Screening Tyrosine Kinase Inhibitors Targeting Pancreatic Cancer: Validation of Assays on Platelet Derived Growth Factor Receptor Gy. Bökönyi 3, E. Várkondi 1, E. Schäfer 1,2, P. Bánhegyi 1, Zs. Székelyhidi 1, T. Gyökeres 2, J. Hamvas 2, Tejeda M 4, L. Őrfi 2, Gy. Kéri 3, R. Schwab 1, Á. Pap 2 Cooperative Res. Centre, Semmelweis Univ. 1 ; Dept. of Gastroenterology MÁV Hospital 2 ; Peptide Biochemistry Res. Gr. of Hung. Acad. Sci. & Semmelweis Univ. 3, Nat Inst Oncol 4, Budapest, Hungary Cooperative Research Center Semmelweis University Budapest, Hungary Dept. of Gastroenterology MÁV Hospital Budapest, Hungary Introduction Over the last 15 years, a significant number of human diseases such as cancers have been attributed to defects in cellular signaling pathways. This observation has dramatically accelerated efforts towards the development of new therapeutic approaches. Changes in the expression of platelet-derived growth factor receptor (PDGFR) have been described in gastrointestinal tumors, and correlated with more aggressive behavior. This finding suggests that blockade of PDGF- dependent growth pathways may be an effective strategy to inhibit growth of these tumors. Aims Materials and Methods Assay was performed in 96-well plate format 1. Substrate Poly-Glu-Tyr (Sigma) was attached to the bottom of the plates 2. Enzyme reaction was performed in the presence of ATP (Sigma) at 37°C for 30 minutes 3. Phosphorylated substrate was detected by HRP-conjugated anti-P-Tyr antibody and OPD I. Principles of assay technology II. Assay details: 1.Enzyme: Recombinant PDGFR was expressed in baculovirus transected Sf9 expression system (ProQinase) 2.Structure of drug-candidate compounds: Conclusions The present ELISA based non-radioactive TK assay offers a reproducible, sensitive and rapid method to measure TK activity and enables large-scale screening of PDGFR inhibitors. Based on the success of the initial screening tests form one nested chemical library, further screens form our extended validation library will be performed along with QSAR optimizations to gain preclinical lead candidates. Results-Summary Recombinant PDGRF- enzyme activity 3.Screening A referenced inhibitor [SU- 6668(oxindol)] was tested in five concentrations (32,8-1,28 µM). The results were expressed as a percentage value of the control (T/C%) Our aim was set-up and characterize a non- radioactive TK assay platform to screen potential drug-candidate compound libraries designed against the ATP binding site of PDGFR receptor, representing a uniform functional target. Criteria for an optimal screening assay »Low-to medium throughput platform »High Sensitivity »Robust (stable assay conditions) »Reproducible (inter and intra-assay) »Relevant (validated molecular targets incorporated) »Informative (to be extrapolated to cellular assays) »Rapid, simple »Cost effective Results I.Results II. Effective compound Recombinant PDGFR-Kinetics SU-6668 –positive control Following optimization and standardization based on individual determination of kinetic parameters and calculation of Km values, reference PDGFR inhibitors were tested. Based on stability, reproducibility and published reference SU-6668 was chosen as internal positive control for further tests. One potent inhibitor was found from a nested chemical library of 20 compounds that will be entered in more detailed QSAR characterizations. Ineffective compound IC 50 =0,06uM Li Sun et al, 1999