TESTING AND CHARACTERISING FOCUSED TYROSINE KINASE INHIBITORY LIBRARIES

1. TESTING AND CHARACTERISING FOCUSED TYROSINE KINASE INHIBITORY LIBRARIES • Györgyi Bökönyi1, Eszter Schafer2, Edit Várkondi2, Edit Z. Szabó1, Frigyes Wáczek2, Zsolt Székelyhidi2, Péter G. Bánhegyi2, 2, György Mészáros2,Dániel Erős2, László Őrfi2-3, Á. Pap2, Richard E. Schwab2, György Kéri1 • Hungarian Academy of Sciences Peptide Biochemistry Research Group 1 • Co-operative Res. Centre2, Dept. Pharm. Chem.3 Semmelweis University, Budapest, Hungary • Cooperative Research Center • Semmelweis University • Budapest, Hungary • Dept. of Gastroenterology • MÁV Hospital • Budapest, Hungary Methods Introduction 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. ELISA-based non-radiactive assay Cellular antiproliferative assays Tyr 96-well mikrotiter plate Poly-Glu-Tyr well Oxidized OPD Deoxidized OPD OPD Panc-1 ATP + ENZYME H2SO4 ELISA reader (490nm)) HRP conj. Ab P-Tyr P-Tyr Aims • 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 Results I. Results I. Lineweaver Plot of EGFR Toxic No effect Reference inhibitor Screening strategy Effective 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. GIF: (Growth Inhibitory Factor) is calculated from the differences of T/C values after 6h and 48h post-treatment, respectively. Kinase assay EGFR Biochemical assays EGFR Cell proliferation Apoptosis MTT Proliferation assays MTT MB Results II. Results II. FACS 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 Mean in vitro efficacy of selected compound(n=4) on EGFRTK activity Mean in vitro efficacy of selected compound(n=8) on EGFRTK activity 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 5 % of the compounds showed superior than 80% efficacy regarding kinase inhibition connected to minimum in vitro toxicity. 10 % of the compounds showed superior than 80% efficacy regarding growths inhibition connected to minimum in vitro toxicity. Summary Time course changes during apoptosis by Flow cytometry Materials 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. 6h 16h 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 24h 48h Calculated logP: 4,64 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 (seelower right plot). 5,6,7,8-Tetrahydro-benzo[4,5]thieno[2,3-d]pyrimidine Calculated logP: 3,31 7H-Pyrrolo[2,3-d]pyrimidine Calculated logP: 3,88 Quinazoline