title: Biosynthesis of radiolabeled curacin A and its rapid and apparently irreversible binding to the colchicine site of tubulin.
abstract: Curacin A is a potent competitive inhibitor of colchicine binding to tubulin, and it inhibits the growth of tumor cells. We prepared [(14)C]curacin A biosynthetically to investigate its interaction with tubulin. Binding was rapid, even at 0 degrees C, with a minimum k(f) of 4.4 x 10(3) M(-1) s(-1). We were unable to demonstrate any dissociation of the [(14)C]curacin A from tubulin. Consistent with these observations, the K(a) value was so high that an accurate determination by Scatchard analysis was not possible. The [(14)C]curacin A was released from tubulin following urea treatment, indicating that covalent bond formation does not occur. We concluded that curacin A binds more tightly to tubulin than does colchicine. Besides high-affinity binding to the colchicine site, we observed significant superstoichiometric amounts of the [(14)C]curacin A bound to tubulin, and Scatchard analysis confirmed the presence of two binding sites of relatively low affinity with a K(a) of 3.2 x 10(-5) M(-1).
authors.affiliation: Developmental Therapeutics Program, National Cancer Institute, Frederick, Maryland, 21702, USA.
authors.forename: P
authors.initials: P
authors.lastname: Verdier-Pinard
authors.forename: N
authors.initials: N
authors.lastname: Sitachitta
authors.forename: J V
authors.initials: JV
authors.lastname: Rossi
authors.forename: D L
authors.initials: DL
authors.lastname: Sackett
authors.forename: W H
authors.initials: WH
authors.lastname: Gerwick
authors.forename: E
authors.initials: E
authors.lastname: Hamel
mesh_terms: D000970:Antineoplastic Agents / Q000138:chemical synthesis / Q000493:pharmacokinetics*; D001665:Binding Sites; D002250:Carbon Radioisotopes; D003078:Colchicine / Q000737:chemistry / Q000493:pharmacokinetics*; D000458:Cyanobacteria / Q000378:metabolism; D003521:Cyclopropanes / Q000138:chemical synthesis / Q000737:chemistry / Q000493:pharmacokinetics*; D007553:Isotope Labeling / Q000379:methods; D007700:Kinetics; D015394:Molecular Structure; D013844:Thiazoles / Q000138:chemical synthesis / Q000737:chemistry / Q000493:pharmacokinetics*; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D002250:Carbon Radioisotopes; D003521:Cyclopropanes; D013844:Thiazoles; D014404:Tubulin; C095843:curacin A; D003078:Colchicine