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 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 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 H 2 O 2 H 2 O 2 H 2 O 2 Substrate: Poly (G lu,Tyr) Phosphorylated Substrate: Poly (G lu,Tyr-P) OPD O xidized O PD H 2 SO 4 H 2 SO 4 H 2 SO 4 Protonised O PD D irectE LIS A assay H R P peroxidase A nti-P-Tyr A ntibody Sigma (PTK-101) 37°C Recombinant PDGRF- enzyme activity Recombinant EGFR- enzyme activity Grefinib (ZD 1893) Iressa- positive control Number of tested compounds PDGFR EGFR Active (IC 50 >10uM) Inactiv e Active (IC 50 >1 uM) Inactiv e 1 20 5 145 Structure of drug-candidate compounds X: NH,NR,S R,R1,R2,R3,R4: H, (cyclo)alkyl, (hetero)aryl or combinated X R2 R3 N N R1 R2 R3 R4 R5 R6 X:NH,NR,S R,R1,R2,R3,R4:H,(cyclo)alkyl,(hetero)aryl,orcom I. II. X N N R1 R2 R3 R4 N N R1 R2 ,R4:H,(cyclo)alkyl,(hetero)aryl,orcombinated I. II. 0 20 40 60 80 100 120 0 5 10 15 20 25 30 35 Inhibitor(uM /w ell) . T/C % . 0 20 40 60 80 100 120 0 5 10 15 20 25 30 35 Inhibitor(uM ) . T/C % . 0,0 0,5 1,0 1,5 2,0 0 20 40 60 80 100 120 recom binantEG FR (ng/w ell) . OD . 0,0 0,5 1,0 1,5 2,0 0 50 100 150 200 250 PDG FR (ng/w ell) . OD . 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
Welcome message from author
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
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. Őrfi3, 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
Cooperative Research CentreSemmelweis University Budapest, Hungary
Dept. of GastroenterologyMÁV 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 plates2. 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 cloning2. Subcloning the insert into Baculovirus transfer vector-E.coli 3. Generating recombinant Baculoviruses by co-transfection 4. Amplifying recombinant virus5. Expressing recombinant protein
Experimental shame of EGFR-GST fusion protein with recombinant technique
H2O2H2O2 H2O2
Substrate:Poly (Glu, Tyr)
PhosphorylatedSubstrate:Poly (Glu, Tyr-P)
OPD
Oxidized OPD
H2SO4
H2SO4
H2SO4
Protonised OPD
DirectELISA assay
HRPperoxidase
Anti-P-Tyr Antibody
Sigma(PTK-101)
37°C
Recombinant PDGRF- enzyme activity Recombinant EGFR- enzyme activity
Grefinib (ZD 1893) Iressa- positive control
Number of tested compounds
PDGFR EGFR
Active (IC50>10uM)
Inactive Active(IC50>1 uM)
Inactive
1 20 5 145
Structure of drug-candidate compounds
X: NH,NR,S
R,R1,R2,R3,R4: H, (cyclo)alkyl, (hetero)aryl or combinated
X N
N
R1
R2
R3 R4N
N
R1
R2
R3
R4
R5
R6
X: NH, NR, SR, R1,R2,R3,R4: H, (cyclo)alkyl, (hetero)aryl, or combinated
I. II.
X N
N
R1
R2
R3 R4N
N
R1
R2
R3
R4
R5
R6
X: NH, NR, SR, R1,R2,R3,R4: H, (cyclo)alkyl, (hetero)aryl, or combinated
I. II.
0
20
40
60
80
100
120
0 5 10 15 20 25 30 35
Inhibitor (uM/well).
T/C %.
0
20
40
60
80
100
120
0 5 10 15 20 25 30 35
Inhibitor (uM).
T/C %.
0,0
0,5
1,0
1,5
2,0
0 20 40 60 80 100 120
recombinant EGFR (ng/well).
OD.
0,0
0,5
1,0
1,5
2,0
0 50 100 150 200 250
PDGFR (ng/well).
OD.
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
Related Documents
