1. TESTING AND CHARACTERISING FOCUSED TYROSINE KINASE INHIBITORY LIBRARIES Györgyi Bökönyi 1, Eszter Schafer 2, Edit Várkondi 2, Edit Z. Szabó 1, Frigyes Wáczek 2, Zsolt Székelyhidi 2, Péter G. Bánhegyi 2, 2, György Mészáros 2,Dániel Erős 2, László Őrfi 2-3, Á. Pap 2, Richard E. Schwab 2, György Kéri 1 Hungarian Academy of Sciences Peptide Biochemistry Research Group 1 Co-operative Res. Centre 2, Dept. Pharm. Chem. 3 Semmelweis University, Budapest, Hungary Cooperative Research Center Semmelweis University Budapest, Hungary Dept. of Gastroenterology MÁV Hospital Budapest, Hungary Introduction Aims ELISA based non-radioactive TK assays offer reproducible, simple and rapid methods to measure kinase activity and enables large-scale screening of TK inhibitors. However, in our hands, methodological burdens seem to limit the sensitivity of ELISA-based approaches (relatively high background). In this respect ECL-based or fluorescent technologies seem to be superior on the „cost of the prize” of the assay. Because of these difficulties we have worked out a screening strategy using A431 EGFR overexpressing cell lines in proliferation assays for prescreening. The assay was carried out in two timepoints to distinguish between apoptotic and necrotic effect. The compounds were screened in core structure groups in order to select the best core structures for further synthetic work. The best compounds were further analyzed in EGF-RTK assay and in specific apoptotic assay with FACS. Analyzing the structures of several active inhibitors revealed that most of the potent compounds contained a condensed bicyclic heteroaryl moiety where the pyrimidine ring seems to be crucial. Criteria for an optimal screening assay »Low-to medium throughput platform »High Sensitivity »Robust (stable assay conditions) »Reproducible (inter and intra-assay) »Relevant (validated mol. targets incorporated) »Informative (to be extrapolated to cell. assays) »Rapid, simple »Cost effective Screening strategy Kinase assay ApoptosisCell proliferation Biochemical assays Proliferation assays FACS EGFR MTT MB MTT Materials Methods Poly-Glu-Tyr P-Tyr OPD ELISA reader (490nm)) 96-well mikrotiter plate Tyr ATP ENZYM E P-Tyr + well Oxidized OPD H 2 SO 4 HRP conj. Ab Deoxidized OPD Results I. Lineweaver Plot of EGFR To determine the optimal concentration of tyrosine kinase enzyme and its substrate, we determined the kinetic parameters (Km and Vmax values) for VEGFR, EGFR and PDGFR. Inhibitory effect of ÖL-57 had the most stable and reproducible inhibitory effect of 90% and thus seems optimal for positive control in future experiments. ELISA-based non-radiactive assay Cellular antiproliferative assays Summary Time course changes during apoptosis by Flow cytometry 7H-Pyrrolo[2,3-d]pyrimidine 5,6,7,8-Tetrahydro- benzo[4,5]thieno[2,3-d]pyrimidine Quinazoline The selected set of compounds can be grouped around 3 core structures. Condensed bicyclic heteroaryl cores containing a pyrimidine ring have been synthesized using ortho-cyanoarylamine building blocks. The resulting compounds and intermediates were characterized via MS and NMR spectroscopy and analyzed by HPLC for purity Tyrosine kinases have been shown to play a crucial role in signal transduction pathways and have been implicated as key players in many „proliferative disorders” including cancer and atherosclerosis. Inhibitors designed against potential novel kinase targets are in the focus of the drug discovery today. Our group has designed and synthesized a large set of new potential inhibitory compounds, competing for the ATP-binding site in the catalytic domain of Protein Tyrosine Kinases (PTK), the leading-structural target of these enzymes nowadays. Our aim was to characterize the efficacy of these compounds by testing them with relevant bioassays: »in vitro cell proliferation assays »Non-radioactive tyrosine kinase assays » flow cytometry Reference inhibitor Results II. Results I. Results II. Toxic Effective No effect GIF: (Growth Inhibitory Factor) is calculated from the differences of T/C values after 6h and 48h post-treatment, respectively. Mean (+/- SD) in vitro antiproliferative effect of tested compounds (n=12) at 6 hours Mean (+/- SD) in vitro antiproliferative effect of tested compounds (n=12) at 48 hours Analysis of apoptosis induction was based on Annexin-V and propidium iodide staining. Early apoptotic cells in this assay are identified as propidium iodide negative + Annexin-V positive cell population (see upper right plot). Normal cells are negative for both dyes (see upper left plot), whereas the necrotic / late apoptotic cell- population is positive for both dyes (see lower right plot). 10 % of the compounds showed superior than 80% efficacy regarding growths inhibition connected to minimum in vitro toxicity. Panc-1 Mean in vitro efficacy of selected compound(n=4) on EGFRTK activity Mean in vitro efficacy of selected compound(n=8) on EGFRTK activity 5 % of the compounds showed superior than 80% efficacy regarding kinase inhibition connected to minimum in vitro toxicity. 6h 16h 24h 48h Calculated logP: 4,64 Calculated logP: 3,31 Calculated logP: 3,88