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 |
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Authors: | Katsuki Masaaki Chuang Victor Tuan Giam Nishi Koji Kawahara Kohichi Nakayama Hitoshi Yamaotsu Noriyuki Hirono Shuichi Otagiri Masaki |
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Institution: | Department of Biopharmaceutics, Graduate School of Medical and Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto, 862-0973, Japan. |
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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. |
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