Phosphohistidine and phosphotyrosine analogues

Patent metadata

Field	Value
Publication number	US-2016207948-A1
Application number	US-201414914994-A
Country	US
Kind code	A1
Filing date	Sep 2, 2014
Priority date	Sep 4, 2013
Publication date	Jul 21, 2016
Grant date	—

Abstract

The invention relates to phosphohistidine analogues. The invention also relates to antibodies that specifically bind to the analogues and methods of generating said antibodies. In one embodiment of the invention there is provided a phosphohistidine analogue of Formula V: (V) wherein W is selected from H, CO 2 H or CONH 2 ; and X is selected from CH or N.

First claim

1 . A phosphohistidine analogue of Formula V or Formula III:

wherein
W is selected from H, CO 2 H or CONH 2 ;
X 1 is selected from CH or N;
X 2 is selected from NH, O or S.

2 . The phosphohistidine analogue according to claim 1 , wherein said analogue is selected from the group consisting of:

3 - 11 . (canceled)

12 . The phosphohistidine analogue according to claim 1 , selected from the group consisting of:

13 . The phosphohistidine analogue of claim 1 wherein W is selected from H or CO 2 H.

14 . The phosphohistidine analogue according to claim 1 , wherein X 1 is CH.

15 . A polypeptide comprising the analogue of claim 1 .

16 . An antibody or fragment thereof, that binds to the analogue of claim 1 wherein said antibody binds to a phosphohistidine amino acid residue or analogue thereof with an affinity of 5-fold greater or more than its affinity for a molecule other than a phosphohistidine amino acid residue or analogue thereof.

17 . (canceled)

18 . The antibody or fragment thereof according to claim 16 , wherein said antibody is a monoclonal antibody.

19 . (canceled)

20 . The antibody or fragment thereof according to claim 16 , wherein said phosphohistidine amino acid residue is a residue in a polypeptide.

21 . A method of producing an anti-phosphohistidine antibody, comprising:
(a) immunizing a non-human animal with an analogue according to claim 1 ; (b) collecting serum from the immunized animal; (c) isolating from the serum a population of polyclonal antibodies that bind to the analogue.

22 . The method according to claim 21 , wherein said analogue is provided as an amino acid residue in a polypeptide; or is coupled to a carrier protein, and wherein said carrier protein is optionally selected from keyhole limpet hemocyanin (KLH), or bovine serum albumin (BSA).

23 - 26 . (canceled)

27 . A polyclonal antibody or fragment thereof or a population of polyclonal antibodies obtained by the method according to claim 21 .

28 . A method of producing an anti-phosphohistidine antibody, comprising:
(a) immunizing a non-human animal with an analogue according to claim 1 ; (b) generating hybridomas by fusing B-cells isolated from the immunized animal with myeloma cells; (c) identifying hybridomas that produce monoclonal antibodies that bind to the analogue; and (d) propagating said identified hybridomas to produce monoclonal antibodies that bind to the analogue.

29 . The method according to claim 28 , wherein said analogue is provided as an amino acid residue in a polypeptide; or is coupled to a carrier protein, and wherein said carrier protein is selected from keyhole limpet hemocyanin (KLH), or bovine serum albumin (BSA).

30 - 32 . (canceled)

33 . A monoclonal antibody or fragment thereof obtained by the method according to claim 28 .

34 . A method of detecting a phosphohistidine-containing polypeptide comprising:
(a) contacting a test polypeptide with an antibody according to claim 16 ; and (b) detecting binding of said antibody to said test polypeptide,
wherein binding of the antibody to the test polypeptide identifies the polypeptide as containing a phosphohistidine residue.

35 - 41 . (canceled)

42 . A polypeptide comprising the analogue of claim 2 .

43 . A polypeptide comprising the analogue of claim 12 .

44 . An antibody or fragment thereof, that binds to the analogue of claim 2 wherein said antibody binds to a phosphohistidine amino acid residue or analogue thereof with an affinity of 5-fold greater or more than its affinity for a molecule other than a phosphohistidine amino acid residue or analogue thereof.

45 . An antibody or fragment thereof, that binds to the analogue of claim 12 wherein said antibody binds to a phosphohistidine amino acid residue or analogue thereof with an affinity of 5-fold greater or more than its affinity for a molecule other than a phosphohistidine amino acid residue or analogue thereof.

46 . An antibody or fragment thereof, that binds to the analogue of claim 1 wherein said antibody binds to a phosphohistidine amino acid residue or analogue thereof with an affinity of 5-fold greater or more than its affinity for phosphotryosine or a phosphotryosine amino acid residue.

47 . An antibody or fragment thereof, that binds to the analogue of claim 2 wherein said antibody binds to a phosphohistidine amino acid residue or analogue thereof with an affinity of 5-fold greater or more than its affinity for phosphotryosine or a phosphotryosine amino acid residue.

48 . An antibody or fragment thereof, that binds to the analogue of claim 12 wherein said antibody binds to a phosphohistidine amino acid residue or analogue thereof with an affinity of 5-fold greater or more than its affinity for phosphotryosine or a phosphotryosine amino acid residue.

Assignees

• Univ Sheffield

Inventors

• Jackson Richard
• Muimo Richard
• Lilley Matthew

Classifications

• C07F9/58

  Pyridine rings · CPC title

• C07F9/655345

  the sulfur atom being part of a five-membered ring · CPC title

• C07F9/65031 Primary

  having the nitrogen atoms in the positions 1 and 2 · CPC title

• G01N33/5308

  for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites · CPC title

• C07K14/001

  by chemical synthesis · CPC title