Systems and devices for protease detection based on engineered polymers and biopolymers and methods of use

Patent metadata

Field	Value
Publication number	US-11268127-B2
Application number	US-201515116750-A
Country	US
Kind code	B2
Filing date	Feb 4, 2015
Priority date	Feb 4, 2014
Publication date	Mar 8, 2022
Grant date	Mar 8, 2022

Abstract

The presently disclosed subject matter relates to modular peptide-substrate protease assays, in particular low cost and reliable methodology for measurement of protease and other enzyme activity that may be detected by optical turbidimetry or visual observation. The presently disclosed subject matter also relates to methods of using the disclosed assays within methods of detecting and monitoring diseases, methods of drug discovery, as well as in detection devices and systems.

First claim

What is claimed:

1. A method for detecting protease activity in a sample comprising
a) contacting the sample with amphiphilic fusion polypeptides, wherein each of the fusion polypeptides comprise repeats of a hydrophobic domain of an elastin-like polypeptide having a lower critical solution temperature and repeats of a hydrophilic domain separated by a protease substrate domain;
b) providing a stimulus in response to which the fusion polypeptides self-assemble into micelles comprising coronae comprising the protease substrate domains;
c) providing conditions in which a protease present in the sample will cleave the protease substrate domain, whereby the hydrophobic domains of the micelles aggregate into particles at a detectable aggregation rate; and
d) detecting the aggregation rate of the particles in the sample;
wherein the aggregation rate of the particles in the sample is positively correlated to the protease activity in the sample.

2. The method of claim 1 , wherein the protease substrate domains comprise a cleavage site for a protease selected from the group consisting of a serine protease, a threonine protease, a cysteine protease, an asparatic protease, a glutamic protease, and a metalloproteinase.

3. The method of claim 1 , wherein detecting the aggregation rate of particles in the sample comprises optical turbidimetry.

4. The method of claim 1 , wherein detecting the aggregation rate of particles in the sample comprises visual observation.

5. The method of claim 1 wherein the hydrophobic domain comprises repeats of the amino acid sequence GVGVP (SEQ ID NO: 23) or VPGVG (SEQ ID NO: 4).

6. The method of claim 5 wherein the hydrophobic domain comprises sixty (60) repeats of the amino acid sequence GVGVP (SEQ ID NO: 23) or VPGVG (SEQ ID NO: 4).

7. The method of claim 5 wherein the hydrophobic domain comprises sixty (60) repeats of the amino acid sequence GVGVP (SEQ ID NO: 23) or VPGVG (SEQ ID NO: 4).

8. The method of claim 1 wherein the hydrophilic domain comprises DEGQQDDEEGY (SEQ ID NO: 11) or repeats of VPGAGVPGGG (SEQ ID NO: 5).

9. The method of claim 1 wherein the hydrophilic domain comprises thirty (30) repeats of VPGAGVPGGG (SEQ. ID NO: 5).

10. The method of claim 1 wherein the hydrophilic domain is located at the C-terminal of the protease substrate domain.

11. The method of claim 1 wherein:
the hydrophobic domain comprises repeats of the amino acid sequence GVGVP (SEQ ID NO: 23) or VPGVG (SEQ ID NO: 4);
the hydrophilic domain comprises DEGQQDDEEGY (SEQ ID NO: 11) or repeats of VPGAGVPGGG (SEQ ID NO: 5); and
the hydrophilic domain is located at the C-terminal of the protease substrate domain.

12. The method of claim 11 wherein the hydrophobic domain comprises sixty (60) repeats of the amino acid sequence GVGVP (SEQ ID NO: 23) or VPGVG (SEQ ID NO: 4) and the hydrophilic domain comprises thirty (30) repeats of VPGAGVPGGG (SEQ ID NO: 5).

13. The method of claim 11 wherein the hydrophobic domain comprises sixty (60) repeats of the amino acid sequence GVGVP (SEQ ID NO: 23) or VPGVG (SEQ ID NO: 4) and the hydrophilic domain comprises thirty (30) repeats of VPGAGVPGGG (SEQ ID NO: 5).

14. The method of claim 1 wherein the hydrophilic domain comprises DEGQQDDEEGY (SEQ ID NO: 11) or repeats of VPGAGVPGGG (SEQ ID NO: 5).

15. The method of claim 1 wherein the hydrophilic domain comprises thirty (30) repeats of VPGAGVPGGG (SEQ ID NO: 5).

16. The method of claim 1 wherein the hydrophilic domain is located at the C-terminal of the protease substrate domain.

17. The method of claim 1 wherein:
the hydrophobic domain comprises repeats of the amino acid sequence GVGVP (SEQ ID NO: 23) or VPGVG (SEQ ID NO: 4);
the hydrophilic domain comprises DEGQQDDEEGY (SEQ ID NO: 11) or repeats of VPGAGVPGGG (SEQ ID NO: 5); and
the hydrophilic domain is located at the C-terminal of the protease substrate domain.

18. A method of predicting or diagnosing a disease in a subject comprising:
determining the activity of a protease in a biological sample of the subject using the method of claim 1 relative to the activity of the protease from a control sample from the subject or a control sample from subjects who do not have the disease;
wherein a significant difference between the activity of the protease in the biological sample and the control sample is indicative that the subject has or is susceptible to developing the disease.

19. The method of claim 18 , wherein the disease is selected from the group consisting of an infectious disease, an inflammatory disease, a cardiovascular disease, and a cancer.

20. The method of claim 18 , wherein the protease is a metalloproteinase.

21. The method of claim 18 , wherein the protease is selected from the group consisting of a serine protease, a threonine protease, a cysteine protease, an asparatic protease, a glutamic protease, and a metalloproteinase.

22. The method of claim 18 wherein an increase in the activity of the protease is indicative of the disease.

23. A method of monitoring the progression or recurrence of a disease in a subject comprising:
determining the activity of a protease in a biological sample of the subject using the method of claim 1 relative to the activity of the protease from a control sample from the subject or a control sample from subjects who do not have the disease;
wherein a significant difference between the activity of the protease in the biological sample and the control sample is indicative of the progression or recurrence of the disease in the subject.

24. The method of claim 23 , wherein the disease is selected from the group consisting of an infectious disease, an inflammatory disease, a cardiovascular disease, and a cancer.

25. The method of claim 23 , wherein the protease is a metalloproteinase.

26. The method of claim 23 , wherein the protease is selected from the group consisting of a serine protease, a threonine protease, a cysteine protease, an asparatic protease, a glutamic protease, and a metalloproteinase.

27. The method of claim 23 wherein an increase in the activity of the protease is indicative of the progression or recurrence of the disease.

28. A method for determining the efficacy of a therapeutic treatment for a disease in a subject undergoing the treatment comprising:
determining the activity of a protease in a biological sample of the subject subjected to therapeutic treatment using the method of claim 1 relative to the activity of the protease from a control sample from the subject or a control sample from subjects who do not have the disease;
wherein a significant difference between the activity of the protease in the biological sample and the control sample is indicative of the efficacy of the therapeutic treatment of the disease in the subject.

29. The method of claim 28 , wherein the disease is selected from the group consisting of an infectious disease, an inflammatory disease, a cardiovascular disease, and a cancer.

30. The method of claim 28 , wherein the protease is a metalloproteinase.

31. The method of claim 28 , wherein the protease is selected from the group consisting of a serine protease, a threonine protease, a cysteine protease, an asparatic protease, a glutamic protease, and a metalloproteinase.

32. The method of claim 28 wherein a decrease in the a

Assignees

• Lopez Gabriel P
• Chilkoti Ashutosh
• Ghoorchian Ali
• Garcia Quiroz Felipe
• Univ Duke

Inventors

• Lopez Gabriel P
• Chilkoti Ashutosh
• Ghoorchian Ali
• Garcia Quiroz Felipe

Classifications

• G01N33/573

  for enzymes or isoenzymes · CPC title

• C12Q1/37 Primary

  involving peptidase or proteinase · CPC title