Self-immolative probes for enzyme activity detection

Patent metadata

Field	Value
Publication number	US-10718005-B2
Application number	US-201816177676-A
Country	US
Kind code	B2
Filing date	Nov 1, 2018
Priority date	Nov 16, 2010
Publication date	Jul 21, 2020
Grant date	Jul 21, 2020

Abstract

Provided is a compound having the structure:

(SIG)-(SI-MOD) m

where SIG is a signaling molecule, SI is a self-immolative structure bound to SIG such that SIG has a reduced signal relative to the signal of SIG without SI, MOD is a moiety bound to SI that is subject to modification by an activator, and m is an integer from 1 to about 10. When MOD is modified by an activator, SI is destabilized and self-cleaved from SIG such that SIG generates an increased signal. Also provided are methods of determining whether a sample, such as a cell, comprises an activator, such as a nitroreducase, using the compound. Further provided are methods of determining whether a mammalian cell is hypoxic using the compound where nitroreductase is the activator. A method of detecting a microorganism that comprises a nitroreductase using the compound where nitroreductase is the activator is also provided.

First claim

What is claimed is:

1. A compound for detecting histone deacetylase activity, said compound consisting of the structure:

wherein SIG is a signaling moiety,
wherein SI is a self-immolative structure bound to SIG such that SIG has a reduced signal relative to the signal of SIG without SI,
wherein m is an integer from 1 to 10, and
wherein, when the compound is modified by histone deacetylase activity, SI is destabilized and self-cleaved from SIG such that SIG generates an increased signal.

2. The compound of claim 1 , wherein SIG is a fluorescent moiety or a luminescent moiety.

3. The compound of claim 1 , wherein m is an integer from 2 to 10.

4. The compound of claim 3 , wherein SIG is a fluorophore selected from a symmetric or asymmetric cyanine, a merocyanine, a styryl moiety, an oxazine, a xanthene, a coumarin, an iminocoumarin, a xanthene, a rhodamine, a rhodamine derivative, a rosamine, a rosamine derivative, a rhodol or a rhodol derivative.

5. The compound of claim 1 , wherein m=1.

6. The compound of claim 5 , wherein SIG is a fluorophore selected from a symmetric or asymmetric cyanine, a merocyanine, a styryl moiety, an oxazine, a xanthene, a coumarin, an iminocoumarin, a xanthene, a rhodamine, a rhodamine derivative, a rosamine, a rosamine derivative, a rhodol or a rhodol derivative.

7. The compound of claim 1 , consisting of the structure:

wherein each R 1 is independently a hydrogen, a halogen, Z, a cyano group, an isocyano group, a thiocyano group, an isothiocyano group, an azido group, a trihalomethyl group; a sulfonate group, a sulfate group, a carboxyl group, a carbonyl group, an ester group, an amido group, a carbamate group, a phosphate group, a phosphonate group, an amino group, an alkoxy group, a thiol group, a sulfoxy group, a sulfone group, a sulfonamide group, a phosphino group, or a silane group,
wherein Z is a reducible nitrogen-containing group, or an amino group bound to an electron deficient moiety, and

wherein n is 0, 1, 2, 3, or 4.

8. The compound of claim 7 , wherein SIG is a fluorescent moiety or a luminescent moiety.

9. The compound of claim 7 , wherein m is an integer from 2 to 10.

10. The compound of claim 9 , wherein SIG is a fluorophore selected from a symmetric or asymmetric cyanine, a merocyanine, a styryl moiety, an oxazine, a xanthene, a coumarin, an iminocoumarin, a xanthene, a rhodamine, a rhodamine derivative, a rosamine, a rosamine derivative, a rhodol or a rhodol derivative.

11. The compound of claim 7 , wherein m=1.

12. The compound of claim 11 , wherein SIG is a fluorophore selected from a symmetric or asymmetric cyanine, a merocyanine, a styryl moiety, an oxazine, a xanthene, a coumarin, an iminocoumarin, a xanthene, a rhodamine, a rhodamine derivative, a rosamine, a rosamine derivative, a rhodol or a rhodol derivative.

13. A method for detecting histone deacetylase activity in a sample, the method comprising
(a) incubating the sample with the compound of claim 1 for a time and under conditions sufficient for the compound to be modified by a histone deacetylase if present in the sample; and
(b) measuring the signal generated by SIG, wherein the amount of signal generated is proportional to the amount of histone deacetylase activity in the sample.

14. The method of claim 13 , wherein the sample is a fluid of an organism or a colony of organisms, or an extract thereof.

15. A method for detecting histone deacetylase activity in a sample, the method comprising
(a) incubating the sample with the compound of claim 7 for a time and under conditions sufficient for the compound to be modified by a histone deacetylase if present in the sample; and
(b) measuring the signal generated by SIG, wherein the amount of signal generated is proportional to the amount of histone deacetylase activity in the sample.

16. The method of claim 15 , wherein the sample is a fluid of an organism or a colony of organisms, or an extract thereof.

17. A compound consisting of the structure:

18. The compound of claim 17 , consisting of the structure:

19. The compound of claim 17 , consisting of the structure:

20. The compound of claim 17 , consisting of the structure:

21. A method for detecting histone deacetylase activity in a sample, the method comprising
(a) incubating the sample with the compound of claim 17 for a time and under conditions sufficient for the compound to be modified by a histone deacetylase if present in the sample; and
(b) measuring the luminescent signal generated by the compound, wherein the amount of signal generated is proportional to the amount of histone deacetylase activity in the sample.

22. The method of claim 21 , wherein the sample is a fluid of an organism or a colony of organisms, or an extract thereof.

Assignees

• Enzo Life Sciences Inc

Inventors

• Lebedeva Irina
• Patton Wayne Forrest
• Szczepanik Maciej
• Xiang Yuejun
• Pande Praveen

Classifications

• G01N2333/92

  Triglyceride splitting, e.g. by means of lipase · CPC title

• G01N2333/918

  Carboxylic ester hydrolases (3.1.1) · CPC title

• C07C271/08

  having oxygen atoms of carbamate groups bound to acyclic carbon atoms · CPC title

• C07D491/052

  the oxygen-containing ring being six-membered · CPC title

• G01N2500/00

  Screening for compounds of potential therapeutic value · CPC title