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J. Biol. Chem., Vol. 280, Issue 2, 1384-1391, January 14, 2005

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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 1-Acid Glycoprotein^*

Masaaki Katsuki, Victor Tuan Giam Chuang, Koji Nishi, Kohichi Kawahara¶, Hitoshi Nakayama¶, Noriyuki Yamaotsu||, Shuichi Hirono||, and Masaki Otagiri**

From the Departments of Biopharmaceutics and ^¶Molecular Cell Function, Graduate School of Medical and Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto, 862-0973, Japan, the Department of Pharmacy, Faculty of Allied Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia, and ^||The School of Pharmaceutical Sciences, Kitasato University, 5-9-1, Shirokane, Minato-ku, Tokyo, 108-8641, Japan

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 1-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.

Received for publication, September 27, 2004 , and in revised form, October 26, 2004.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed: Dept. of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto, 862-0973, Japan. Tel.: 81-96-3714150; Fax: 81-96-3627690; E-mail: otagirim{at}gpo.kumamoto-u.ac.jp.

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