Method for identifying modulators of notch signaling

Patent metadata

Field	Value
Publication number	US-10274481-B2
Application number	US-201615017986-A
Country	US
Kind code	B2
Filing date	Feb 8, 2016
Priority date	Dec 21, 2011
Publication date	Apr 30, 2019
Grant date	Apr 30, 2019

Abstract

The present invention relates to use of inhibitors of Notch signalling pathway selected from the group consisting of 6-(4-Tert-Butylphenoxy)Pyridin-3-Amine (I3), its derivatives, in treating and/or preventing cancers.

First claim

The invention claimed is:

1. A method for identifying modulators of NOTCH signalling in a cell culture, comprising
(i) co-culturing mammalian cells expressing a NOTCH ligand and mammalian cells expressing a NOTCH receptor to form a co-culture, wherein said mammalian cells expressing a NOTCH ligand are HeLa cells that have been transfected with a NOTCH ligand expression system and said mammalian cells expressing a NOTCH receptor are HeLa cells that have been transfected with a NOTCH receptor expression system;
(ii) incubating the co-culture with and without a candidate modulator of NOTCH signalling to be tested
(iii) quantifying the amount of a NOTCH intracellular domain (NICD) that has been released from a NOTCH receptor; and/or
(iv) quantifying the NICD-mediated modulation of a NOTCH signalling specific reporter system, wherein a reduction in the signal of the NOTCH signalling specific reporter system, a reduction of the amount of NICD, the absence of NICD and/or the absence of the signal of the NOTCH signalling specific reporter system in said co-culture that has been incubated with a candidate modulator of NOTCH signalling, compared to the signal of the NOTCH signalling specific reporter system and/or the amount of NICD in said co-culture that has been incubated without a candidate modulator of NOTCH signalling, is indicative of NOTCH signalling modulation.

2. The method according to claim 1 , wherein the expressed NOTCH ligand is selected from the group consisting of Delta 1 (DL1), Delta 3 (DL3), Delta 4 (DL4), Jagged 1 and Jagged 2.

3. The method according to claim 2 , wherein the expressed NOTCH ligand is DL4.

4. The method according to claim 1 , wherein the expressed NOTCH receptor is selected from the group consisting of a NOTCH1 receptor, a NOTCH2 receptor, a NOTCH3 receptor and a NOTCH4 receptor.

5. The method according to claim 4 , wherein the expressed NOTCH receptor is a NOTCH1 receptor or a NOTCH2 receptor.

6. The method according to claim 1 , wherein the NICD is released from the NOTCH receptor which is expressed by said mammalian cells expressing a NOTCH receptor.

7. The method according to claim 1 , wherein the mammalian cells expressing a NOTCH ligand and the mammalian cells expressing a NOTCH receptor are co-cultured in a ratio of 1:5 to 5:1.

8. The method according to claim 1 , wherein the mammalian cells expressing a NOTCH ligand and the mammalian cells expressing a NOTCH receptor are co-cultured in a ratio of 1:1.

9. The method according to claim 1 , wherein the mammalian cells expressing a NOTCH ligand and the mammalian cells expressing a NOTCH receptor have about 100% confluency prior to incubation with and without a candidate modulator of NOTCH signalling to be tested.

10. The method according to claim 1 , wherein said mammalian cells that have been transfected with a NOTCH ligand expression system have been stably transfected with a NOTCH ligand expression system; and/or
wherein said mammalian cells that have been transfected with a NOTCH receptor expression system have been stably transfected with a NOTCH receptor expression system.

11. The method according to claim 1 , wherein said mammalian cells that have been transfected with a NOTCH ligand expression system have been transiently transfected with a NOTCH ligand expression system; and/or
wherein said mammalian cells that have been transfected with a NOTCH receptor expression system have been transiently transfected with a NOTCH receptor expression system.

12. The method according to claim 1 , wherein said mammalian cells expressing a NOTCH receptor have been transfected with at least one plasmid encoding a NOTCH signalling specific reporter system.

13. The method according to claim 12 , wherein the NOTCH signalling specific reporter system comprises a C promoter binding factor 1 (CBF1), suppressor of hairless, Lag1 (CSL) transcription factor binding site in the promoter region and a reporter protein.

14. The method according to claim 13 , wherein the reporter protein is selected from the group consisting of a reporter luciferase, a reporter beta-galactosidase and a reporter fluorescent protein.

15. The method according to claim 14 , wherein said reporter fluorescent protein is selected from the group consisting of GFP, eGFP, tomato, cherry, dsRED, Venus and Cyan.

16. The method according to claim 13 , wherein the reporter protein is a luciferase.

17. The method according to claim 13 , wherein the reporter protein is a firefly ( Photinus pyralis ) luciferase.

18. The method according to claim 12 , wherein said mammalian cells expressing a NOTCH receptor have been further transfected with at least one plasmid encoding a further reporter system.

19. The method according to claim 18 , wherein said further reporter system comprises a Renilla ( Renilla reniformis ) luciferase.

20. The method according to claim 1 , wherein said NOTCH ligand expression system is a lentivirus containing a NOTCH ligand cDNA and/or wherein said NOTCH receptor expression system is a lentivirus containing a plasmid comprising mouse NOTCH receptor cDNA.

21. The method according to claim 1 , wherein the cells of said cell culture are lysed prior to
quantifying the amount of a NOTCH intracellular domain (NICD) that has been released from a NOTCH receptor; and/or
quantifying the NICD-mediated modulation of a NOTCH signalling specific reporter system.

22. The method according to claim 1 , wherein the amount of NICD in (iii) is quantified using Western blotting.

23. A method for identifying modulators of NOTCH signalling by high throughput screening of a library comprising candidate modulators of NOTCH signalling comprising the steps of:
(a) providing an assay in wells of a microwell plate wherein the assay comprises a co-culture comprising mammalian cells expressing a NOTCH ligand and mammalian cells expressing a NOTCH receptor, wherein said mammalian cells expressing a NOTCH ligand are HeLa cells that have been transfected with a NOTCH ligand expression system and said mammalian cells expressing a NOTCH receptor are HeLa cells that have been transfected with a NOTCH receptor expression system;
(b) incubating the co-culture of said assay with and without a candidate modulator of NOTCH signalling to be tested; and
(c)(i) quantifying the amount of a NOTCH intracellular domain (NICD) that has been released from the NOTCH receptor; and/or
(c)(ii) quantifying the NICD-mediated modulation of a NOTCH signalling specific reporter system;
wherein a reduction in the signal of the NOTCH signalling specific reporter system, a reduction of the amount of NICD, the absence of NICD and/or the absence of the signal of the NOTCH signalling specific reporter system in said co-culture that has been incubated with a candidate modulator of NOTCH signalling, compared to the signal of the NOTCH signalling specific reporter system and/or the amount of NICD in said co-culture that has been incubated without a candidate modulator of NOTCH signalling, is indicative of NOTCH signalling modulation.

24. The method according to claim 23 , wherein incubation in step (b) lasts from 30 min to 48 h.

25. The method according to claim 23 , wherein incubation in step (b) lasts at least 12 hours.

Assignees

• Ecole Polytechnique Fed Lausanne Epfl

Inventors

• Radtke Freddy
• Lehal Rajwinder
• Reinmüller Viktoria
• Zhu Jieping

Classifications

• G01N33/57595

  involving intracellular compounds · CPC title

• G01N33/5759

  involving compounds localised on the membrane of tumour or cancer cells · CPC title

• G01N33/5011 Primary

  for testing antineoplastic activity · CPC title

• G01N33/57492

  Physics · mapped topic

• C12N15/09

  Recombinant DNA-technology · CPC title