Binding compounds and methods for identifying binding compounds

• US 20030167129A1
• Filed: 10/26/2001
• Published: 09/04/2003
• Est. Priority Date: 10/27/2000
• Status: Abandoned Application

• Current Assignee: IWA, LLC
• Original Assignee: IWA, LLC
• Inventors: Krstenansky, John, Kates, Steven A., Wilson, Carol J., Nestor, John J. Jr., Tan Hehir, Christina A.
• Application Number:
  US10/014,322
• Publication Number:
  US 20030167129A1
• Time in Patent Office: Days
• US Class Current: 702/19
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 1/047 Simultaneous synthesis of different peptide species; Peptide libraries
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C07K 14/7158 for chemokines
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 7/06 having 5 to 11 amino acids
• CPC Class Codes: G01N 2333/726 G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
• CPC Class Codes: G01N 2500/20 cell-free systems
• CPC Class Codes: G01N 33/6863 Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
• CPC Class Codes: G01N 33/74 involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors

Abstract

Binding compounds for CXC chemokine receptor 4 and methods for identifying binding compounds for CXC chemokine receptor 4 are provided. Also provided are therapeutic agents comprising such compounds.

Claims

1. A method of identifying a binding compound for CXC chemokine receptor 4 comprising the steps of:

a) providing a library of two or more molecules;

b) providing a molecule having a binding property corresponding to CXC chemokine receptor 4;

c) binding a molecule from said library of two or more molecules to said molecule having a binding property corresponding to CXC chemokine receptor 4;

d) separating said bound molecule from said library of two or more molecules; and

e) identifying said bound molecule as a binding compound for CXC chemokine receptor 4.

2. The method of claim 1, wherein said library of two or more molecules is selected from the group consisting of linear peptides, cyclic peptides, natural amino acids, unnatural amino acids, peptidomimetic compounds and small molecule compounds.

3. The method of claim 1, wherein said molecule having a binding property corresponding to CXC chemokine receptor 4 is a partially purified CXC chemokine receptor.

4. The method of claim 1, wherein at least one of said two or more molecules is selected from a group consisting of a peptide, a peptidomimetic or small molecule that can substitute for a protein capable of binding to receptors, enzymes or other proteins.

5. The method of claim 1, further comprising the step of solubilizing said molecule having a binding property corresponding to CXC chemokine receptor 4 substantially in the absence of sodium chloride.

6. The method of claim 1, further comprising the step of solubilizing said molecule having a binding property corresponding to CXC chemokine receptor 4 using a buffer having a low salt concentration.

7. The method of claim 1, wherein at least one of said two or more molecules comprises a molecule having an antagonistic effect on CXC chemokine receptor 4 binding activity.

8. The method of claim 1, wherein said library comprises a phage library.

9. The method of claim 1, wherein said steps a, b, c, and d are repeated at least once prior to said step e.

10. The method of claim 1, wherein said molecule having a binding property corresponding to CXC chemokine receptor 4 comprises a CXC chemokine receptor 4 molecule and a tag selected from the group consisting of GST, FLAG, 6×

His, C-MYC, MBP, V5, Xpress, CBP, and HA).

11. A binding compound for CXC chemokine receptor 4 identified according to the method of claim 1.

12. A method of preventing HIV infection in a patient, the method comprising administering to said patient a therapeutic composition comprising the compound of claim 1 in a physiological carrier.

13. A method of treating or preventing AIDS in a patient, the method comprising administering to said patient a therapeutic composition comprising the compound of claim 1 in a physiological carrier.

14. A method of treating or preventing AIDS in a patient, the method comprising administering to said patient a therapeutic composition comprising the compound of claim 1 in a controlled release injectable formulation.

19. A binding compound identified according to the method of claim 1 having a sequence selected from the group consisting of P-A-H-Y-P-M-L (SEQ ID NO:

73), Q-Y-A-T-P-N-K (SEQ ID NO;

     74), Q-Q-R-S-T-A-F (SEQ ID NO;

     75), P-F-R-A-T-T-E (SEQ ID NO;

     76), T-D-K-L-L-L-D (SEQ ID NO;

     77), H-T-Q-H-V-R-T (SEQ ID NO;

     78), L-G-V-K-A-P-S (SEQ ID NO;

     79), D-L-Q-A-R-Y-S (SEQ ID NO;

     80), S-L-T-E-P-S-L (SEQ ID NO;

     81), S-T-W-P-L-A-Q (SEQ ID NO;

     82), and R-T-T-S-D-A-L (SEQ ID NO;

     83).

35. A binding compound identified according to the method of claim 1 having the sequence comprising ARSLI(2-Nal)R(Tic)ARR(2-Nal)RR (SEQ ID NO:

72).

36. A binding compound identified according to the method of claim 1 having the sequence comprising ARSLI(2-Nal)RPARR(2-Nal)RR (SEQ ID NO:

60).

37. A binding compound identified according to the method of claim 1 having the sequence comprising KKKARSLI(2-Nal)RLARR(2-Nal)RR (SEQ ID NO:

48).

38. A binding compound identified according to the method of claim 1 having the sequence comprising ARSLI(2-Nal)RAARR(2-Nal)RR (SEQ ID NO:

29).

39. A binding compound identified according to the method of claim 1 having the sequence comprising RRARSLI(2-Nal)RAARR(2-Nal)RR (SEQ ID NO:

44).

40. A binding compound identified according to the method of claim 1 having the sequence comprising H-ARSLI(2-Nal)RHARR(2-Nal)RR (SEQ ID NO:

47).

41. A binding compound identified according to the method of claim 1 having the sequence comprising Cyclo (Glu⁰, Lys⁴) EMARKLI(2-Nal)R(Tic)ARR(2-Nal)RR (SEQ ID NO:

123).

42. A binding compound identified according to the method of claim 1 having the sequence comprising Cyclo (Glu⁸, Lys¹²) ARSLI(2-Nal)E(Tic)RAK(2-Nal)RR (SEQ ID NO:

124).

43. A binding compound identified according to the method of claim 1 having the sequence comprising Cyclo (D-Cys⁸, Cys¹¹) ARSLI(2-Nal)c(Tic)RCR(2-Nal)RR.

44. A binding compound identified according to the method of claim 1 having the sequence comprising Cyclo (Glu⁰, Lys⁴) EMARKLIWRPAKAKKK (SEQ ID NO:

101).

45. A method of treating disease in a patient, the method comprising administering to said patient a therapeutic composition comprising the compound of claim 1 in a physiological carrier.

15. A computer-aided method for identifying relative binding affinity of a test molecule to CXC chemokine receptor 4, comprising the steps of:

a) entering input data characterizing CXC chemokine receptor 4 into a computer program;

b) entering input data characterizing at least one test peptide-like molecule, each of known sequence but unknown binding affinity;

c) analyzing each applied test peptide-like molecule using the computer program to generate a prediction of a relative binding affinity for each test peptide-like molecule, and outputting such prediction.

16. A method for determining an amino acid sequence motif for an interaction site of a binding compound for CXC chemokine receptor 4, comprising the steps of:

a) contacting a peptide library with a molecule having a binding property corresponding to CXC chemokine receptor 4 under conditions which allow for interaction between said molecule having a binding property corresponding to CXC chemokine receptor 4 and said peptide library;

b) allowing said molecule having a binding property corresponding to CXC chemokine receptor 4 to interact with said peptide library such that a complex is formed between said molecule having a binding property corresponding to CXC chemokine receptor 4 and a subpopulation of library members capable of interacting with said molecule having a binding property corresponding to CXC chemokine receptor 4;

c) separating said subpopulation of library members capable of interacting with said molecule having a binding property corresponding to CXC chemokine receptor 4 from library members that are incapable of interacting with said molecule having a binding property corresponding to CXC chemokine receptor 4;

d) determining a relative abundance of different amino acid residues at each degenerate position within said subpopulation of library members; and

e) determining an amino acid sequence motif for an interaction site of said molecule having a binding property corresponding to CXC chemokine receptor 4, based upon said relative abundance of different amino acid residues at each degenerate position within the library members.

17. An amino acid sequence motif for a binding compound for CXC chemokine receptor 4 identified according to the method of claim 16.

18. An amino acid sequence motif identified according to the method of claim 16 having sequence M-A-R-S-L-1-W-R-P-A-K-A-K-K-K (SEQ ID NO:

1).

20. A binding compound having the amino acid sequence motif for CXC chemokine receptor 4 determined by the method of claim 16.

21. A binding compound identified according to the method of claim 16 having the sequence comprising A′

-B′

-C′

-D′

-E′

-E′

-F′

-C′

-G′

-F′

/C′

/B′

-C′

-B′

/C′

-F′

/C′

-C′

-C′

.

22. The method of claim 16, wherein at least one member of said peptide library comprises at least one unnatural amino acid.

23. The method of claim 16, wherein said molecule having a binding property corresponding to CXC chemokine receptor 4 is selected from the group consisting of linear peptides, cyclic peptides, natural amino acids, unnatural amino acids, peptidomimetic compounds and small molecule compounds.

24. The method of claim 16, wherein said peptide library comprises at least one molecule selected from the group consisting of linear peptides, cyclic peptides, natural amino acids, unnatural amino acids, peptidomimetic compounds and small molecule compounds.

25. The method of claim 16, wherein said peptide library is selected from a group consisting of M-X-X-X-X-R-X-X-X-X-A, M-A-X-X-X-X-R-X-X-X-X-K-K-K (SEQ ID NO:

68), M-A-X-X-X-X-W-X-X-X-X-A-K-K-K (SEQ ID NO;

     69), M-A-R-X-X-1-W-R-X-X-X-A-K-K-K (SEQ ID NO;

     70), M-X-X-X-X-W-X-X-X-X-A-K-K-K (SEQ ID NO;

     71), cyclo(M-X-X-X-X-R-X-X-X-X-N), and cyclo(M-K-X-D-H-R-X-X-K-N) (SEQ ID NO;

     61).

26. The method of claim 16, wherein said peptide library is selected from a pre-determined CPI peptide sequence.

27. A library comprising members based upon an amino acid sequence motif for an interaction site of a binding compound for CXC chemokine receptor 4, the motif being determined by permitting at least one peptide member from a peptide library to interact with said binding compound for CXC chemokine receptor 4, and determining an amino acid sequence of at least one peptide that interacts with said binding compound for CXC chemokine receptor 4.

28. A method of solubilizing or immobilizing a compound corresponding to the binding property of CXC chemokine receptor 4, wherein the solubilization or immobilization is conducted substantially in the absence of sodium chloride when determining a compound corresponding to the binding of CXC chemokine receptor 4.

29. A method of solubilizing or immobilizing a compound corresponding to the binding property of CXC chemokine receptor 4, wherein the solubilization or immobilization is conducted by a using a low salt concentration when determining a compound corresponding to the binding of CXC chemokine receptor 4.

30. The method of claim 29, wherein said low salt concentration comprises a predetermined amount of magnesium and calcium.

31. A CXC chemokine 4 transfer vector comprising a CXC chemokine receptor 4 molecule and a tag selected from the group consisting of GST, FLAG, 6×

His, C-MYC, MBP, V5, Xpress, CBP, and HA.

32. A method of using a three-dimensional structure of CXC chemokine receptor 4 in a drug screening assay comprising:

a) selecting a potential drug by performing rational drug design with the three-dimensional structure, wherein said selecting step is performed in conjunction with computer modeling;

b) contacting the potential drug with a first molecule comprising a first CXC chemokine receptor 4; and

c) detecting the binding of the potential drug with said first molecule;

wherein a potential drug is selected as a drug if the potential drug binds to said first molecule.

33. The method of claim 32, wherein said first molecule is labeled.

34. The method of claim 32, wherein said first molecule is bound to a solid support.