A general method for screening of peptidomimetic libraries by ELISA based tyrosine kinase assay Gy. Bökönyi 1, E. Schäfer 2, E. Várkondi 2, Edit Z. Szabó 1, F. Wáczek 2,P. Bánhegyi 2, Zs. Székelyhidi 2, B. Hegymegi-Barakonyi 2,L. Őrfi 3, T. Vántus 1, R. E. Schwab 2,Gy. Kéri 1 Peptide Biochemistry Res. Gr.of Hung. Acad. Sci.& Semmelweis Univ. 1, Cooperative Res. Centre, Semmelweis Univ. 2, Vichem Chemie Ltd. 3 Budapest, Hungary Cooperative Research Centre 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. Tyrosine kinases have been shown to play a crucial role in signal transduction pathways and have been implicated as kay players in many „proliferative disorders” including cancer. Inhibitors designed against potential novel kinase targets are in in the focus of the drug discovery today. 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. Enzymes: Recombinant PDGFR was expressed in baculovirus transected Sf9 expression system (ProQinase) EGFR-GST with recombinant technique 4. Phosphorylated substrate was detected by HRP-conjugated anti-P-Tyr antibody and OPD Conclusions The present ELISA based non-radioactive TK assay offers a reproductable, sensitive and rapid method to measure TK activity and enables large-scale screening of PDGFR and EGFR 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 Screening A referenced inhibitors [SU- 6668(oxindol) PDGFR, Grefitinib (ZD 1893) EGFR] 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 to screen large numbers of inhibitory compounds with an ELISA- based non-radioactive tyrosine kinase assay. To meet the needs of high amount enzyme arising in this assay technology, custom production of the recombinant enzyme was nessesary. We establised a recombinant expression system. Mass production of recombinant proteins will enable scale up in the testing processes with future potential of full automation. Criteria for an optimal screening assay Results I. Results II. SU-6668 –positive control 1. Preparing target DNA insert for cloning 2. Subcloning the insert into Baculovirus transfer vector-E.coli 3. Generating recombinant Baculoviruses by co-transfection 4. Amplifying recombinant virus 5. Expressing recombinant protein Experimental shame of EGFR-GST fusion protein with recombinant technique Recombinant PDGRF- enzyme activity Recombinant EGFR- enzyme activity Grefinib (ZD 1893) Iressa- positive control Number of tested compounds PDGFREGFR Active (IC 50 >10uM) InactiveActive (IC 50 >1 uM) Inactive Structure of drug-candidate compounds X: NH,NR,S R,R1,R2,R3,R4: H, (cyclo)alkyl, (hetero)aryl or combinated Principles of assay technology »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