Labeling methodology comprising oligopeptides

• US 20060134691A1
• Filed: 12/04/2003
• Published: 06/22/2006
• Est. Priority Date: 12/05/2002
• Status: Abandoned Application

• Current Assignee: Jan-Marcus Seifert, Manfred Auer, Nicole-Claudia Meisner
• Original Assignee: Jan-Marcus Seifert, Manfred Auer, Nicole-Claudia Meisner
• Inventors: Seifert, Jan-Marcus, Meisner, Nicole-Claudia, Auer, Manfred
• Application Number:
  US10/534,966
• Publication Number:
  US 20060134691A1
• Time in Patent Office: Days
• US Class Current: 435/7.100
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

A method for providing a labeled target protein or labeled target peptide in high purity, which method comprises the steps of a. contacting a chemical molecule, which chemical molecule comprises a target protein or a target peptide residue beside amino acid residues, spacer residues, and/or linker residues, an affinity tagging residue, and a labeling residue which labeling residue may be detected by physical means, with an affinity support, to obtain said chemical molecule bound to the affinity support via the affinity tag of the affinity tagging residue, b. removing impurities in the reaction mixture surrounding the affinity support to which the chemical molecule according to step a. is bound, and c. cleaving or eluting said chemical molecule from said affinity support; and compounds useful in such method. Such method is e.g. useful for (ultra) HTS-screening, especially spectroscopic HTS systems at single molecule resolution.

Claims

1. A method for providing a labeled target protein or labeled target peptide, which method comprises the steps of:

a. contacting a chemical molecule, which chemical molecule comprises, spacer and/or linker residues, and optionally comprises the following parts

a target protein or a target peptide residue which is covalently bound as one of both terminal groups of said chemical molecule to a chemical backbone, which chemical backbone consists of amino acids and/or spacers and optionally linkers, covalently bound to each other, and which backbone contains a spacer and/or linker residues, and optionally contains

one amino acid or spacer residue as the second of both terminal groups of said chemical molecule covalently bound to a reversible or irreversible affinity tagging residue, with the proviso that in case that the affinity tagging residue is an irreversible affinity tagging residue, there is at least one linker residue covalently bound to said amino acid or spacer residue, which amino acid or spacer residue is on the other hand covalently bound to the irreversible affinity tagging residue, and

one amino acid or spacer residue covalently bound to a labeling residue which labeling residue may be detected by physical means,

with an affinity support, to obtain said chemical molecule bound to the affinity support via the affinity tag of the affinity tagging residue,

b. removing impurities in the reaction mixture surrounding the affinity support to which the chemical molecule according to step a. is bound, and

c. cleaving or eluting said chemical molecule from said affinity support to obtain

(i) in case of an irreversible affinity tagging residue, a labeled target protein or labeled peptide comprising a chemical backbone, which backbone contains a spacer and/or linker residues, and optionally contains one amino acid or spacer residue covalently bound to a labeling residue which labeling residue may be detected by physical means,

(ii) in case of a reversible affinity tagging residue, a chemical backbone covalently bound to a protein or a peptide residue, which backbone contains a spacer and/or linker residues, and optionally contains

one amino acid or spacer residue covalently bound to a labeling residue which labeling residue may be detected by physical means,

one amino acid or spacer residue covalently bound to a labeling residue, which labeling residue may be detected by physical means, and which labeling residue or spacer residue is covalently bound to a reversible affinity tagging residue.

2. A method for providing a labeled target protein or labeled target peptide, which method comprises a method of claim 1, comprising the steps of

a. reacting a compound, which compound comprises, a said spacer and/or linker residues, and optionally comprises the following parts

one amino acid or spacer residue as one of both terminal groups of said chemical molecule covalently bound to a reversible or irreversible affinity tagging residue, with the proviso that in case that the affinity tagging residue is an irreversible affinity tagging residue, there is at least one linker residue covalently bound to said amino acid or spacer residue, which amino acid or spacer residue is on the other hand covalently bound to the irreversible affinity tagging residue,

one amino acid or spacer residue covalently bound to a labeling residue which labeling residue may be detected by physical means,

one amino acid or spacer residue covalently bound to a reactive group containing residue, which residue is covalently bound as one of both terminal groups of said compound, and wherein the reactive group is able to react with a functional group contained in a target protein or target peptide,

with a target peptide or target protein,

b. contacting a chemical molecule obtained in step 1. with an affinity support, to obtain said chemical molecule bound to the affinity support via the affinity tag of the affinity tagging residue,

c. removing impurities in the reaction mixture surrounding the affinity support to which the chemical molecule according to step a. is bound, and

d. cleaving or eluting said chemical molecule from said affinity support to obtain

(i) in case of an irreversible affinity tagging residue, a labeled target protein or labeled target peptide comprising a chemical backbone, which backbone contains, optionally beside spacer and/or linker residues, one amino acid or spacer residue covalently bound to a labeling residue which labeling residue may be detected by physical means,

(ii) in case of a reversible affinity tagging residue, a chemical backbone covalently bound to a protein or a peptide residue, which backbone contains, said spacer and/or linker residues, and optionally contains

one amino acid or spacer residue covalently bound to a labeling residue which labeling residue may be detected by physical means, and

one amino acid or spacer residue covalently bound to a reversible affinity tagging residue.

3. A compound, which compound comprises the following parts

a. a reactive group containing residue, which residue is covalently bound as one of both terminal groups of said compound, and wherein the reactive group is selected from the group consisting of thiol, halogen, imine, aldehyde, the double bond in a vinylsulfonyl or a maleimido group and a group which forms together with a carbonyl group of said residue a carboxylic acid derivative;

b. a label containing residue, which label may be detected by physical means;

c. an affinity tagging group containing residue, which residue is covalently bound as the second of both terminal residues in a CHEMICAL COMPOUND, and wherein said affinity tagging group is a reversible affinity tagging group or an irreversible affinity tagging group, with the proviso that in case that the affinity tagging group is an irreversible affinity tagging group, there is at least one linker residue covalently bound to an amino acid or spacer residue, which carries the irreversible affinity tagging group, and

which residue of any of a., b., or c. is selected from

1 to 10 amino acids, which amino acids are covalently bound to each other via amide bonds of the carboxylic acid group of one amino acid and the amine group of a second amino acid,

1 to 10 spacers, which spacers are covalently bound to each other via functional groups of said spacers, and

1 to 10 amino acids and spacers, which amino acid and spacers are covalently bound to each other either via amide bonds of the carboxylic acid group of one amino acid and the amine group of a second amino acid, or via functional groups of said spacers, or via an amine group or a carboxylic acid group of an amino acid with a functional group of a spacer;

d. optionally further spacer residues; and

e. optionally linker residues.

4. A compound according to claim 3, which is a compound of formula

A-Y-D-Y′

—

X—

Y″

-E 

 

I

wherein

A is

a group of formula

wherein x is 1 to 5, and

R is (C_1-4)alkyl carboxylic acid ester, e.g. propionic acid ethyl ester, or a group of formula

wherein y is 2 to 4, e.g. 2;

D is

a group of formula

or a group of formula

or a group of formula

or a group of formula

or a group of formula

LABEL is a group of formula

or a group of formula

or a group of formula

or a group of formula

wherein ANION is a negatively charged group, e.g. an anion, such as CF₃COO⁻

,

X is a group of formula

wherein

R₁ is H or CH₃, R₂ is H or —

OCH₃ and z is 1 to 5;

E is a group of formula

or a group of formula

wherein to one of the amine groups a group AFF is bound and wherein n is 1 to 4, and

EITHER the affinity tag is bound to a carboxylic acid residue, e.g. the carboxylic residue of (ADO1)n and is a group of formula

or of formula

or of formula

OR the affinity tag is bound to an amino group, e.g. to the ε

-amino group of the lysine residue in LYS-GLY-NT1 or LYS-GLY-NT2;

or to an amino group of (ADO1)n and is a group of formula

or of formula

or of formula

and

Y, Y′

, Y″

independently of each other are a group of formula

wherein n is 1 to 4,

or a group of formula

with the proviso, that

IF A is CYS or MAL,

THEN

D is LYS1, LYS1 (GLY), GLY-LYS1, LYS-GLY1 or (ADO1)_n-LYS1,

X is X1,

E is LYS-GLY-NT1 or (ADO1)n,

AFF is AFF1, AFF2, AFF3 or AFF4, and

Y, Y′

and Y″

independently of each other are GLY1 or (ADO1)n, wherein n is 1 to 4, and

IF A is THIOESTER,

THEN

D is LYS2, GLY-LYS1, LYS-GLY2 or (ADO2)_n-LYS1,

X is X2,

E is LYS-GLY-NT2 or (ADO2)n and n is 1 to 4,

AFF is AFF1, AFF5 or AFF6, and

Y, Y′ and

Y″

independently of each other are GLY2 or (ADO2)n, wherein n is 1 to 4.

5. A compound according to claim 4 wherein A, Y, D, Y′

, X, Y″ and

E are as defined in TABLE 1 below, wherein in X1 and X2, R₁ is methyl, R₂ is methoxy and z=3, and in (ADO1)n and (ADO2)n n=1, except for EX 12 wherein n=3; and

for EX 11, 15, and 26 to 29 wherein n=2;

6. A compound according to claim 3, which is covalently bound to the residue of a target protein or a target peptide via a bond originating from the reaction of a reactive group with a functional group of said target protein or target peptide.

7. A compound according to claim 6, which compound is further bound to an affinity support via an affinity tagging group.

8. A compound according to claim 3 in the form of a salt.

9. A compound according to claim 3 bound to a solid support via a terminal functional group.

10. The use of a compound of according to claim 3 for the labeling of a target protein or target peptide.

11. The use of a target protein or target peptide bound to a compound according to claim 6, in a high throughput screening assay.

12. A kit comprising a compound of claim 3 and instructions for using the kit.

13. A method for identifying an agent that modulates the activity or characteristic of a target protein or a target peptide comprising the steps

a. contacting a target protein or target peptide bound to a compound according to claim 6, with a candidate compound, which is expected to modulate the activity or characteristics of said target protein or target peptide,

b. measuring a signal,

(i) in the absence of such candidate compound, and

(ii) in the presence of such candidate compound

and determining whether there is a difference in the signals measured in steps (i) and (ii), and

c. choosing an agent determined in step b.

14. A pharmaceutical composition comprising an agent identified in a method of claim 13 in association with at least one pharmaceutical excipient, e.g. appropriate carrier and/or diluent, e.g. including fillers, binders, disintegrators, flow conditioners, lubricants, sugars and sweeteners, fragrances, preservatives, stabilizers, wetting agents and/or emulsifiers, solubilizers, salts for regulating osmotic pressure and/or buffers, e.g. further comprising another pharmaceutically active agent.

15. An agent identified by a method of claim 13 for use as a pharmaceutical.

17. A kit comprising a compound of claim 6, and instructions for using the kit.

16. A method of treatment disorders mediated by a target protein or target peptide in vivo, comprising administering to a subject in need of such treatment an effective amount of an agent identified by a method according to the present invention.