TESTING AND CHARACTERISING FOCUSED TYROSINE KINASE

INHIBITORY LIBRARIESGyörgyi Bökönyi1, Eszter Schafer2, Edit Várkondi2, Edit Z. Szabó1, Frigyes Wáczek2, Zsolt Székelyhidi2, Péter

G. Bánhegyi2, 2, György Mészáros2,Dániel Erős2, László Őrfi2-3, Á. Pap2, Richard E. Schwab2, György Kéri1

Hungarian Academy of Sciences Peptide Biochemistry Research Group 1

Co-operative Res. Centre2, Dept. Pharm. Chem.3 Semmelweis University, Budapest, Hungary

Cooperative Research CenterSemmelweis University

Budapest, Hungary

Dept. of GastroenterologyMÁV Hospital

Budapest, Hungary

Introduction

Aims

ELISA based non-radioactive TK assays offer reproducible, simple and rapid methods to measure kinase activity and enables large-scale screening of TK inhibitors. However, in our hands, methodological burdens seem to limit the sensitivity of ELISA-based approaches (relatively high background). In this respect ECL-based or fluorescent technologies seem to be superior on the „cost of the prize” of the assay. Because of these difficulties we have worked out a screening strategy using A431 EGFR overexpressing cell lines in proliferation assays for prescreening. The assay was carried out in two timepoints to distinguish between apoptotic and necrotic effect. The compounds were screened in core structure groups in order to select the best core structures for further synthetic work. The best compounds were further analyzed in EGF-RTK assay and in specific apoptotic assay with FACS. Analyzing the structures of several active inhibitors revealed that most of the potent compounds contained a condensed bicyclic heteroaryl moiety where the pyrimidine ring seems to be crucial.

Criteria for an optimal screening assay

»Low-to medium throughput platform»High Sensitivity»Robust (stable assay conditions)»Reproducible (inter and intra-assay)»Relevant (validated mol. targets incorporated)»Informative (to be extrapolated to cell. assays)»Rapid, simple»Cost effective

Screening strategy

Kinase assay

ApoptosisCell proliferation

Biochemical assays

Proliferation assays

FACS

EGFR

EGFR

MTTMB

MTT

Materials

Methods

Poly-Glu-Tyr

P-Tyr

OPD

ELISAreader

(490nm))

96-well mikrotiter plate

Tyr

ATP ENZYME

P-Tyr

+

well

Oxidized OPD

H2SO4

HRP conj. Ab

Deoxidized OPD

Results I.

Lineweaver Plot of EGFR

To determine the optimal concentration of tyrosine kinase enzyme and its substrate, we determined the kinetic parameters (Km and Vmax values) for VEGFR, EGFR and PDGFR. Inhibitory effect of ÖL-57 had the most stable and reproducible inhibitory effect of 90% and thus seems optimal for positive control in future experiments.

ELISA-based non-radiactive assayCellular antiproliferative assays

SummaryTime course changes during apoptosis

by Flow cytometry

7H-Pyrrolo[2,3-d]pyrimidine

5,6,7,8-Tetrahydro-benzo[4,5]thieno[2,3-d]pyrimidine

Quinazoline

The selected set of compounds can be grouped around 3 core structures.Condensed bicyclic heteroaryl cores containing a pyrimidine ring have been synthesized using ortho-cyanoarylamine building blocks. The resulting compounds and intermediates were characterized via MS and NMR spectroscopy and analyzed by HPLC for purity

Tyrosine kinases have been shown to play a crucial role in signal transduction pathways and have been implicated as key players in many „proliferative disorders” including cancer and atherosclerosis. Inhibitors designed against potential novel kinase targets are in the focus of the drug discovery today.Our group has designed and synthesized a large set of new potential inhibitory compounds, competing for the ATP-binding site in the catalytic domain of Protein Tyrosine Kinases (PTK), the leading-structural target of these enzymes nowadays.

Our aim was to characterize the efficacy of these compounds by testing them with relevant bioassays:»in vitro cell proliferation assays»Non-radioactive tyrosine kinase assays» flow cytometry

y = 7750,9x + 338,16

R2 = 0,9996Km=22,7

y = 3503,3x + 130,3

R2 = 0,9956Km=27

-200

200

600

1000

1400

1800

-0,1 0 0,1 0,2

1/[PGT-Substrate] (ug/ml)1/v

50ng EGFR

25ng EGFR

Lineáris(25ng EGFR)Lineáris(50ng EGFR)

Reference inhibitor

Results II.

Single cellsuspension

MTT* ELISA Reader(570/640 nm)

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

4,0

60.000 30.000 15.000 7.500 3.750 1875 937 469 243 117 Blank

cell No.

OD

Cells: A431, Panc-1

Cell counting

Cell Inoculation andSample Treatment

*Carmichael J et al. Cancer Res. 47(4): pp. 936-42, 1987**Oliver M.H. et al. J. Cell. Sci. 92(3): pp. 513-18, 1989

ELISA Reader(590 nm)

•40.000 cell/well = 6h

•10.000 cell/well = 48h

MB**

Solubilization

37ºC, 5 %, CO2, 10% FCS

Results I.

Results II.

0

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120

1 10 20 30 40

uMol/L

T/C

%

6h

48h

0

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120

1 10 20 30 40 50

uMol/L

T/C

%

0

20

40

60

80

100

120

1 10 20 30 40

uMol/L

T/C

%

6h

48h

GIF%

Toxic

Effective

No effect

GIF: (Growth Inhibitory Factor) is calculated from the differences of T/C values after 6h and 48h post-treatment, respectively.

Mean (+/- SD) in vitro antiproliferative effect of tested compounds (n=12) at 6

hours

Mean (+/- SD) in vitro antiproliferative effect of tested compounds (n=12) at 48

hours

Analysis of apoptosis induction was based on Annexin-V and propidium iodide staining. Early apoptotic cells in this assay are identified as propidium iodide negative + Annexin-V positive cell population (see upper right plot). Normal cells are negative for both dyes (see upper left plot), whereas the necrotic / late apoptotic cell-population is positive for both dyes (see lower right plot).

10 % of the compounds showed superior than 80% efficacy regarding growths inhibition connected to minimum in vitro toxicity.

Panc-1

Mean in vitro efficacy of selected compound(n=4) on EGFRTK activity

Mean in vitro efficacy of selected compound(n=8) on EGFRTK activity

0

20

40

60

80

100

120

140

160

50 10 2 0,4

Inbitor(uM)

T/C

(%

)

0

20

40

60

80

100

120

140

50 10 2 0,4

Inhibitor (uM)

T/C

(%

)

5 % of the compounds showed superior than 80% efficacy regarding kinase inhibition connected to minimum in vitro toxicity.

-20

0

20

40

60

80

100

0,1 1 10 100

log ÖL57 (uM)

T/C

%

100ng EGFR75ng EGFR

50ng EGFR

6h 16h

24h48h

Calculated logP:4,64

Calculated logP:3,31

Calculated logP:3,88

0

20

40

60

80

100

120

50 10 2 0,4 0,08

Inhibitor (uM)

T/C

(%

)

0

20

40

60

80

100

120

50 10 2 0,4 0,08

Inhibitor (uM)

T/C

(%

)