INTRACELLULAR ORGANELLE PEPTIDE TARGETED ENZYME SUBSTRATES

• US 20150219654A1
• Filed: 04/17/2015
• Published: 08/06/2015
• Est. Priority Date: 03/11/2009
• Status: Active Grant

• Current Assignee: Marker Gene Technologies Incorporated (Abcam Plc)
• Original Assignee: Marker Gene Technologies Incorporated (Abcam Plc)
• Inventors: Naleway, John Joseph, Harlan, Fiona Karen, Lusk, Jason Scott
• Granted Patent: US 9,841,426 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 31/445 Non condensed piperidines, e.g. piperocaine
• CPC Class Codes: A61K 47/64 Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent peptidic linkers A61K47/65
• CPC Class Codes: A61K 47/645 Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
• CPC Class Codes: C07K 7/06 having 5 to 11 amino acids
• CPC Class Codes: C07K 7/08 having 12 to 20 amino acids gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68
• CPC Class Codes: C12Q 1/34 involving hydrolase
• CPC Class Codes: G01N 33/5076 involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
• CPC Class Codes: G01N 33/531 Production of immunochemical test materials
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

This invention relates to substrates and methods for the visualization of intracellular organelles, such as the lysosome, peroxiosome, nucleus, Endoplasmic Reticulum and Golgi Apparatus, based upon organelle enzyme activity. Such compounds represent a novel combination of chemically distinct enzyme substrates with targeting and detection substrates which are activated by enzyme activity inside target organelles to produce a detectable signal. The organelle targeted enzyme substrates of this invention are designed to provide high fluorescence at lower pH values found in some organelles and can be used for monitoring enzyme activity inside cells at very low concentrations.

Claims

1. A method of detecting enzyme activity in cellular organelles, comprising:

a) combining a sample that comprises an isolated organelle or a living cell containing the organelle with a biocompatible solution comprising a substrate of the formula;

T−

LINK−

F(R)−

BLOCK(R′

)

where;

1) T represents a targeting group that is a peptide containing a sequence compound that partitions the substrate to the target organelle;

2) F represents a reporter that has further elaboration with one or more substituents R to provide for a visible signal;

3) BLOCK is a monovalent moiety adapted to be cleaved from the remainder of the substrate by action of a specific enzyme native to said organelle, resulting in a visible signal at the site of enzyme reaction; and

b) said sample and substrate are contacted for a time sufficient for said native enzyme to remove BLOCK and produce said visible signal.

2. The method of claim 1, wherein said organelle is selected from the group consisting of lysosomes, nucleus, peroxisomes, the Golgi Apparatus and the Endoplasmic Reticulum.

3. The method of claim 1, wherein said visible signal is selected from the group consisting of fluorescence, chemiluminescence and chromogenic staining.

4. The method of claim 1, wherein said, wherein F is selected from the group consisting of an anthracene, a benzphenalenone, a coumarin, a fluorescein, a naphthofluorescein, a naphthalene, a phenalenone, a pyrene, a resorufin, a dioxetane, an indole, a luminol and a rhodamine.

5. The method of claim 1, wherein said BLOCK is selected from the group consisting of:

(i) a monovalent moiety derived by removal of a hydroxy group from phosphate or sulfate, (ii) a biologically compatible salt of (i);

(iii) a monovalent moiety derived by removal of a hydroxy group from a carboxy group of an aliphatic, aromatic or amino acid or of a peptide; and

(iv) a monovalent moiety derived by removal of an anomeric hydroxy group from a mono- or polysaccharide.

6. The method of claim 1, wherein said LINK comprises from 1 to 20 nonhydrogen atoms selected from the group consisting of C, N, O and S.

7. The method of claim 1, wherein said LINK comprises from 1 to 6 carbon atoms.

8. The method of claim 1 where LINK has the formula —

(CH2)a(CONH(CH2)b)z-, where a is an integer 0-5, b is 1-5 and z is 1.

9. The method of claim 1, further comprising adding an additional detection reagent to said sample.

10. The method of claim 7, wherein said additional detection reagent is selected from the group consisting of a membrane probe, a mitochondrial stain and a nucleic acid stain.

11. The method of claim 1, wherein said sample comprises an abnormal cell.

12. The method of claim 9 wherein the presence of said visible signal is indicative of a disease state present in said abnormal cell.

13. The method of claim 11, further comprising sorting said cells based on the fluorescent response.

14. The method of claim 1, wherein the presence of said visible signal is indicative of a metabolic response to drug activity in said acidic organelle.

15. The method of claim 1, wherein T represents a targeting group comprising a peptide selected from the group consisting of -AKL-, -KKKRK-, -KDEL- and -SCYQRL-.

16. The method of claim 1, wherein T represents a targeting group comprising a peptide having an amino acid sequence selected from the group consisting of:

SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

18, SEQ ID NO;

19, SEQ ID NO;

20, SEQ ID NO;

21, SEQ ID NO;

22, SEQ ID NO;

23, SEQ ID NO;

25, and SEQ ID NO;

26.

17. The method of claim 1, wherein said substrate comprises a formula selected from the group consisting of:

18. A method of detecting enzyme activity in cellular organelles, comprising:

a) combining a sample that comprises an isolated organelle or a living cell containing the organelle with a biocompatible solution comprising a substrate of the formula;

T−

LINK−

F(R)−

BLOCK(R′

)

where;

1) T represents a targeting group that is a peptide having an amino acid sequence selected from the group consisting of;

SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

18, SEQ ID NO;

19, SEQ ID NO;

20, SEQ ID NO;

21, SEQ ID NO;

22, SEQ ID NO;

23, SEQ ID NO;

25, and SEQ ID NO;

26;

2) F represents a reporter that has further elaboration with one or more substituents R to provide for a visible signal;

3) BLOCK is a monovalent moiety adapted to be cleaved from the remainder of the substrate by action of a specific enzyme native to said organelle, resulting in a visible signal at the site of enzyme reaction; and

b) said sample and substrate are contacted for a time sufficient for said native enzyme to remove BLOCK and produce said visible signal.

19. A substrate for detecting enzyme activity in cellular organelles, said substrate comprising the formula:

T−

LINK−

F(R)−

BLOCK(R′

)

where;

1) T represents a targeting group that is a peptide containing a sequence compound that partitions the substrate to the target organelle;

2) F represents a reporter that has further elaboration with one or more substituents R to provide for a visible signal;

3) BLOCK is a monovalent moiety adapted to be cleaved from the remainder of the substrate by action of a specific enzyme native to said organelle, resulting in a visible signal at the site of enzyme reaction.

20. The substrate of claim 19 wherein said targeting group is a peptide having an amino acid sequence selected from the group consisting of:

SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

18, SEQ ID NO;

19, SEQ ID NO;

20, SEQ ID NO;

21, SEQ ID NO;

22, SEQ ID NO;

23, SEQ ID NO;

25, and SEQ ID NO;

26.