JETO HomeIssue Contents

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
Yan Wang, Michael Malkowski, Judity Hailey, Tammy Turek-Etienne, Tom Tripodi and Jonathan A. Pachter

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 µM. C100 also inhibited IGF-1R autophosphorylation, AKT and MAPK activations in cells. This inhibitor provides a useful tool for studying IGF-1R in cells.

Full Text (IP)