Specific binding sites in collagen for integrins and use thereof

• US 20070099244A1
• Filed: 06/05/2006
• Published: 05/03/2007
• Est. Priority Date: 06/03/2005
• Status: Active Grant

• Current Assignee: Texas A&M University System
• Original Assignee: Texas A&M University System
• Inventors: Xu, Xuejun, Hook, Magnus, Kim, Jiyeun
• Granted Patent: US 7,514,531 B2
• Time in Patent Office: Days
• US Class Current: 435/7.2
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/04 specific for metastasis
• CPC Class Codes: C07K 14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C07K 14/78 Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
• CPC Class Codes: C07K 7/06 having 5 to 11 amino acids
• CPC Class Codes: G01N 2333/70546 Integrin superfamily, e.g. VLAs, leuCAM, GPIIb/GPIIIa, LPAM
• CPC Class Codes: G01N 2333/78 Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
• CPC Class Codes: G01N 2500/02 Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
• 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: G01N 33/6887 from muscle, cartilage or connective tissue

Abstract

The present invention identified a high affinity binding sequence in collagen type III for the collagen-binding integrin I domains. Provided herein are the methods used to characterize the sequence, the peptides comprising this novel sequence and the use of the peptides in enabling cell adhesion. Also provided herein are methods to identify specific integrin inhibitors, sequences of these inhibitors and their use in inhibiting pathophysiological conditions that may arise due to integrin-collagen interaction.

Claims

1. A binding motif for collagen-binding I domains comprising an amino acid sequence GROGER (SEQ ID NO:

4).

2. The binding motif of claim 1, wherein said motif binds the I domains of integrins α

₁, and α

₂.

3. The binding motif of claim 1, wherein said motif comprises a charged amino acid in second position.

4. The binding motif of claim 3, wherein said charged amino acid forms a hydrogen bond with carbonyl background of glutamine at position 215 in α

₂I.

5. The binding motif of claim 4, wherein said charged amino acid is arginine.

6. The binding motif of claim 1, wherein the motif is present on N-terminal of human type III collagen or in the α

₁ and α

₂ chain of human type I collagen.

7. A recombinant collagen or collagen-like protein comprising binding motif of claim 1.

8. The recombinant collagen or collagen-like protein of claim 7, wherein the protein has sequence of SEQ ID NO:

15 or SEQ ID NO;

21.

9. An expression vector, comprising:

a DNA sequence encoding the recombinant collagen or collagen-like protein of claim 7.

10. A host cell comprising and expressing the expression vector of claim 8.

11. A synthetic collagen or collagen-like peptide comprising the binding of claim 1.

12. The synthetic peptide of claim 11, wherein the peptide has sequence of SEQ ID NO:

15 or SEQ ID NO;

21.

13. The synthetic peptide of claim 11, wherein the peptide has a triple helical structure.

14. The synthetic peptide of claim 11, wherein said peptide binds I domains of integrins α

₁ and α

₂.

15. A method of identifying an inhibitor of an integrin-collagen interaction, comprising:

designing a test compound comprising a sequence that is specifically recognized by the I domain of the integrin, wherein said design is based on computer-aided molecular modeling; and

comparing the level of binding of the integrin to the collagen in the presence or absence of the test compound, wherein a decrease in binding of the integrin to the collagen in presence of the test compound indicates that the test compound is an inhibitor of the integrin-collagen interaction.

16. The method of claim 15, wherein said integrin is an α

₁β

₁, α

₁β

₁, α

₁₀β

₁ or α

₁₁β

₁.

17. The method of claim 15, wherein said collagen is a type I, type II or type III collagen.

18. The method of claim 15, wherein the inhibitor has a triple helical structure comprising an amino acid sequence GFPGER (SEQ ID NO:

6), GFOGEN (SEQ ID NO;

     7), GFOGEK (SEQ ID NO;

     8), GNOGER (SEQ ID NO;

     9), GSOGER (SEQ ID NO;

     10), GVOGER (SEQ ID NO;

     11) or GPOGER (SEQ ID NO;

     12).

19. An inhibitory compound identified by the method of claim 15.

20. A recombinant collagen or collagen like protein comprising the inhibitory sequence identified by method of claim 15.

21. An expression vector, comprising:

a DNA sequence encoding the recombinant collagen or collagen-like protein of claim 20.

22. A host cell comprising and expressing the expression vector of claim 21.

23. A synthetic collagen or collagen-like peptide comprising the inhibitory sequence identified by method of claim 15.

24. The synthetic peptide of claim 23, wherein the inhibitory sequence in a triple helical structure is GFPGER (SEQ ID NO:

6), GFOGEN (SEQ ID NO;

     7), GFOGEK (SEQ ID NO;

     8), GNOGER (SEQ ID NO;

     9), GSOGER (SEQ ID NO;

     10), GVOGER (SEQ ID NO;

     11) or GPOGER (SEQ ID NO;

     12).

25. The synthetic peptide of claim 24, wherein the peptide is specific for α

₁β

₁, α

₂β

₁, α

₁₀β

₁ or α

₁₁β

₁ integrins.

26. A method of inhibiting integrin-collagen interaction, comprising:

contacting a sample comprising the integrin and the collagen with the peptide of claim 23, wherein said peptide binds the integrin with a greater affinity than the collagen, thereby inhibiting the integrin-collagen interaction.

27. The method of claim 26, wherein the integrin-collagen interaction contributes to cell adhesion, cell migration, cell proliferation, cell differentiation, angiogenesis, platelet aggregation or extracellular matrix assembly.

28. A pharmaceutical composition, comprising:

the inhibitory compound of claim 19 and a pharmaceutically acceptable carrier.

29. A method of treating an individual having a pathophysiological condition resulting from integrin-collagen interaction, comprising the step of:

administering to the individual a pharmacologically effective amount of the composition of claim 28 such that said composition inhibits the integrin-collagen interaction thereby treating the individual with the pathophysiological condition.

30. The method of claim 29, wherein the pathophysiological condition is inflammation, tumor growth, metastasis or angiogenesis.