Nucleic acid molecules encoding a transmembrane serine protease 10, the encoded polypeptides and methods based thereon

• US 7,112,430 B2
• Filed: 05/14/2002
• Issued: 09/26/2006
• Est. Priority Date: 05/14/2001
• Status: Expired due to Fees

• Current Assignee: Dendreon Pharmaceuticals, Inc. (Ya Fei Yuan,)
• Original Assignee: Dendreon Corporation (Bausch Health Cos., Inc.)
• Inventors: Madison, Edwin L., Yeh, Jiunn-Chern
• Primary Examiner(s): Nashed, Nashaat T.
• Assistant Examiner(s): Moore, William W.
• Application Number:
  US10/147,211
• Publication Number:
  US 20030235900A1
• Time in Patent Office: 1,596 Days
• Field of Search: 435/226, 435/23, 530/350
• US Class Current: 435/226
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 13/08 of the prostate
• CPC Class Codes: A61P 15/08 for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 9/6424 Serine endopeptidases (3.4.21)
• CPC Class Codes: Y10S 436/809 Multifield plates or multicontainer arrays

Abstract

Provided herein are type II transmembrane serine protease 10 (MTSP10) polypeptides. Zymogen and activated forms of these polypeptides as well as single and two chain forms of the protease domain are also provided. Methods using the polypeptides to identify compounds that modulate the activity of an MTSP10 are provided.

Claims

1. A substantially purified single or two chain protease comprising an amino acid sequence having at least 99% identify to the sequence of the MTSP10protease amino acid sequence set forth in SEQ ID No. 23.

2. The substantially purified MTSP10 protease of claim 1 which is a two chain protease.

3. The substantially purified MTSP10 protease of claim 1 which is a single chain protease.

4. The MTSP10 protease of claim 1 wherein a free cysteine in the protease domain is replaced with another amino acid.

5. The MTSP10 protease of claim 4, wherein the replacing amino acid is a serine.

6. The substantially purified protease of claim 1, wherein catalytic activity includes in vitro proteolysis of CH₃SO₂-D-HHT-Gly-Arg-pNA.AcOH (spectrozyme t-PA).

15. A conjugate, comprising the protease of claim 1, and a targeting agent linked to the protease directly or via a linker.

16. The conjugate of claim 15, wherein the targeting agent permits

affinity isolation or purification of the conjugate;

attachment of the conjugate to a surface;

detection of the conjugate;

or

targeted delivery of the conjugate to a selected tissue or cell.

21. A solid support comprising two or more proteases of claim 1 linked thereto either directly or via a linker.

22. The support of claim 21, wherein the proteases comprise an array.

23. The support of claim 22, wherein the array further comprises a plurality of different protease domains.

24. A method for identifying compounds that inhibit the protease activity of a MTSP10 protease of claim 1 or claim 11 comprising:

contacting a protease of claim 1 or claim 11 with a substrate that is proteolytically cleaved by the protease, and, either simultaneously, before or after, adding a test compound or plurality thereof;

measuring the amount of substrate cleaved in the presence of the test compound; and

selecting test compounds that decrease the amount of substrate cleaved compared to a control,

thereby identifying compounds that inhibit the activity of the protease.

25. The method of claim 24, wherein the test compounds are small molecules, peptides, peptidomimetics, natural products, antibodies or fragments thereof that inhibit the activity of the protease.

26. The method of claim 24, wherein a plurality of the test compounds are screened simultaneously.

27. The method of claim 24, wherein the control measures the amount of substrate cleaved in the absence of the test compound.

28. The method of claim 26, wherein a plurality of the proteases are linked to a solid support, either directly or via a linker.

29. The method of claim 28, wherein the proteases comprise an array.

30. A method for identifying compounds that inhibit the protease activity of the MTSP10 protease of claim 2, comprising:

contacting the MTSP10 protease of claim 2 with a substrate that is proteolytically cleaved by the protease, and, either simultaneously, before or after, adding a test compound or plurality thereof;

measuring the amount of substrate cleaved in the presence of the test compound; and

selecting test compounds that decrease the amount of substrate cleaved compared to a control,

thereby identifying compounds that inhibit the activity of the protease.

34. A method of identifying a compound that specifically binds to a single-chain and/or two-chain MTSP10 protease of claim 1 or claim 11, comprising:

contacting an MTSP10 protease of claim 1 or claim 11 with a test compound or plurality thereof under conditions conducive to binding of the test compound to the protease; and

selecting test compounds that remain bound specifically to the protease compared to a control,

thereby identifying test compounds that specifically bind a single chain and/or two chain form of the protease.

35. The method of claim 34, wherein the protease is linked either directly or indirectly via a linker to a solid support.

36. The method of claim 34, wherein the test compounds are small molecules, peptides, peptidomimetics, natural products, antibodies or fragments thereof.

37. The method of claim 34, wherein a plurality of the test compounds are screened simultaneously.

38. The method of claim 34, wherein a plurality of the proteases are linked to a solid support.

40. A method for identifying activators of the zymogen form of an MTSP10 protease of claim 1 or claim 11, comprising:

contacting a zymogen form of an MTSP10 protease of claim 1 or claim 11 with a substrate of the activated form of the protease;

detecting cleavage of the substrate, thereby identifying compounds that activate the zymogen.

41. The method of claim 40, wherein the substrate is a chromogenic substrate.

42. The method of claim 40, wherein the substrate is a L-pyroglutamyl-L-prolyl-L-arginine-p-nitroaniline hydrochloride.

43. The method of claim 40, wherein the test compound is a small molecule, a nucleic acid or a protease.

7. A substantially purified single or two chain MTSP10 protease selected from the group consisting of a protease that comprises the sequence of amino acid residues set forth in SEQ ID No. 23 and a MTSP10 protease that comprises a sequence of amino acid residues encoded by a nucleic acid molecule having the sequence of nucleotides set forth in SEQ ID No. 22.

8. The substantially purified MTSP10 protease of claim 7 which is a two chain protease.

9. The substantially purified protease of claim 7, wherein catalytic activity includes in vitro proteolysis of CH₃SO₂-D-HHT-Gly-Arg-pNA.AcOH (spectrozyme t-PA).

10. The protease of claim 7 that consists of the sequence of amino acid residues set forth in SEQ ID No. 23.

31. A method for identifying test compounds that inhibit the protease activity of the MTSP10 protease of claim 7 comprising:

contacting the MTSP10 protease of claim 7 with a substrate proteolytically cleaved by the protease, and, either simultaneously, before or after, adding a test compound or plurality thereof;

measuring the amount of substrate cleaved in the presence of the test compound; and

selecting test compounds that decrease the amount of substrate cleaved compared to a control,

thereby identifying compounds that inhibit the activity of the protease.

32. A method for identifying test compounds that inhibit the protease activity of a the MTSP10 protease of claim 8, comprising:

contacting the MTSP10 protease of claim 8 with a substrate that is proteolytically cleaved by the protease; and

,

either simultaneously, before or after, adding a test compound or plurality thereof;

measuring the amount of substrate cleaved in the presence of the test compound; and

selecting test compounds that decrease the amount of substrate cleaved compared to a control,

thereby identifying compounds that inhibit the activity of the protease.

39. A method of identifying a compound that binds to the single-chain and/or two-chain form of the MTSP10 protease of claim 7, comprising:

contacting the MTSP10 protease of claim 7 with a test compound or plurality thereof under conditions conducive to binding of the test compound to the protease; and

selecting test compounds that remain bound specifically to the protease compared to a control,

thereby identifying test compounds that specifically bind a single chain and/or two chain form of the protease.

11. A substantially purified single or two chain MTSP10 protease that comprises one or more amino acid substitutions at positions selected from the group of positions consisting of 67, 272, 336, 383, 409, 418, 462–

467 and 573 in the sequence set forth in SEQ ID No. 23.

17. A conjugate comprising the protease of claim 11 and a targeting agent linked to the protease directly or via a linker.

18. The conjugate of claim 17, wherein the targeting agent permits

affinity isolation or purification of the conjugate,

attachment of the conjugate to a surface;

detection of the conjugate;

or

targeted delivery of the conjugate to a selected tissue or cell.

12. A substantially purified MTSP10 protease that consists of the sequence of amino acids at positions 1–

230 of SEQ ID No. 6.

19. A conjugate comprising the protease of claim 12 and a targeting agent linked to the protease directly or via a linker.

20. The conjugate of claim 19, wherein the targeting agent permits

affinity isolation or purification of the conjugate;

attachment of the conjugate to a surface;

detection of the conjugate;

or

targeted delivery of the conjugate to a selected tissue or cell.

33. A method for identifying test compounds that inhibit the protease activity of the MTSP10 protease of claim 12, comprising:

contacting a the MTSP10 protease of claim 12 with a substrate that is proteolytically cleaved by the protease; and

,

either simultaneously, before or after, adding a test compound or plurality thereof;

measuring the amount of substrate cleaved in the presence of the test compound; and

selecting test compounds that decrease the amount of substrate cleaved compared to a control,

thereby identifying compounds that inhibit the activity of the protease.

13. A substantially purified single chain MTSP10 protease that comprises an amino acid substitution at one or more positions selected from the group of positions consisting of 1–

6 or 112 of SEQ ID No. 6.

14. A substantially purified two chain MTSP10 protease that comprises an amino acid substitution at one or more positions selected from the group of positions consisting of 1–

6 or 112 of SEQ ID No. 6.