title: Interaction of the antitumor compound cryptophycin-52 with tubulin.
abstract: Cryptophycin-52 (LY355703) is currently undergoing clinical evaluation for cancer chemotherapy. It is a potent suppressor of microtubule dynamics in vitro, and low picomolar concentrations appear to inhibit cancer cell proliferation at mitosis by stabilizing spindle microtubules. In the present study, using [(3)H]cryptophycin-52, we found that the compound bound to tubulin at a single high-affinity site [apparent K(a) (3.6 +/- 1) x 10(6) L/mol, 34 degrees C]. The binding of cryptophycin-52 to tubulin was rapid, not appreciably temperature-dependent, and very poorly reversible. However, we could remove [(3)H]cryptophycin-52 from [(3)H]cryptophycin-52-tubulin complex by denaturing the complex with either urea treatment or boiling. These data suggest that the binding of cryptophycin-52 to tubulin is not covalent. A van't Hoff plot of the binding data indicated that the binding of cryptophycin-52 to tubulin is primarily entropy-driven with a minimum enthalpy contribution. In addition, cryptophycin-52 perturbed the far-ultraviolet circular dichroic spectrum of tubulin and it inhibited the colchicine-induced guanosine triphosphatase activity of tubulin, indicating that its binding to tubulin induces a conformational change in the tubulin. Competition experiments with vinblastine suggest that the binding site for crytophycin-52 may overlap with the vinblastine binding site.
authors.affiliation: Department of Molecular, The Interdepartmental Program in Biochemistry and Molecular Biology, University of California, Santa Barbara, California 93106, USA.
authors.forename: D
authors.initials: D
authors.lastname: Panda
authors.forename: V
authors.initials: V
authors.lastname: Ananthnarayan
authors.forename: G
authors.initials: G
authors.lastname: Larson
authors.forename: C
authors.initials: C
authors.lastname: Shih
authors.forename: M A
authors.initials: MA
authors.lastname: Jordan
authors.forename: L
authors.initials: L
authors.lastname: Wilson
mesh_terms: D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000737:chemistry* / Q000378:metabolism*; D001667:Binding, Competitive; D002417:Cattle; D047630:Depsipeptides; D007700:Kinetics; D007769:Lactams / Q000737:chemistry* / Q000378:metabolism*; D007783:Lactones / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism*; D014747:Vinblastine / Q000737:chemistry / Q000378:metabolism; D014748:Vinca Alkaloids / Q000737:chemistry / Q000378:metabolism
chemical_list: D000903:Antibiotics, Antineoplastic; D047630:Depsipeptides; D007769:Lactams; D007783:Lactones; D014404:Tubulin; D014748:Vinca Alkaloids; D014747:Vinblastine; C114033:cryptophycin 52