Incorporation of phosphorylation sites

Details Incorporation of phosphorylation sites Incorporation of phosphorylation sites

1998-10-16

2002-01-01

Russel, Jeffrey E. (Department: 1651)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

C435S068100, C530S329000, C530S345000

Reexamination Certificate

active

06335176

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to the incorporation in compounds of sites that are substrates for phosphorylating enzymes. The compounds that are phosphorylated by these enzymes, for example proteins and peptides, are particularly useful in assays, such as those used in drug discovery.
BACKGROUND OF THE INVENTION
Ligand binding techniques, which measure molecular interactions, have been fundamental in the elucidation of cellular signaling processes and in the discovery of drugs. As the understanding of the molecular basis of disease has matured, the number of potential molecular targets for pharmaceutical intervention has dramatically increased. As a result, there is a need in the drug discovery process for new methods to measure novel biomolecular interactions. Studies using [
125
I]-ACTH (Lefkowitz et al., (1970)
Proc. Natl. Acad Sci. U.S.A.
65(3): 745-752) and [
125
I]-insulin (Cuatrecasas, (1971)
Proc. Natl. Acad. Sci.
68(6): 1264-8) allowed the direct detection of G protein-coupled receptors and cytokine receptors, respectively, for the first time. These methodologies have since been widely employed in the field of signal transduction biochemistry.
The Bolton-Hunter reagent (Bolton and Hunter, (1973)
Biochem. J.
133: 529-539) subsequently allowed the introduction of
125
I into proteins using a mild acylation reaction, thus expanding the use of
125
I-labeling to proteins that are sensitive to oxidative iodination. The Bolton-Hunter reagent also allowed labeling of proteins or peptides that lack tyrosine residues by linking to reactive amine groups.
Fluorescent ligands, which allow detection sensitivities to be achieved that are similar to those achieved using
125
I, are now also routinely used (Hemmila and Webb (1997)
Drug Discovery Today
2:373-381; Inglese et. al. (1998)
Biochemistry
37:2372-2377. Fluorescent labeling procedures are safer and more stable than radiolabeling procedures employing
125
I.
However, radiolabeled ligands remain useful because of the unique properties of radioisotopes, which allow important assay techniques such as the scintillation proximity assay (Hart and Greenwald (1979)
Mol. Immunol.
16: 265-267; Bosworth and Towers (1989)
Nature
341: 167-168) and autoradiography to be carried out. Nevertheless, to employ such assays advantageously, a variety of labeling methods are required.
Phosphorylation reactions can be carried out to introduce radioactive phosphorus into compounds. In proteins, this has been done by incorporating cAMP-dependent protein kinase A (PKA) consensus sites. For example, labeling of receptor ligands and fusion proteins with
32
P has been accomplished by recombinantly engineering PKA consensus sites into the amino acid sequences of those ligands and proteins (Li et al. (1989)
Proc. Natl. Acad. Sci. USA
86:558-562). Such recombinant methods, however, must be individually designed for each target protein. Furthermore, when the phosphorylation site is recombinantly introduced into the protein sequence it can adversely affect protein function or become inaccessible after the protein has folded. Also, recombinantly introducing the phosphorylation site normally allows only one site to be incorporated.
There is a need for a method to chemically modify already existing proteins and peptides so that they become substrates for protein kinase. Such a method would be used to radioactively phosphorylate proteins that have already been synthesized, such as those that are commonly commercially available.
There is also a need for a method that allows introduction of multiple phosphorylation sites in proteins, and which normally does not interfere with the protein's function, or become inaccessible as a result of protein folding.
SUMMARY OF THE INVENTION
This present invention relates to a reagent for incorporating phosphorylation sites into compounds, particularly into proteins and peptides. The reagent has the structure
A—B—C
wherein A is a moiety that is specifically reactive with a reactive side chain in the compound, B is a linking moiety, and C is a peptide sequence that contains a kinase substrate.
The invention also relates to a method involving reacting the reagent with the compound to produce a phosphorylatable product, and phosphorylating the product using a kinase specific for the kinase substrate.
In another aspect, the present invention relates to the phosphorylatable or phosphorylated product resulting from the method of the invention. dr
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A
shows a coomassie stained gel of proteins either treated (lanes 2,4,6,8) or untreated (lanes 1,3,5,7) with the reagent of the invention and phosphorylated with PKA using [&ggr;-
32
P]ATP.
FIG. 1B
shows the same gel exposed to a phosphoimager screen to detect [
32
P] incorporation.
FIG. 2
shows a protein (R) modified with the reagent of the invention, and then enzymatically phosphorylated with [g-
32
P]ATP using the catalytic subunit of cAMP dependent protein kinase A (PKA).
FIG. 3
shows phosphorylated neurokinin (NKA) according to the invention. The phosphorylated product (R=PO
3
) and unphosphorylated precursor (R=H) are shown.
FIG. 4A
shows a coomassie stained gel of an SDS-PAGE analysis of compounds modified using the reagent of the invention.
FIG. 4B
shows an autoradiograph of lanes corresponding to those in FIG.
4
A.
FIG. 4C
shows a coomassie stained gel of an SDS-PAGE analysis of leptin that was modified using the reagent of the invention.
FIG. 4D
shows an autoradiograph of lanes corresponding to those in of FIG.
4
C.
FIG. 5A
shows an SDS-PAGE analysis of an Fc-fusion of erythropoietin (Epo) that was modified using the reagent of the invention.
FIG. 5B
shows a coomassie stained gel of an SDS-PAGE analysis of
32
P-labeled Epo-Fc.
FIG. 5C
shows an autoradiograph of lanes that correspond to those in FIG.
5
B.
FIG. 6
shows an ethylenediamine derivative of biotin modified with the reagent of the invention.
FIGS. 7A and 7B
, and
7
C show the structures of alprenolol (FIG.
7
A), an alprenolol derivative (
FIG. 7B
) and the alprenolol derivative modified with the reagent of the invention (FIG.
7
C).
FIG. 8
shows Table 1, containing results for ligand-receptor systems assayed using compounds labeled according to the invention.
FIG. 9
shows results of saturation binding experiments for four ligands labeled with the reagent of the invention.
FIG. 10
is a Table (Table 2) showing the estimated dissociation (K
D
) and inhibition (K
i
) constants determined for four ligands based on results shown in FIG.
9
and FIG.
11
. The calculated constants are compared with published values for the corresponding
125
I or Eu
3+
-labeled ligands.
FIG. 11A
shows a binding plot for competition of NKA-[
33
P]labeled ligand of the invention by NKA for NK2 receptor on CHO cells.
FIG. 11B
shows a binding plot for competition of IL8-[
33
P]labeled ligand of the invention for CXC2 receptor on CHO cells.
FIG. 11C
shows a binding plot for competition of Epo-[
33
P]labeled ligand of the invention for EpoR-Fc surface-coated plates.
FIG. 11D
shows a binding plot for competition of leptin [
33
P]labeled ligand of the invention for ObR-Fc surface-coated plates.


REFERENCES:
patent: 5106955 (1992-04-01), Endo et al.
patent: 5165923 (1992-11-01), Thorpe et al.
patent: 5459240 (1995-10-01), Foxwell et al.
patent: 5583212 (1996-12-01), Foxwell et al.
patent: 5599681 (1997-02-01), Epstein et al.
patent: 5986061 (1999-11-01), Pestka
patent: 6066462 (2000-05-01), Goueli
Pierce Immuno Technology Catalog & Handbook p. A-6, 1992.*
Foxwell et al. Conjugation of Monoclonal Antibodies to a Synthetic . . . Br. J. Cancer, vol. 57, pp. 489-493, 1988.*
Cell Biology: Bo-Liang Li et al., Creation of Phosphorylation Sites in Proteins: Construction of a Phosphorylatable Human Interferon &agr;,Proc. Natl. Acad. Sci. USA,vol. 86, pp. 558-562, Jan. 1989.
Michael Brinkley, A Brief Survey of Methods for Preparing Protein Conjugate

Affiliated with

Also associated with

Rating

Incorporation of phosphorylation sites does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Incorporation of phosphorylation sites, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Incorporation of phosphorylation sites will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2859073