title: Screening for small molecule inhibitors of insulin-like growth factor receptor (IGF-1R) kinase: comparison of homogeneous time-resolved fluorescence and 33P-ATP plate assay formats.
abstract: Insulin-like growth factor receptor 1 (IGF-1R) plays a critical role in oncogenic transformation (1). IGF-1R is overexpressed in some tumors including breast, lung, cervical, and Wilms' tumors (2-6). Upon binding of IGF-I or IGF-II, IGF-1R, a tyrosine kinase, phosphorylates tyrosine residues on two major substrates, IRS-1 and Shc, which subsequently signal through the Ras/Raf and PI 3-kinase/AKT pathways (7). Extensive literature has shown that when the IGF-1R signaling pathway is blocked by antisense, dominant negative truncation or neutralizing antibodies, cellular transformation and tumor formation in mice is inhibited (8-18). Small molecule kinase inhibitors represent a valid approach to inhibit activity and downstream signalling of IGF-1R. To date, few potent and selective small molecule inhibitors of IGF-1R kinase activity have been reported. We expressed the tyrosine kinase domain of IGF-1R (IGF-1R/TK) in insect cells and subsequently purified the partially activated IGF-1R/TK. A compound library has been screened using a homogeneous time-resolved fluorescence (HTRF) assay. The hits generated by HTRF were then evaluated in a 33P ATP streptavidin-Flashplate assay (Flashplate). There was approximately 78% hit congruence between the two assay formats. One compound, C100, inhibited the IGF-1R kinase activity with an IC50 of 1 microM. C100 also inhibited IGF-1R autophosphorylation, AKT and MAPK activations in cells. This inhibitor provides a useful tool for studying IGF-1R in cells.
authors.affiliation: Department of Tumor Biology, Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA. yan.wang@spcorp.com
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.forename: Michael
authors.initials: M
authors.lastname: Malkowski
authors.forename: Judith
authors.initials: J
authors.lastname: Hailey
authors.forename: Tammy
authors.initials: T
authors.lastname: Turek-Etienne
authors.forename: Tom
authors.initials: T
authors.lastname: Tripodi
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Pachter
mesh_terms: D001943:Breast Neoplasms; D045744:Cell Line, Tumor; D019411:Clinical Laboratory Techniques; D005453:Fluorescence; D006801:Humans; D009368:Neoplasm Transplantation / Q000502:physiology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D017526:Receptor, IGF Type 1 / Q000037:antagonists & inhibitors*; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D047428:Protein Kinase Inhibitors; D011505:Protein-Tyrosine Kinases; D017526:Receptor, IGF Type 1