affiliation: Program in Physical Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Adrian
authors.initials: A
authors.lastname: Begaye
authors.forename: Shana
authors.initials: S
authors.lastname: Trostel
authors.forename: Zhiming
authors.initials: Z
authors.lastname: Zhao
authors.forename: Richard E
authors.initials: RE
authors.lastname: Taylor
authors.forename: David C
authors.initials: DC
authors.lastname: Schriemer
authors.forename: Dan L
authors.initials: DL
authors.lastname: Sackett
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000378:metabolism*; D045744:Cell Line, Tumor; D041961:Deuterium Exchange Measurement; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D059565:G2 Phase Cell Cycle Checkpoints; D006801:Humans; D007783:Lactones / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism; D009154:Mutation; D017434:Protein Structure, Tertiary; D014404:Tubulin / Q000235:genetics / Q000378:metabolism*
chemical_list: D019086:Bridged Bicyclo Compounds, Heterocyclic; D007783:Lactones; D014404:Tubulin; C414085:peloruside A