A Novel Pim-1 Kinase Inhibitor Targeting Residues That Bind the Substrate Peptide

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• 作者：Keiko Tsuganezawa¹ ; Hisami Watanabe¹ ; Lorien Parker¹ ; Hitomi Yuki¹ ; Shigenao Taruya¹ ; Yukari Nakagawa¹ ; Daisuke Kamei¹ ; Masumi Mori¹ ; Naoko Ogawa¹ ; Yuri Tomabechi¹ ; Noriko Handa¹ ; Teruki Honma¹ ; Shigeyuki Yokoyama¹ ; ² ; ³ ; Hirotatsu Kojima⁴ ; Takayoshi Okabe⁴ ; Tetsuo Nagano⁴ ; Akiko Tanaka¹ ; ⁴ ; ^{aktanaka@riken.jp}
• 关键词：FCS ; fluorescent correlation spectroscopy ; BIM1 ; bisindolylmaleimide ; TAMRA ; 5-carboxytetramethylrhodamine succinimidyl ester ; SSM ; Secondary Structure Matching ; PI ; propidium iodide ; FITC ; fluorescein isothiocyanate ; PDB ; Protein Data Bank ; DMSO ; dimethy
• 刊名：Journal of Molecular Biology
• 出版年：2012
• 期刊代码：102_00222836
• 类别：imm
• 出版时间：30 March, 2012
• 卷：417
• 期：3
• 页码：240-252
• 文件大小：981 K

摘要

A new screening method using fluorescent correlation spectroscopy was developed to select kinase inhibitors that competitively inhibit the binding of a fluorescently labeled substrate peptide. Using the method, among approximately 700 candidate compounds selected by virtual screening, we identified a novel Pim-1 kinase inhibitor targeting its peptide binding residues. X-ray crystal analysis of the complex structure of Pim-1 with the inhibitor indicated that the inhibitor actually binds to the ATP-binding site and also forms direct interactions with residues (Asp128 and Glu171) that bind the substrate peptide. These interactions, which cause small side-chain movements, seem to affect the binding ability of the fluorescently labeled substrate. The compound inhibited Pim-1 kinase in vitro, with an IC₅₀ value of 150聽nM. Treatment of cultured leukemia cells with the compound reduced the amount of p21 and increased the amount of p27, due to Pim-1 inhibition, and then triggered apoptosis after cell-cycle arrest at the G₁/S phase. This screening method may be widely applicable for the identification of various new Pim-1 kinase inhibitors targeting the residues that bind the substrate peptide.