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

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-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)

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Inhibitor (uM)

T/C%

Recombinant PDGFR

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Inhibitor (uM)

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