Pharmacophore recombination for the identification of small molecule drug lead compounds

• US 6,344,334 B1
• Filed: 03/26/1999
• Issued: 02/05/2002
• Est. Priority Date: 03/27/1998
• Status: Expired due to Term

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Ellman, Jonathan A., Choong, Ingrid
• Primary Examiner(s): Celsa, Bennett
• Assistant Examiner(s): BAKER, MAURIE GARCIA
• Application Number:
  US09/277,461
• Time in Patent Office: 1,047 Days
• Field of Search: 435/7.1, 435/7.2, 435/6, 436/501, 436/518, 424/1.11, 424/9.1, 424/178.1, 424/193.1, 530/345, 530/389.1, 530/402, 530/807, 514/449-458, 549/283-290
• US Class Current: 435/7.1
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: G01N 2500/04 Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
• CPC Class Codes: G01N 33/531 Production of immunochemical test materials
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures
• CPC Class Codes: Y10S 530/807 Hapten conjugated with peptide or protein

Abstract

Method for identifying a drug lead compound that inhibits binding of target bioogical molecules (TBM) by contacting TBM with members of a library of candidate cross-linked target binding fragments (CXBF), each CXBF having at least two candidate target bindig fragments (CTBF), which are inhibitors of binding, linked to a cross-linker, and selecting CXBF that inhibit the binding of the TBM to a greater extent than either of the individual CTBF linked to said cross-linker, wherein the library of CXBF is produced by: (a) screening a population of CTBF capable of being chemically cross-linked by a cross-linker to identify a subpopulation of the CTBF that inhibit binding of the TBM; and (b) chemically cross-linking members of the subpopulation of CTBF or structurally related analogs thereof with a cross-linker to provide a library of CXBF, wherein at least one linking group comprises an oxime ether linking group.

Claims

1. A method for identifying a drug lead compound that inhibits binding of members of a complementary pair of target biological molecules (TBM) of interest, the method comprising:

contacting both members of said pair of TBM with individual members of a library of candidate cross-linked target binding fragments (CXBF) either sequentially or simultaneously, each CXBF comprising at least two candidate target binding fragments (CTBF) that are inhibitors of binding linked to a cross-linker, and selecting CXBF that inhibit the binding of said pair of TBM to a greater extent than either of the individual CTBF linked to said cross-linker, wherein said library of CXBF is produced by;

(a) screening a population of CTBF capable of being chemically cross-linked by a cross-linker to identify a subpopulation of said CTBF that inhibit binding of said pair of TBM;

(b) chemically cross-linking members of the subpopulation of CTBF or structurally related analogs thereof with a cross-linker to provide a library of CXBF;

wherein at least one linking group comprises an oxime ether linking group;

and wherein the CXBF are represented by the formulae;

 

wherein

TBF_m represents the linked residue of a first CTBF;

TBF_n represents the linked residue of a second CTBF;

TBF_m-part A and B represent the parts of a linked residue of a CTBF having two fragments bonded to a single atom in LG₃;

TBF_n-part C and D represent the parts of a linked residue of a CTBF having two fragments bonded to a single atom in LG₄;

XL represents a cross-linker of the formula

2. The method according to claim 1, wherein at least one member of the population of CTBF binds to the TBM with a K_d of from about 5 mM to about 0.05 mM.

3. The method according to claim 1, wherein at least one of said selected CXBF that inhibits the binding pair of TBM to a greater extent than either of the individual CTBF linked to said 1 linker element binds to the TBM with a K_d of 500 nM or lower.

4. The method according to claim 1, wherein step (a) is accomplished by an in vitro biological assay.

5. The method according to claim 1, wherein the library of CXBF comprises either homodimeric or heterodimeric CXBF or a mixture thereof.

6. The method according to claim 1, wherein the library of CXBF comprises CXBF with a molecular weight of less than about 750 daltons.

7. The method according to claim 1, wherein each of said pair of TBM is a human protein or human pathogen.

8. The method according to claim 1, wherein one of said proteins is an enzyme, a human hormone, cytokine or chemokine, a human receptor or fragments thereof.

9. The method of claim 1, wherein the sinking groups of said cross-linker are the same or are different.

10. The method of claim 1, wherein each of said at least two CTBF independently binds to the TBM with a K_d of from about 3 mM to about 100 μ

M.

11. The method of claim 1, wherein the in vitro biological assay is a competitive assay comprising:

contacting one member of said pair of TBM with an assay molecule that binds to said TBM, in the presence of one of said CTBF and measuring the inhibition of the binding of the assay molecule to said TBM.

12. The method of claim 11, wherein the assay molecule is an antibody to the TBM.

13. The method of claim 11, wherein the assay is an ELISA assay.

14. The method of claim 1, wherein selecting CXBF that inhibit the binding of said pair of TBM to a greater extent than either of the individual CTBF linked to said linker element is accomplished by an in vitro biological assay.

15. The method of claim 11, wherein said CTBF are the same or are different.