PDGFR TK assay for screening inhibitory compound-libararies

Introduction: Changes in the expression of platelet-derived growth factor receptor (PDGFR) have been described in gastrointestinal tumors, and correlated with more aggressive behaviour. This finding suggests that blockade of PDGF-dependent growth pathways may be an effective strategy to inhibit growth of these tumors.

Our Aim was to set-up and characterize a non-radioactive TK assay platform to screen potential drug-candidate compound libraries designed against the ATP binding site of PDGF receptor, representing a uniform functional target.

Methods: An assay based on detection of phosphorylated substrate was established. Recombinant PDGFR alpha derived from baculovirus transfected insect-cell (Sf9) expression systems (ProQinase) and a synthetic substrate polyGluTyr Σ was used. Detection was performed utilizing anti-phosphotyrosine monoclonal antibody (Clone PT-66 Sigma) conjugated with HRPeroxidase and OPD as peroxidase substrate. Absorbances were measured with ELISA reader and correlated with the phosphorylated substrate thus the enzyme activity. Results: Following optimization and standardization based on individual determination of kinetic parameters and calculation of Km values, 2 reference PDGFR inhibitors were tested. One of them had more stable and reproducible inhibitory effect of 90% that was well-comparable to the literature data and thus seems optimal for positive control in future experiments.

Conclusion: The present ELISA based non-radioactive TK assay offers a reproducible, sensitive, simple and rapid method to measure TK activity and enables large-scale screening of PDGFR inhibitors.