screening tyrosine kinase inhibitors targeting pancreatic cancer: validation of assays on platelet...
TRANSCRIPT
Screening Tyrosine Kinase Inhibitors Targeting Pancreatic Cancer:
Validation of Assays on Platelet Derived Growth Factor Receptor
Gy. Bökönyi3, E. Várkondi1, E. Schäfer1,2, P. Bánhegyi1, Zs. Székelyhidi1,T. Gyökeres2, J. Hamvas2, Tejeda M4, L. Őrfi2, Gy. Kéri3, R. Schwab1, Á. Pap2
Cooperative Res. Centre, Semmelweis Univ.1; Dept. of Gastroenterology MÁV Hospital2; Peptide Biochemistry Res. Gr. of Hung. Acad. Sci. & Semmelweis Univ.3, Nat Inst Oncol4,
Budapest, Hungary
Cooperative Research CenterSemmelweis 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.
Changes in the expression of platelet-derived growth factor receptor (PDGFR) have been described in gastrointestinal tumors, and correlated with more aggressive behavior. This finding suggests that blockade of PDGF-dependent growth pathways may be an effective strategy to inhibit growth of these tumors.
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. Phosphorylated substrate was detected by HRP-conjugated anti-P-Tyr antibody and OPD
H2O2H2O2 H2O2
Substrate:Poly (Glu, Tyr)
PhosphorylatedSubstrate:Poly (Glu, Tyr-P)
OPD
OxidizedOPD
H2SO4
H2SO4
H2SO4
ProtonisedOPD
DirectELISA assay
HRPperoxidase
Anti-P-Tyr Antibody
Sigma(PTK-101)
37°C
I. Principles of assay technology
II. Assay details:
1. Enzyme: Recombinant PDGFR was expressed in baculovirus transected Sf9 expression system (ProQinase)
2. Structure of drug-candidate compounds:
Conclusions
The present ELISA based non-radioactive TK assay offers a reproducible, sensitive and rapid method to measure TK activity and enables large-scale screening of PDGFR 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
Recombinant PDGRF- enzyme activity
3. Screening A referenced inhibitor [SU- 6668(oxindol)] 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 set-up and characterize a non-radioactive TK assay platform to screen potential drug-candidate compound libraries designed against the ATP binding site of PDGFR receptor, representing a uniform functional target.
Criteria for an optimal screening assay
» 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
Results I. Results II.
Effective compound
Recombinant PDGFR-Kinetics
SU-6668 –positive control
Following optimization and standardization based on individual determination of kinetic parameters and calculation of Km values, reference PDGFR inhibitors were tested.
Based on stability, reproducibility and published reference SU-6668 was chosen as internal positive control for further tests.
One potent inhibitor was found from a nested chemical library of 20 compounds that will be entered in more detailed QSAR characterizations.
Ineffective compound
IC50=0,06uM
Li Sun et al, 1999
N
N R2
R1
R3
R4
R5
R6
R1, R2: alkyl, cycloalkyl, aryl, heteroaryl, or combinatedR3, R6: hydrogen, halogeneR4, R5: hydrogen, or nitro group
Benzo[g]quinoxaline derivates
y = 336,72x + 225,06
R2 = 0,9876Km=1,49
y = 146,81x + 113,65
R2 = 0,987Km= 1,3
-200
0
200
400
600
800
1000
-1,5 -1 -0,5 0 0,5 1 1,5 2 2,5
1/PGT (ug/ml)
1/v
50 ng PDGFR
20 ng PDGFR
Lineáris (20 ngPDGFR)Lineáris (50 ngPDGFR)
0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
1,6
1,8
0 50 100 150 200 250
PDGFR (ng/well)
0
20
40
60
80
100
120
140
0 5 10 15 20 25 30 35
Inhibitor (uM)
T/C%
Recombinant PDGFR
0
10
20
30
40
50
60
70
80
90
100
0 5 10 15 20 25 30 35
Inhibitor (uM)
T/C % Recombinant PDGFR
0
10
20
30
40
50
60
70
80
90
100
0 0,2 0,4 0,6 0,8 1 1,2 1,4
Inhibitor (uM)
T/C %
0
20
40
60
80
100
120
140
160
180
0 5 10 15 20 25 30 35
Inhibitor (uM)
T/C%