Establishment of the active catalytic domain of human PDGFRβ tyrosine kinase-based ELISA assay for inhibitor screening

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 PDGFRβ as a soluble active kinase using Bac-to-Bac expression system, and studied the correlations between PDGFRβ 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 PDGFRβ kinase. Of 500 RTK target-based compounds, TKI-30 was identified as a small molecule potential inhibitor of PDGFRβ  (IC50 = 0.34 μM). Further studies indicated that TKI-30 blocked PDGF-BB-induced autophosphorylation of PDGFRβ 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 PDGFRβ-based ELISA assay is a new method available for screening inhibitors targeting PDGFRβ kinase and TKI-30 is a potential novel anti-cancer agent worthy of being further investigated.