Methods for Identifying Compounds that Modulate Enzymatic Activities by Employing Covalently Bonded Target-Extender Complexes with Ligand Candidates

• US 20080220536A1
• Filed: 04/16/2007
• Published: 09/11/2008
• Est. Priority Date: 06/26/1998
• Status: Abandoned Application

• Current Assignee: Daniel A. Erianson, Robert S. Mcdowell, Stig Hansen
• Original Assignee: Daniel A. Erianson, Robert S. Mcdowell, Stig Hansen
• Inventors: Hansen, Stig, Erianson, Daniel A., McDowell, Robert S.
• Application Number:
  US11/735,944
• Publication Number:
  US 20080220536A1
• Time in Patent Office: Days
• US Class Current: 436/501
• CPC Class Codes: C07K 1/1072 by covalent attachment of residues or functional groups
• CPC Class Codes: G01N 2333/916 acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
• 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 33/573 for enzymes or isoenzymes

Abstract

The present invention relates to the use of “tethering” to identify compounds that modulate enzymatic activity.

Claims

1. A method comprising:

a) providing a target having a reactive thiol located outside of a site of interest;

b) contacting the target with an extender thereby forming a target-extender complex wherein the extender comprises a first functionality that forms a first covalent bond with the thiol and a second functionality that is capable of forming a second covalent bond;

c) contacting the target-extender complex with a candidate ligand that comprises a group that is capable of forming a second covalent bond with the second functionality;

d) forming a second covalent bond between the target-extender complex and the candidate ligand thereby forming a target-extender-ligand conjugate; and

, e) identifying the candidate ligand present in the target-extender-ligand conjugate;

wherein at least the second covalent bond formed is a disulfide bond.

5. The method of claim 1, wherein the first covalent bond formed is a thioether bond and the second covalent bond is a disulfide bond, whereby the target-extender-ligand complex formed has a thioether bond between the target and extender and a disulfide bond between the extender and the ligand candidate.

6. The method of claim 5, wherein the second functionality is a disulfide.

7. The method of claim 5, wherein the second functionality is a thioester.

8. The method of claim 1, wherein the first covalent bond formed is a first disulfide bond and the second covalent bond formed is a second disulfide bond, whereby the target-extender-ligand complex formed has a first disulfide bond between the target and the extender and a second disulfide bond between the extender and the ligand candidate.

9. The method of claim 8, wherein the second functionality is a disulfide.

10. The method of claim 8, wherein the second functionality is a thioester.

11. The method of claim 1 wherein the extender includes a double or triple bond adjacent to a carbonyl, imide, quinine, CN, NO₂, —

S(═

O)—

or vinyl sulfone.

12. The method of claim 1 wherein the extender is selected from the group consisting of

13. The method of claim 12 wherein is selected from the group consisting of unsubstituted C₁-C₂₀ aliphatic, substituted C₁-C₂₀ aliphatic, unsubstituted aryl and substituted aryl.

14. The method of claim 1, wherein the reactive thiol on the target is a naturally occurring —

SH from a cysteine that is part of a naturally occurring protein sequence.

15. The method of claim 1 wherein the reactive thiol on the target is from a cysteine where mutagenesis was used to replace a naturally occurring amino acid.

16. The method of claim 1 wherein the reactive thiol is a masked a disulfide.

17. The method of claim 1 wherein the target-extender complex is contacted with a candidate ligand in the presence of a reducing agent.

18. The method of claim 17 wherein the reducing agent is selected from the group consisting of:

cysteine, cysteamine, dithiothreitol, dithioerythritol, glutathione, 2-mercaptoethanol, 3-mercaptoproprionic acid, a phosphine and sodium borohydride.

2. (canceled)

3. A method for identifying a candidate ligand comprising:

a) providing a target having a first reactive nucleophile located inside of a site of interest and a second reactive nucleophile located outside of the site of interest;

b) contacting the target with an extender thereby forming a target-extender complex, the extender comprising a first functionality and a latent second functionality, a cleavable linker and a binding determinant wherein the first functionality forms a first covalent bond with the second reactive nucleophile and the binding determinant binds to the site of interest;

c) cleaving the extender at the cleavable linker thereby forming a modified target-extender complex thereby exposing the second functionality and releasing the binding determinant from the site of interest;

d) contacting the modified target-extender complex with a candidate ligand that comprises a group that is capable of forming a second covalent bond with the second functionality;

e) forming a second covalent bond between the modified target-extender complex and the candidate ligand thereby forming a target-extender-ligand conjugate; and

,

f) identifying the candidate ligand present in the target-extender-ligand conjugate.

4. (canceled)

19. A method comprising:

a) providing a target having a reactive nucleophile located outside of a site of interest;

b) contacting the target with an extender thereby forming a target-extender complex wherein the extender comprises a first functionality that reacts with the nucleophile in the target to form a covalent bond with the nucleophile and a second functionality that is capable of forming a disulfide bond, wherein the extender is selected from the group consisting of

20. The method of claim 19 wherein the candidate ligand present in the target-extender-ligand conjugage is identified using mass spectrometry.

21. The method of claim 19 wherein the candidate ligand present in the target-extender-ligand conjugage is identified using a labeled probe.

22. The method of claim 19 wherein the candidate ligand present in the target-extender-ligand conjugage is identified using a functional assay.

23. The method of claim 19 wherein the candidate ligand present in the target-extender-ligand conjugage is identified using chromatography.

24. A method comprising:

a) providing a target having a first reactive thiol located inside of a site of interest and a second reactive thiol located outside of the site of interest;

b) contacting the target with an extender thereby forming a target-extender complex, the extender comprising a first functionality and a latent second functionality, a cleavable linker and a binding determinant wherein the first functionality forms a thioether bond with the second reactive thiol and the binding determinant binds to the site of interest;

c) cleaving the extender at the cleavable linker thereby forming a modified target-extender complex by exposing the second functionality and releasing the binding determinant from the site of interest;

d) contacting the modified target-extender complex with a candidate ligand that comprises a group that is capable of forming a disulfide bond with the second functionality;

e) forming a disulfide bond between the modified target-extender complex and the candidate ligand thereby forming a target-extender-ligand conjugate; and

f) identifying the candidate ligand present in the target-extender-ligand conjugate.

25. The method of claim 24 wherein the extender includes a double or triple bond adjacent to a carbonyl, imide, quinine, CN, NO₂, —

S(═

O)—

or vinyl sulfone.

26. The method of claim 24 wherein the extender is selected from the group consisting of

27. The method of claim 26 wherein is selected from the group consisting of unsubstituted C₁-C₂₀ aliphatic, substituted C₁-C₂₀ aliphatic, unsubstituted aryl and substituted aryl.