Molecular sensor and methods of use thereof

Patent metadata

Field	Value
Publication number	US-9696310-B2
Application number	US-201514738894-A
Country	US
Kind code	B2
Filing date	Jun 14, 2015
Priority date	Dec 31, 2012
Publication date	Jul 4, 2017
Grant date	Jul 4, 2017

Abstract

The present invention is directed to fluorescent molecular sensor based on Thiazole Orange for protein detection. Interaction of the protein target with the molecular sensors of this invention results in a significant increase in the fluorescence emission. The generation of light output signal enables one to detect protein biomarkers associated with different diseases or detecting the protein of interest also in living cells.

First claim

What is claimed:

1. A fluorescent monomolecular sensor, wherein said sensor is a Thiazole Orange-based protein identifier (TOPI) comprising a Thiazole Orange (TO) derivative and at least-one selective protein binders; wherein said sensor is represented by the structure of formula IX:

wherein
n, o, p and q are independently integers between 0 to 15;
An − is a counter ion, selected from tosylate (p-toluenesulfonate; CH 3 C 6 H 4 SO 3 − ), PF 6 − , CF 3 COO − , I − , Cl − , Br − , or F − ;
G 1 and G 2 are independently a bond, carbamate, amide, amine, C 1 -C 12 alkyl amine, ester, ketone, O, N, S, carbonate, —O-alkyl-NH—, carbamoyl phosphate or phosphate;
T 1 is hydrogen or

 wherein, n′ is between 0 and 15;
T 2 is hydrogen or

 wherein q′ is between 0 and 15;
V 1 , V 1′ , V 2 and V 2′ are independently a bond, a triazole, an amide [—C(O)NH or —NHC(O)], —C(O)O—, —OC(O)—, O, NH, N-alkyl, S, —PO 4 H—, C 1 -C 12 alkyl ether, —NH-alkyl-NH—, —O-alkyl-NH—, —NH-alkyl-O— wherein said alkyl is optionally substituted and wherein said alkyl is optionally interrupted by an heteroatom consisting of O, N, P, S or combination thereof
X 1 , X 1 ′, X 2 and X 2′ are independently a bond or C 1 -C 12 alkyl, —C(O)NH—, —NHC(O), —C(O)O—, —OC(O)—, O, NH, N-alkyl, S, —PO 4 H—, C 1 -C 12 alkyl ether, C 1 -C 12 -alkyl-NH, -alkyl-NHC(O)-alkyl, -alkyl-C(O)NH-alkyl, —NH-alkyl-NH—, —O-alkyl-NH—, —NH-alkyl-O— wherein said alkyl is optionally substituted and wherein said alkyl is optionally interrupted by an heteroatom consisting of O, N, P, S or combination thereof;
L 1 , L 1′ , L 2 and L 2′ are independently a bond or C 1 -C 12 alkyl, C(O), —C(O)NH—, —NHC(O)—, —C(O)O—, —OC(O)—, O, NH, C 1 -C 12 N-alkyl, S, —PO 4 H, —PO 4 H—{[(CH 2 ) y O] x } z —PO 3 H—, wherein y is between 1-5, x is between 1-10 and z is between 1-10, —PO 4 H—PEG, C 1 -C 12 alkyl ether, C 1 -C 12 alkylamine, C 1 -C 12 alkyl-NH, —C(O)NH-alkyl, —NHC(O)-alkyl, NH-alkyl-NH—, —O— alkyl-NH—, —NH-alkyl-O—, wherein said alkyl is optionally substituted and wherein said alkyl is optionally interrupted by an heteroatom consisting of O, N, P, S or combination thereof; and
R 1 , R 1′ , R 2 and R 2′ are independently hydrogen, halide, SO 3 − , CN, NO 2 , phosphate, SO 3 − or a selective protein binder;
wherein at least one of R 1 , R 1′ , R 2 and R 2′ is a protein selective binder.

2. The sensor of claim 1 , wherein said TOPI comprises two selective protein binders.

3. The sensor of claim 2 , wherein said protein is a homodimer.

4. The sensor of claim 1 , wherein said selective binder comprises: marimastat, ethacrynic acid, bisethacrynic acid, metal complex of nitrilotriacetic acid (NTA), metal complex of his NTA, metal complex of tris-NTA, PDGF-BB, heparin, FGF aptamer, biotin, tacrine, estrogen, DNA aptamer, RNA aptamer, peptide aldehyde, estrogen, suberoylanilidehydroxamic acid (SAHA), a peptide binder or any combination thereof.

5. The sensor according to claim 1 , represented by the structure of formula XIII:

6. The sensor of claim 5 , represented by the structure of formula X:

7. The sensor of claim 6 , wherein said sensor is represented by the structure of formula XI:

8. The sensor of claim 1 , wherein T 1 and T 2 are hydrogens.

9. The sensor of claim 1 , wherein said G 1 and G 2 are each independently a carbamate or an amide.

10. The sensor of claim 1 , wherein said V 1 and V 2 are each independently a triazole, an O, an NH or a bond.

11. The sensor of claim 1 , wherein said X 1 and X 2 are each independently a C 1 -C 12 alkyl, a C 1 -C 12 alkyl-NH, a C 1 -C 12 alkylether, -alkyl-C(O)NH-alkyl or a bond.

12. The sensor of claim 1 , wherein said L 1 and L 2 are each independently a bond, —PO 4 H—{[(CH 2 ) y O] x } z —PO 3 H—, wherein y is 2, x is 3 and z is 6; —PO 4 H-PEG; C 1 -C 12 alkyl-NH or a C 1 -C 12 alkyl.

13. The sensor of claim 1 , wherein said o and p are each independently 2 or 3.

14. The sensor of claim 1 , wherein said n and q are each independently 0, 1, 2 or 5.

15. The sensor of claim 1 , wherein R 1 and R 2 are both a selective binder.

16. The sensor of claim 1 , wherein said V 1 and V 2 are identical; X 1 and X 2 are identical; L 1 and L 2 are identical; R 1 and R 2 are identical; o and p are identical; and n and q are identical.

17. The sensor of claim 1 , wherein said sensor is compound 14, 140, 20, 26, 33, 34, 35, 36, or 37:

18. A method of detecting a protein of interest (POI) in a biological medium, said method comprise:
a. measuring an optical signal of the sensor of claim 1 ;
b. placing said sensor in said biological medium;
c. re-measuring the optical signal of said sensor in said biological medium,
wherein an enhancement in the optical signal of said sensor indicates on the presence of said POI in said biological medium.

19. The method of claim 18 , wherein said optical signal is fluorescence emission.

20. The method of claim 18 , wherein said POI is matrix metalloproteases (MMPs) protein and its isoforms, glutathione S-Transferase (GSTs) protein and its isoforms, platelet derived growth factor, Histidine-tagged proteins, estrogen receptor, fibroblast growth factor (FGF), caspases, PSA, fibronecin, lysozyme, β-amyloid, avidin, streptavidin, Acetylcholinesterase or histone deacetylases (HDACs).

21. The method of claim 18 , wherein said biological medium is blood, tissue, serum, or urine.

22. The method of claim 20 , wherein said GST is a biomarker for cancer, renal tubular injury and for monitoring graft failure or regeneration following living donor liver transplantation.

23. The method of claim 22 , wherein said GST comprises isoforms GSTA1, GSTA2, GSTM1, GSTK1, GSTO1, GSTZ1, GSTT1 and GSTP1 and said method further differentiates between said isoforms.

24. The method of claim 18 , wherein said POI is achetylcholine esterase (AChE), wherein said AChE is a biomarker for Alzheimer disease.

25. The method of claim 18 , wherein said sensor is compound 14, 140, 20, 26, 33, 34, 35, 36, or 37:

Assignees

• Yeda Res & Dev

Inventors

• Margulies David
• Motiei Leila
• Unger Linor

Classifications

• G01N33/575

  for cancer · CPC title

• C09K2211/1029

  containing one nitrogen atom as the heteroatom · CPC title

• G01N2333/96494

  Matrix metalloproteases, e. g. 3.4.24.7 · CPC title

• C07K7/06

  having 5 to 11 amino acids · CPC title

• C07D417/14

  containing three or more hetero rings · CPC title