Cysteine mutants and methods for detecting ligand binding to biological molecules

• US 20030194745A1
• Filed: 08/05/2002
• Published: 10/16/2003
• Est. Priority Date: 06/26/1998
• Status: Abandoned Application

• Current Assignee: Sunesis Pharmaceuticals, Inc. (Viracta Therapeutics, Inc.)
• Original Assignee: Sunesis Pharmaceuticals, Inc. (Viracta Therapeutics, Inc.)
• Inventors: Flanagan, W. Michael, McDowell, Robert S.
• Application Number:
  US10/214,419
• Publication Number:
  US 20030194745A1
• Time in Patent Office: Days
• US Class Current: 435/7.1
• CPC Class Codes: C07K 1/00 General methods for the preparation of peptides , i.e. processes for the organic chemical preparation of peptides or proteins of any length
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 14/4746 p53
• CPC Class Codes: C07K 14/4747 Apoptosis related proteins
• CPC Class Codes: C07K 14/525 Tumour necrosis factor [TNF]
• CPC Class Codes: C07K 14/5406 IL-4
• CPC Class Codes: C07K 14/5437 IL-13
• CPC Class Codes: C07K 14/55 IL-2
• CPC Class Codes: C07K 14/70521 CD28, CD152
• CPC Class Codes: C07K 14/70532 B7 molecules, e.g. CD80, CD86
• CPC Class Codes: C07K 14/70575 NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154 NGF C07K14/48, TNF C07K14/525
• CPC Class Codes: C07K 14/70589 CD45
• CPC Class Codes: C07K 14/70596 Molecules with a "CD"-designation not provided for elsewhere
• CPC Class Codes: C07K 14/71 for growth factors; for growth regulators
• CPC Class Codes: C07K 14/7155 for interleukins [IL]
• CPC Class Codes: C07K 14/745 Blood coagulation or fibrinolysis factors
• CPC Class Codes: G01N 33/53 Immunoassay; Biospecific binding assay; Materials therefor
• CPC Class Codes: G16B 15/00 ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
• CPC Class Codes: G16B 15/30 Drug targeting using structural data; Docking or binding prediction

Abstract

The present invention relates generally to variants of target biological molecules (“TBMs”) and to methods of making and using the same to identify ligands of TBMs. More specifically, the invention relates to individual variant TBMs and sets of variant TBMs, each of which represents a modified version of a protein of interest where a thiol has been introduced at or near a site of interest. Ligands of TBMs are identified in part through the formation of a covalent bond between a potential ligand and a reactive thiol on the TBM.

Claims

1. A method comprising:

a) obtaining a set of coordinates of a three dimensional structure of a protein TBM having n number of residues;

b) selecting a candidate residue i on the three dimensional structure of the TBM wherein the candidate residue i is the ith residue where i is a number between 1 and n and residue i is not a cysteine;

c) selecting a residue j where residue j is adjacent to residue i in sequence;

d) determining a candidate reference value wherein the candidate reference value is a spatial relationship between residue i and residue j;

e) obtaining a database comprising sets of coordinates of disulfide-containing protein fragments wherein each fragment comprises at least a disulfide-bonded cysteine and a first adjacent residue where the disulfide-bonded cysteine and the first adjacent residue share the same sequential relationship as residue i and residue j;

f) determining a comparative reference value for each fragment wherein the comparative reference value is the corresponding spatial relationship between the disulfide-bonded cysteine and the first adjacent residue as the candidate reference value is between residue i and j; and

,

g) determining a score wherein the score is a measure of the number of fragments in the database that possess a comparative reference value that is the same or similar to the candidate reference value.

2. The method of claim 1 further comprising:

selecting a residue k where residue k is adjacent to residue i in sequence and k is not j; and

wherein

the candidate reference value is a spatial relationship between residue i, residue j, and residue k;

each fragment comprises at least a disulfide-bonded cysteine, a first adjacent residue, and a second adjacent residue where the disulfide-bonded cysteine and the first and second adjacent residues share the same sequential relationship as residue i, residue j, and residue k; and

the comparative reference value is the corresponding spatial relationship between the disulfide bonded cysteine, the first adjacent residue, and the second adjacent residue as the candidate reference value is between residue i, residue j, and residue k.

4. The method of any one of claims 1-3 wherein the spatial relationship comprises a dihedral angle.

5. The method of any one of claims 1-3 wherein the spatial relationship comprises a pair of phi psi angles.

6. The method of any one of claims 1-3 wherein the spatial relationship comprises a plurality of distances between atoms of two residues.

7. The method of any one of claims 1-3 wherein residue i is at least partially surface accessible.

8. The method of claim 7 wherein residue i has an accessible surface area of at least about 20 Å

².

9. The method of any one of claims 1-3 wherein residue i does not participate in a hydrogen bond interaction with a backbone atom of the TBM.

3. A method comprising:

a) obtaining a set of coordinates of a three dimensional structure of a protein TBM having n number of residues;

b) selecting a candidate residue i on the three dimensional structure of the TBM wherein the candidate residue i is the ith residue where i is a number between 1 and n and residue i is not a cysteine;

c) selecting residue j and residue k wherein residue j and residue k are both adjacent in sequence to residue i;

d) determining a candidate reference value wherein the candidate reference value is a spatial relationship of at least one backbone atom from each of residue i, residue j, and residue k;

e) obtaining a database comprising sets of coordinates of disulfide-containing protein fragments wherein each fragment comprises at least a disulfide-bonded cysteine, a first adjacent residue, and a second adjacent residue where the disulfide-bonded cysteine, the first adjacent residue, and the second adjacent residue share the same sequential relationship as residue i, residue j, and residue k;

f) determining a comparative reference value for each fragment wherein the comparative reference value is the corresponding spatial relationship between the disulfide-bonded cysteine, the first adjacent residue, and the second adjacent residue as the candidate reference value is between residue i, residue j, and residue k; and

,

g) determining a score wherein the score is a measure of the number of fragments in the database that possess a comparative reference value that is the same or similar to the candidate reference value.

10. A method comprising:

a) obtaining a three dimensional structure of a TBM having n number of residues and a site of interest;

b) selecting a candidate residue i that is at or near the site of interest wherein the candidate residue i is the ith residue where i is a number between 1 and n and residue i is not a cysteine;

c) generating a set of mutated TBM structures wherein each mutated TBM structure possesses a cysteine residue instead of residue i and wherein the cysteine residue is placed in a standard rotamer conformation; and

,

d) evaluating the set of mutated TBM structures.

11. The method of claim 10 wherein the cysteine residue is capped with a S-methyl group.

12. The method of claim 10 wherein the standard rotamer conformation for cysteine comprises:

a chi1 angle selected from the group consisting of about 60°

, about 180°

, and about 300°

; and

a chi2 angle selected from the group consisting of about 60°

, about 120°

, about 180°

, about 270°

, and about 300°

.

13. The method of claim 10 wherein evaluation step comprises determining whether each rotamer conformation makes an unfavorable steric contact with the TBM.

14. The method of claim 10 wherein the evaluation step comprises a force field calculation.

15. The method of claim 11 wherein the evaluation step comprises determining whether each rotamer conformation places the methyl carbon of the S-methyl group closer to the site of interest than the C_β

16. A set of variant proteins, said proteins each being a mutated version of a TBM wherein a naturally occurring non-cysteine residue of the TBM is mutated into a cysteine.

17. The set of claim 16 comprising at least 3 cysteine mutants.

18. The set of claim 16 wherein one or more naturally occurring cysteines of the TBM is mutated to a non-cysteine residue.

19. The set of claim 16 wherein the TBM is a cell surface or soluble receptor.

20. The set of claim 16 wherein the TBM is a cytokine.

21. The set of claim 16 wherein the TBM is an enzyme.

22. The set of claim 16 wherein the TBM is selected from the group consisting of IL-2;

IL-4;

TNF-α

;

IL-1 receptor;

caspase-3;

PTP-1B;

HIV integrase;

BACE1;

MEK-1;

Cat-S;

caspase-1;

IL-13;

CD40L;

BAFF;

P53;

mdm2;

bcl-x;

bax;

CDC25A;

CD28;

B7;

C5A;

AKT;

CD45;

HER2;

GSK-3;

alpha-E/beta-7;

tissue factor; and

Factor VII.