Quantitative chemical proteomics for identifying candidate drug targets.
We have developed a systematic strategy for drug target identification. This consists of the following sequential steps: (1) enrichment of total binding proteins using two differential affinity matrixes upon which are immobilized positive and negative chemical structures for drug activity, respectively; (2) covalent labeling of the proteins with a new cleavable isotope-coded affinity tag (ICAT) reagent, followed by proteolysis of the combined proteins; (3) isolation, identification, and relative quantification of the tagged peptides by liquid chromatography-mass spectrometry; (4) array-based transcription profiling to select candidate proteins; and (5) confirmation of direct interaction between the activity-associated structure and the selected proteins by using surface plasmon resonance. We present a typical application to identify the primary binding protein of a novel class of anticancer agents exemplified by E7070. Our results suggest that this approach provides a new aspect of quantitative proteomics to find specific binding proteins from protein mixture and should be applicable to a wide variety of biologically active small molecules with unidentified target proteins.
Laboratory of Seeds Finding Technology, Eisai Co., Ltd, Tokodai 5-1-3, Tsukuba, Ibaraki 300-2635, Japan. y-oda@hhc.eisai.co.jp
Yoshiya
Y
Oda
Takashi
T
Owa
Toshitaka
T
Sato
Brian
B
Boucher
Scott
S
Daniels
Hidenori
H
Yamanaka
Yasuhiro
Y
Shinohara
Akira
A
Yokoi
Junro
J
Kuromitsu
Takeshi
T
Nagasu
D000970:Antineoplastic Agents / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D016503:Drug Delivery Systems; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D040901:Proteomics / Q000379:methods*; D013449:Sulfonamides / Q000494:pharmacology; D020349:Surface Plasmon Resonance; D013778:Tetrazolium Salts; D013844:Thiazoles; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; C439829:N-(3-chloro-7-indolyl)-1,4-benzenedisulphonamide; D013449:Sulfonamides; D013778:Tetrazolium Salts; D013844:Thiazoles; C022616:thiazolyl blue