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1
A general method for screening of peptidomimetic
libraries by ELISA based tyrosine kinase assay
Gy. Bökönyi1, E. Schäfer2 , E. Várkondi2, Edit
Z. Szabó1, F. Wáczek2,P. Bánhegyi2, Zs.
Székelyhidi2, B. Hegymegi-Barakonyi2,L. Orfi3, T.
Vántus1, R. E. Schwab2,Gy. Kéri1 Peptide
Biochemistry Res. Gr.of Hung. Acad. Sci.
Semmelweis Univ.1, Cooperative Res. Centre,
Semmelweis Univ.2, Vichem Chemie Ltd.3Budapest,
Hungary
Dept. of Gastroenterology MÁV Hospital Budapest,
Hungary
Cooperative Research Centre Semmelweis
University Budapest, Hungary
Introduction
Aims
Criteria for an optimal screening assay
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.

• 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

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.
Materials and Methods
Experimental shame of EGFR-GST fusion protein
with recombinant technique
Principles of assay technology
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
37C 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
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
Structure of drug-candidate compounds
X NH,NR,S R,R1,R2,R3,R4 H, (cyclo)alkyl,
(hetero)aryl or combinated
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)
Results II.
Results I.
Results-Summary
Recombinant PDGRF- enzyme activity
Recombinant EGFR- enzyme activity
Number of tested compounds Number of tested compounds Number of tested compounds Number of tested compounds
PDGFR PDGFR EGFR EGFR
Active (IC50gt10uM) Inactive Active (IC50gt1 uM) Inactive
1 20 5 145
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.
SU-6668 positive control
Grefinib (ZD 1893) Iressa- positive control