Whole cell assay systems for cell surface proteases

• US 7,045,311 B2
• Filed: 10/25/2002
• Issued: 05/16/2006
• Est. Priority Date: 10/25/2001
• Status: Active Grant

• Current Assignee: Monogram Biosciences, Inc. (Laboratory Corporation Of America Holdings)
• Original Assignee: Monogram Biosciences, Inc. (Laboratory Corporation Of America Holdings)
• Inventors: Gibbons, Ian, Ciambrone, Gary J.
• Primary Examiner(s): Leary, Louise N.
• Application Number:
  US10/281,458
• Publication Number:
  US 20030108978A1
• Time in Patent Office: 1,299 Days
• Field of Search: 435/23, 435/24, 435/29, 435/4, 435/968, 435/975
• US Class Current: 435/23
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: G01N 2333/4709 Amyloid plaque core protein
• CPC Class Codes: G01N 2333/726 G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
• CPC Class Codes: G01N 2500/10 involving cells
• CPC Class Codes: G01N 33/561 Immunoelectrophoresis
• CPC Class Codes: G01N 33/566 using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 435/975 Kit

Abstract

The activity of a cell surface protease, particularly an ADAM, is determined in a rapid and sensitive assay employing a whole cell system. The assays are effective to identify effector molecules that affect the activity of a cell surface protease directly or indirectly, and to screen for therapeutic agents that modulate those effector molecules. The assays can also be used to screen for therapeutic agents that modulate the activity of a cell surface protease associated with a disease or medical condition. Kits comprising at least one component of the present assays are also provided.

Claims

1. A method of assaying for the activity of an ADAM (a disintegrin and metalloprotease) in a whole cell system, comprising:

(a) selecting a soluble substrate that ADAM specifically cleaves;

(b) combining the soluble substrate with the whole cell system under conditions that allow processing of the substrate to a product by the ADAM;

(c) separating the substrate and the product; and

(d) determining the amount of the product as an indication of the ADAM activity.

2. The method of claim 1, wherein the substrate contains a detectable label.

3. The method of claim 2, wherein the substrate is cleavable by the ADAM to release a product comprising the detectable label.

4. The method of claim 1, wherein said step of separating the substrate and the product is performed by chromatographic means or by electrophoresis.

5. The method of claim 4, wherein the step of separating is performed using capillary electrophoresis or electrophoresis on a microfluidic device.

6. The method of claim 1, wherein the cells have endogenous levels of the ADAM.

7. The method of claim 1, wherein the whole cell system comprises less than about 10⁴ cells per assay.

8. The method of claim 1, wherein the whole cell system comprises less than about 10³ cells per assay.

9. The method of claim 1, wherein the whole cell system comprises less than about 500 cells per assay.

10. The method of claim 1, wherein the whole cell system comprises less than about 50 cells per assay.

11. The method of claim 2, wherein the ADAM is selected from the group consisting of ADAM 10 and ADAM 17.

12. A method of assaying the activities of a plurality of ADAMs in a whole cell system, comprising

selecting a plurality of soluble substrates, said plurality comprising at least one substrate that each ADAM specifically cleaves to a product to be assayed,

combining said substrates with said whole cell system, under conditions that allow processing of the substrates to products by the corresponding ADAMs, such that each ADAM produces a different product;

and determining the amount of each product, as an indication of the corresponding ADAM activity.

13. The method of claim 12, wherein each substrate is an electrophoretic probe comprising an electrophoretic tag, said tag having distinct optical or separation properties with respect to the electrophoretic tag of every other probe used in the assay;

and each said probe is cleavable by the corresponding ADAM to release the electrophoretic tag.

14. The method of claim 13, wherein each said electrophoretic probe is of the form S-L-(D-M), where:

S is a substrate peptide cleavable by the corresponding ADAM;

(D-M) represents an electrophoretic tag, where

D is a detectable group and

M is a mobility modifier effective to impart to the electrophoretic tag a known electrophoretic mobility different from the mobility of the electrophoretic tag of any other probe employed in the assay; and

L is a bond or linking group connecting the substrate peptide to the electrophoretic tag.

15. A method for determining the effect of a candidate effector on the activity of an ADAM in a whole cell system, comprising:

(a) selecting a soluble substrate that ADAM specifically cleaves;

(b) preparing two mixtures of the whole cell system and the soluble substrate, wherein only one of the mixtures contains the candidate effector;

(c) incubating the mixtures under conditions that allow processing of the substrate to a product by the ADAM, if the ADAM is active;

(d) determining the amount of the product formed in each mixture; and

(e) comparing the amount of product formed in the separate mixtures to determine the effect of the candidate effector on the ADAM activity.

16. The method of claim 15, wherein the candidate effector is a receptor.

17. The method of claim 16, wherein the receptor is a G-protein coupled receptor (GPCR).

18. The method of claim 15, wherein the substrate contains a detectable label.

19. The method of claim 18, wherein the substrate is cleavable to release a product comprising the detectable label.

20. The method of claim 19, further comprising the step of separating the substrate and the product before determining the amount of the product.

21. The method of claim 20, wherein the step of separating is performed using capillary electrophoresis.

22. The method of claim 15, wherein the cells have endogenous levels of said ADAM.

23. The method of claim 15, for use in determining the effect of a candidate effector on the activity of a plurality of ADAMs from a whole cell system,

wherein a plurality of said soluble substrates, comprising at least one substrate that is specifically cleavable to a product by each ADAM to be assayed, is combined with said whole cell system in two mixtures, where one of the mixtures contains the candidate effector;

and the amount of each product formed in the two mixtures is compared, to determine the effect of the candidate effector on the activity of each ADAM.

24. The method of claim 23, wherein each substrate is an electrophoretic probe comprising an electrophoretic tag, said tag having distinct optical or separation properties with respect to the electrophoretic tag of every other probe used in the assay;

and each said probe is cleavable by the corresponding ADAM to release the electrophoretic tag.

25. The method of claim 24, wherein each said electrophoretic probe is of the form S-L-(D-M), where:

S is a substrate peptide cleavable by the corresponding ADAM;

(D-M) represents an electrophoretic tag, where

D is a detectable group and

M is a mobility modifier effective to impart to the electrophoretic tag a known electrophoretic mobility different from the mobility of the electrophoretic tag of any other probe employed in the assay; and

L is a bond or linking group connecting the substrate peptide to the electrophoretic tag.

26. A method for determining the effect of a candidate ligand on a receptor in a whole cell system comprising an ADAM, wherein the receptor is known to activate or inhibit the activity of said ADAM, comprising:

(a) selecting a soluble substrate that ADAM specifically cleaves;

(b) preparing two mixtures of the whole cell system and the soluble substrate, wherein only one of the mixtures contains the candidate ligand;

(c) incubating the mixtures under conditions that allow processing of the substrate to a product by the ADAM, if the ADAM is active;

(d) determining the amount of the product formed in each mixture; and

(e) comparing the amount of product formed in the separate mixtures, to determine the effect of the candidate ligand on the receptor.

27. The method of claim 26, wherein the receptor is a GPCR.

28. The method of claim 27, wherein the substrate contains a detectable label.

29. The method of claim 28, wherein the substrate is cleavable to release a product comprising the detectable label.

30. The method of claim 29, further comprising the step of separating the substrate and the product before determining the amount of the product.

31. The method of claim 30, wherein the step of separating is performed using capillary electrophoresis.

32. The method of claim 26, wherein the cell system has endogenous levels of the ADAM.

33. A method of determining the effect of a G-protein coupled receptor (GPCR) on the activity of an ADAM in a whole cell system, comprising:

(a) selecting a ligand known to activate or inhibit activity of the GPCR and a soluble substrate that ADAM specifically cleaves;

(b) preparing two mixtures of the whole cell system and the soluble substrate, wherein only one of the mixtures contains the ligand;

(c) incubating the mixtures under conditions that allow processing of the substrate to a product by the ADAM, if the ADAM is active;

(d) determining the amount of the product formed in each mixture; and

(e) comparing the amount of product formed in the separate mixtures to determine the effect of the GPCR on the ADAM activity.

34. The method of claim 33, wherein the cells have endogenous levels of said ADAM.

35. The method of claim 33, for determining the effect of a G-protein coupled receptor (GPCR) on the activity of a plurality of ADAMs in a whole cell system,

wherein a plurality of soluble substrates, comprising at least one substrate that each ADAM specifically cleaves to be assayed, is added to said whole cell system in two mixtures, of which only one contains said ligand;

and the amount of each product formed in the separate mixtures is compared, to determine the effect of the GPCR on the activity of each ADAM.

36. The method of claim 35, wherein each substrate is an electrophoretic probe comprising an electrophoretic tag, said tag having distinct optical or separation properties with respect to the electrophoretic tag of every other probe used in the assay;

and each said probe is cleavable by the corresponding ADAM to release the electrophoretic tag.

37. The method of claim 36, wherein each said electrophoretic probe is of the form S-L-(D-M), where:

S is a substrate peptide cleavable by the corresponding ADAM;

(D-M) represents an electrophoretic tag, where

D is a detectable group and

M is a mobility modifier effective to impart to the electrophoretic tag a known electrophoretic mobility different from the mobility of the electrophoretic tag of any other probe employed in the assay; and

L is a bond or linking group connecting the substrate peptide to the electrophoretic tag.

38. A method for determining the effect of a compound on cleavage of the β

-amyloid precursor protein (APP) at the α

-secretase site in a whole cell system, comprising;

(a) selecting an enzyme that cleaves the β

-amyloid precursor protein at the α

-secretase site in the whole cell system;

(b) selecting a soluble substrate having a cleavable α

-secretase site;

(c) preparing two mixtures of the soluble substrate and the whole cell system, wherein only one of the mixtures contains the compound;

(d) incubating the mixtures under conditions that allow cleavage of the substrate at the α

-secretase site;

(e) determining the amount of cleavage of the substrate in each mixture; and

(f) comparing the amount of cleavage in the separate mixtures, to determine the effect of the compound on cleavage of APP.

39. The method of claim 38, wherein the substrate contains a detectable label.

40. The method of claim 39, wherein the substrate is cleavable to release a product comprising the detectable label.

41. The method of claim 38, wherein the enzyme is ADAM 17 or ADAM 10.

42. The method of claim 38, wherein the substrate having a cleavable α

-secretase site is a fragment of human β

-amyloid precursor protein.

43. A kit comprising

a soluble substrate for an ADAM and

a candidate effector for ADAM activity.

44. The kit of claim 43, comprising

a plurality of soluble substrates, each substrate being specific for a different ADAM, and

a plurality of candidate effectors for ADAM activity.

45. The kit of claim 44, where each said substrate further comprises an electrophoretic tag, having a detectable group and a mobility modifier effective to impart to the electrophoretic tag a known electrophoretic mobility different from the mobility of the electrophoretic tag of any other substrate employed in the assay.

46. A method of assaying for the activity of a plurality of cell surface ADAM proteases in a whole cell system, comprising:

(a) providing, for each protease being assayed, a soluble substrate the protease specifically cleaves,

(b) combining the substrates with the whole cell system under conditions that allow processing of each substrate to a product by the respective protease; and

(c) electrophoretically separating and determining the amount of each product, as an indication of the activity of the corresponding protease;

wherein each substrate is an electrophoretic probe comprising an electrophoretic tag, said tag having distinct optical or separation properties with respect to the electrophoretic tag of every other probe used in the assay; and

each said probe is cleavable by the corresponding protease to release the electrophoretic tag.

47. The method of claim 46, wherein the whole cell system has endogenous levels of said proteases.