title: Two photoaffinity analogues of the tripeptide, hemiasterlin, exclusively label alpha-tubulin.
abstract: A synthetic analogue of the tripeptide hemiasterlin, designated HTI-286, depolymerizes microtubules, is a poor substrate for P-glycoprotein, and inhibits the growth of paclitaxel-resistant tumors in xenograft models. Two radiolabeled photoaffinity analogues of HTI-286, designated 4-benzoyl-N,beta,beta-trimethyl-l-phenylalanyl-N(1)-[(1S,2E)-3-carboxy-1-isopropylbut-2-enyl]-N(1),3-dimethyl-l-valinamide (probe 1) and N,beta,beta-trimethyl-l-phenylalanyl-4-benzoyl-N-[(1S,2E)-3-carboxy-1-isopropyl-2-butenyl]-N,beta,beta-trimethyl-l-phenylalaninamide (probe 2), were made to help identify HTI-286 binding sites in tubulin. HTI-286, probe 1, and probe 2 had similar affinities for purified tubulin [apparent K(D(app)) = 0.2-1.1 microM], inhibited polymerization of purified tubulin approximately 80%, and were potent inhibitors of cell growth (IC(50) = 1.0-22 nM). Both radiolabeled probes labeled exclusively alpha-tubulin. Labeling by [(3)H]probe 1 was inhibited by probe 1, HTI-286, vinblastine, or dolastatin 10 (another peptide antimitotic agent that depolymerizes microtubules) but was either unaffected or enhanced (at certain temperatures) by colchicine or paclitaxel. [(3)H]Probe 1 also labeled exclusively tubulin in cytosolic extracts of whole cells. The major, if not exclusive, contact site for probe 1 was mapped to residues 314-339 of alpha-tubulin and corresponds to the sheet 8 and helix 10 region. This region is known to (1) have longitudinal interactions with beta-tubulin across the interdimer interface, (2) have lateral interactions with adjacent protofilaments, and (3) contact the N-terminal region of stathmin, a protein that induces depolymerization of tubulin. Binding of probe 1 to this region may alter the conformation of tubulin outside the labeling domain, since enzymatic removal of the C-terminus of only alpha-tubulin by subtilisin after, but not before, photolabeling is blocked by probe 1. These results suggest that hemiasterlin is in close contact with alpha-tubulin and may span the interdimer interface so that it contacts the vinblastine- and dolastatin 10-binding sites believed to be in beta-tubulin. In addition, we speculate that antimitotic peptides mimic the interaction of stathmin with tubulin.
authors.affiliation: Oncology Research, Chemical and Screening Sciences, Radiosynthesis Group, and Bioorganic Enzymology, Wyeth Research, 401 North Middletown Road, Pearl River, New York 10965, USA. nunesm@wyeth.com
authors.forename: Maria
authors.initials: M
authors.lastname: Nunes
authors.forename: Joshua
authors.initials: J
authors.lastname: Kaplan
authors.forename: Joseph
authors.initials: J
authors.lastname: Wooters
authors.forename: Malathi
authors.initials: M
authors.lastname: Hari
authors.forename: Albert A
authors.initials: AA
authors.lastname: Minnick
authors.forename: Michael K
authors.initials: MK
authors.lastname: May
authors.forename: Celine
authors.initials: C
authors.lastname: Shi
authors.forename: Sylvia
authors.initials: S
authors.lastname: Musto
authors.forename: Carl
authors.initials: C
authors.lastname: Beyer
authors.forename: Girija
authors.initials: G
authors.lastname: Krishnamurthy
authors.forename: Yongchang
authors.initials: Y
authors.lastname: Qiu
authors.forename: Frank
authors.initials: F
authors.lastname: Loganzo
authors.forename: Semiramis
authors.initials: S
authors.lastname: Ayral-Kaloustian
authors.forename: Arie
authors.initials: A
authors.lastname: Zask
authors.forename: Lee M
authors.initials: LM
authors.lastname: Greenberger
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D002417:Cattle; D003600:Cytosol / Q000378:metabolism; D047630:Depsipeptides; D006131:Growth Inhibitors / Q000737:chemistry / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000037:antagonists & inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D007624:KB Cells; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010449:Peptide Mapping; D019920:Photoaffinity Labels / Q000378:metabolism*; D011485:Protein Binding; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014404:Tubulin / Q000378:metabolism*; D050257:Tubulin Modulators; D014747:Vinblastine / Q000378:metabolism
chemical_list: D047630:Depsipeptides; D006131:Growth Inhibitors; C473896:HTI-286; D009842:Oligopeptides; D019920:Photoaffinity Labels; D021122:Protein Subunits; D014404:Tubulin; D050257:Tubulin Modulators; D014747:Vinblastine; C467070:hemiasterlin; D006160:Guanosine Triphosphate; C064570:dolastatin 10