title: Use of photoaffinity labeling and site-directed mutagenesis for identification of the key residue responsible for extraordinarily high affinity binding of UCN-01 in human alpha1-acid glycoprotein.
abstract: 7-Hydroxystaurosporine (UCN-01) is a protein kinase inhibitor anticancer drug currently undergoing a phase II clinical trial. The low distribution volumes and systemic clearance of UCN-01 in human patients have been found to be caused in part by its extraordinarily high affinity binding to human alpha1-acid glycoprotein (hAGP). In the present study, we photolabeled hAGP with [3H]UCN-01 without further chemical modification. The photolabeling specificity of [3H]UCN-01 was confirmed by findings in which other hAGP binding ligands inhibited formation of covalent bonds between hAGP and [3H]UCN-01. The amino acid sequence of the photolabeled peptide was concluded to be SDVVYTDXK, corresponding to residues Ser-153 to Lys-161 of hAGP. No PTH derivatives were detected at the 8th cycle, which corresponded to the 160th Trp residue. This strongly implies that Trp-160 was photolabeled by [3H]UCN-01. Three recombinant hAGP mutants (W25A, W122A, and W160A) and wild-type recombinant hAGP were photolabeled by [3H]UCN-01. Only mutant W160A showed a marked decrease in the extent of photoincorporation. These results strongly suggest that Trp-160 plays a prominent role in the high affinity binding of [3H]UCN-01 to hAGP. A docking model of UCN-01 and hAGP around Trp-160 provided further details of the binding site topology.
authors.affiliation: Department of Biopharmaceutics, Graduate School of Medical and Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto, 862-0973, Japan.
authors.forename: Masaaki
authors.initials: M
authors.lastname: Katsuki
authors.forename: Victor Tuan Giam
authors.initials: VT
authors.lastname: Chuang
authors.forename: Koji
authors.initials: K
authors.lastname: Nishi
authors.forename: Kohichi
authors.initials: K
authors.lastname: Kawahara
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Nakayama
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Yamaotsu
authors.forename: Shuichi
authors.initials: S
authors.lastname: Hirono
authors.forename: Masaki
authors.initials: M
authors.lastname: Otagiri
mesh_terms: D000595:Amino Acid Sequence; D001667:Binding, Competitive; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D009961:Orosomucoid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019920:Photoaffinity Labels; D011485:Protein Binding; D011487:Protein Conformation; D019311:Staurosporine / Q000031:analogs & derivatives* / Q000378:metabolism*; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism
chemical_list: D009961:Orosomucoid; D019920:Photoaffinity Labels; C054852:7-hydroxystaurosporine; D014357:Trypsin; D019311:Staurosporine