HNQO1-activatable fluorescent probe for imaging cancer cells in-vitro and in-vivo

Patent metadata

Field	Value
Publication number	US-11957766-B2
Application number	US-202016950107-A
Country	US
Kind code	B2
Filing date	Nov 17, 2020
Priority date	Nov 26, 2019
Publication date	Apr 16, 2024
Grant date	Apr 16, 2024

Abstract

The present invention includes a probe, an assay, a method of detecting, a human NAD(P)H quinone oxidoreductase-1 (hNQO1) enzyme activity with a fluorescent probe comprising a quinone propionic acid (QPA) conjugated to dicyanoisophorone (DCP), wherein the hNQO1 reduces the probe to releases a fluorescent DCP, and a method of making the same.

First claim

What is claimed is:

1. An assay to detect human NAD(P)H quinone oxidoreductase-1 (hNQO1) enzyme activity comprising:
contacting an hNQO1 enzyme with a probe comprising a quinone propionic acid (QPA) conjugated to dicyanoisophorone (DCP), wherein the hNQO1 reduces the probe and releases a fluorescent DCP.

2. The assay of claim 1 , wherein the probe is defined as further comprising an ester or amide bond between the QPA and the DCP.

3. The assay of claim 1 , wherein the probe has at least one of: a large stokes shift, a sensitivity and selectivity against hNQO1, no cytotoxicity up to 100 μM, or cell permeability.

4. The assay of claim 1 , the probe has the formula:

5. The assay of claim 1 , wherein a redox activity of hNQO1 reduces a quinone moiety of QPA into an o-hydroxydihydrocinnamic acid derivative that undergoes lactonization under physiological conditions to yield dihydrocoumarin and fluorescent DCP.

6. The assay of claim 1 , wherein the hNQO1 enzyme is in a cell, a tissue, an organ, or a cancer.

7. The assay of claim 1 , wherein the hNQO1 enzyme is in a breast, lung, prostate, stomach, colon, pancreatic, brain, or head and neck cancer.

8. A probe comprising a quinone propionic acid (QPA) conjugated to a dicyanoisophorone (DCP).

9. The probe of claim 8 , wherein the quinone propionic acid (QPA) is conjugated to the dicyanoisophorone (DCP) by an ester or amide bond.

10. The probe of claim 8 , wherein the probe has the formula:

11. The probe of claim 8 , wherein the probe has at least one of: a large stokes shift, a sensitivity and selectivity against hNQO1, no cytotoxicity up to 100 μM, or cell permeability.

12. The probe of claim 8 , wherein the probe is reduced by a human NAD(P)H quinone oxidoreductase-1 (hNQO1) enzyme.

13. The probe of claim 8 , wherein the probe is a dicyanoisophorone (DCP) fluorophore conjugated to a NQO1 substrate quinone propionic acid (QPA).

14. The probe of claim 8 , wherein a redox activity of hNQO1 reduces a quinone moiety of QPA into an o-hydroxydihydrocinnamic acid derivative that undergoes lactonization under physiological conditions to yield dihydrocoumarin and fluorescent DCP.

15. The probe of claim 8 , wherein the hNQO1 enzyme is in a cell, a tissue, an organ, or a cancer.

16. The probe of claim 8 , wherein the hNQO1 enzyme is in a breast, lung, prostate, stomach, colon, pancreatic, brain, or head and neck cancer.

17. A method of detecting a cancer cell that expresses a human NAD(P)H quinone oxidoreductase-1 (hNQO1) enzyme comprising:
contacting the cancer cell with a probe comprising a quinone propionic acid (QPA) conjugated to dicyanoisophorone (DCP), wherein the hNQO1 reduces the probe and releases a fluorescent DCP; and detecting fluorescence in the cancer cell.

18. The method of claim 17 , wherein the quinone propionic acid (QPA) is conjugated to the dicyanoisophorone (DCP) by an ester or amide bond.

19. The method of claim 17 , wherein the quinone propionic acid (QPA) conjugated to dicyanoisophorone (DCP) has the formula:

20. The method of claim 17 , wherein the probe has at least one of: a large stokes shift, a sensitivity and selectivity against hNQO1, no cytotoxicity up to 100 μM, or cell permeability.

21. The method of claim 17 , wherein a redox activity of hNQO1 reduces a quinone moiety of QPA into an o-hydroxydihydrocinnamic acid derivative that undergoes lactonization under physiological conditions to yield dihydrocoumarin and fluorescent DCP.

22. The method of claim 17 , wherein the fluorescence is detected in vivo.

23. The method of claim 17 , further comprising the step of obtaining a sample from a subject and measuring the activity of the hNQO1 in the sample.

24. The method of claim 17 , wherein the cancer is a breast, lung, prostate, stomach, colon, pancreatic, brain, or head and neck cancer.

25. A method of making a probe comprising a quinone propionic acid (QPA) conjugated to dicyanoisophorone (DCP) comprising:
carrying out the following reaction:

wherein the conditions are:
(a) ammonium acetate, acetic anhydride, acetic acid, toluene, reflux, 12 h;
(b) piperidine, acetonitrile, 50° C., 6 h;
(c) SnCl 2 , HCl, ethyl acetate, 8 h;
(d) methyl 3,3-dimethylacrylate, methanesulfonic acid, 70° C., 2 h;
(e)N-Bromosuccinimide, acetonitrile, water, room temperature, 1 h; and
(f) 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, pyridine, rt, 10 h.

Assignees

• Univ Texas Tech System

Inventors

• Srivenugopal Kalkunte S
• Punganuru Surendra R

Classifications

• G01N33/57595

  involving intracellular compounds · CPC title

• G01N33/575

  for cancer · CPC title

• A61K49/0052 Primary

  Small organic molecules (oligomers, polymers, dendrimers A61K49/0054) · CPC title

• C07C255/38

  the carbon skeleton being further substituted by esterified hydroxy groups · CPC title

• C07C255/44

  at least one of the singly-bound nitrogen atoms being acylated · CPC title