title: Establishment of the active catalytic domain of human PDGFRbeta tyrosine kinase-based ELISA assay for inhibitor screening.
abstract: Tyrosine kinases are emerging as frequent targets of primary oncogenic events and therefore represent an optimal focus of therapeutic intervention. In an effort towards therapeutic PDGFR inactivation, we expressed the catalytic domain of PDGFRbeta as a soluble active kinase using Bac-to-Bac expression system, and studied the correlations between PDGFRbeta activity and enzyme concentration, ATP concentration, substrate concentration and divalent cation type. And a convenient, effective and non-radioactive ELISA screening model is then established for identification of the potential inhibitors targeting PDGFRbeta kinase. Of 500 RTK target-based compounds, TKI-30 was identified as a small molecule potential inhibitor of PDGFRbeta (IC(50)=0.34 microM). Further studies indicated that TKI-30 blocked PDGF-BB-induced autophosphorylation of PDGFRbeta in a dose-dependent manner in Swiss 3T3 cells and human umbilical vein smooth muscle cells (HUVSMCs). Moreover, it dose-dependently suppressed the PDGF-BB-induced proliferation in HUVSMCs and tube formation of HUVEC. Our data collectively indicated that PDGFRbeta-based ELISA assay is a new method available for screening inhibitors targeting PDGFRbeta kinase and TKI-30 is a potential novel anti-cancer agent worthy of being further investigated.
authors: Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, 555 Zu Chong Zhi Road, Zhangjiang Hitec Park, Shanghai, 201203 PR China.
authors: Xiu-Hua
authors: XH
authors: Zhang
authors: Xiao-Ning
authors: XN
authors: Guo
authors: Li
authors: L
authors: Zhong
authors: Xiao-Min
authors: XM
authors: Luo
authors: Hua-Liang
authors: HL
authors: Jiang
authors: Li-Ping
authors: LP
authors: Lin
authors: Jian
authors: J
authors: Ding
mesh_terms: D000818:Animals; D020134:Catalytic Domain; D002460:Cell Line; D004353:Drug Evaluation, Preclinical; D042783:Endothelial Cells / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D009036:Moths; D047428:Protein Kinase Inhibitors / Q000032:analysis / Q000302:isolation & purification*; D011725:Pyridines / Q000494:pharmacology*; D011743:Pyrimidines / Q000494:pharmacology*; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D047428:Protein Kinase Inhibitors; D011725:Pyridines; D011743:Pyrimidines; D011993:Recombinant Fusion Proteins; C524096:TKI-30; D008345:Manganese; D020797:Receptor, Platelet-Derived Growth Factor beta; D008274:Magnesium