Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Tripeptides – e.g. – tripeptide thyroliberin – etc.
Reexamination Certificate
1998-04-29
2002-02-19
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Tripeptides, e.g., tripeptide thyroliberin , etc.
C514S018700, C514S019300
Reexamination Certificate
active
06348572
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the use of mutant phosphatase and protease enzymes in a competitive binding assay. Specific examples are the enzymes, tyrosine phosphatase and cysteine protease, e.g. Cathepsin K, and the assay specifically described is a scintillation proximity assay using a radioactive inhibitor to induce scintillation.
BACKGROUND OF THE INVENTION
The use of the scintillation proximity assay (SPA) to study enzyme binding and interactions is a new type of radioimmunoassay and is well known in the art. The advantage of SPA technology over more conventional radioimmunoassay or ligand-binding assays, is that it eliminates the need to separate unbound ligand from bound ligand prior to ligand measurement. See for example,
Nature
, Vol, 341, pp. 167-178 entitled “Scintillation Proximity Assay ” by N. Bosworth and P. Towers,
Anal. Biochem
. Vol. 217, pp. 139-147 (1994) entitled “Biotinylated and Cysteine-Modified Peptides as Useful Reagents For Studying the Inhibition of Cathepsin G” by A. M. Brown, et al.,
Anal. Biochem
. Vol. 223, pp. 259-265 (1994) entitled “Direct Measurement of the Binding of RAS to Neurofibromin Using Scintillation Proximity Assay” by R. H. Skinner et al. and
Anal. Biochem
. Vol. 230, pp. 101-107(1995) entitled “Scintillation Proximity Assay to Measure Binding of Soluble Fibronectin to Antibody-Captured alpha
5
&bgr;
1
Integrin” by J. A. Pachter et al.
The basic principle of the assay lies in the use of a solid support containing a scintillation agent, wherein a target enzyme is attached to the support through, e.g., a second enzyme-antienzyme linkage. A known tritiated or I
125
iodinated binding agent, i.e., radioligand inhibitor ligand for the target enzyme is utilized as a control, which when bound to the active site in the target enzyme, is in close proximity to the scintillation agent to induce a scintillation signal, e.g., photon emission, which can be measured by conventional scintillation/radiographic techniques. The unbound tritiated (hot) ligand is too far removed from the scintillation agent to cause an interfering measurable scintillation signal and therefore does not need to be separated, e.g., filtration, as in conventional ligand-binding assays.
The binding of an unknown or potential new ligand (cold, being non-radioactive) can then be determined in a competitive assay versus the known radioligand, by measuring the resulting change in the scintillation signal which will significantly decrease when the unknown ligand also possesses good binding properties.
However, a problem arises when utilizing a target enzyme containing a cysteine group, having a free thiol linkage, —SH, (or present as —S
−
) which is in the active site region or is closely associated with the active site and is important for enzyme-ligand binding. If the unknown ligand or mixture, e.g. natural product extracts, human body fluids, cellular fluids, etc. contain reagents which can alkylate, oxidize or chemically interfere with the cysteine thiol group such that normal enzyme-ligand binding is disrupted, then false readings will occur in the assay.
What is needed in the art is a method to circumvent and avoid the problem of cysteine interference in the scintillation proximity assay (SPA) procedure in enzyme binding studies.
An inhibitor of PTP 1B has the potential to improve insulin-sensitivity and will have utility in preventing or treating Type 1 and Type 2 diabetes, to improve glucose tolerance, to improve insulin-sensitivity when there is insulin-resistance and to treat or prevent obesity. In addition, it may be of use to treat or prevent cancer, neurodegenerative diseases etc.
SUMMARY OF THE INVENTION
We have discovered that by substituting serine for cysteine in a target enzyme, where the cysteine plays an active role in the wild-type enzyme-natural ligand binding process, usually as the catalytic nucleophile in the active binding site, a mutant is formed which can be successfuly employed in a scintillation proximity assay without any active site cysteine interference.
This discovery can be utilized for any enzyme which contains cysteine groups important or essential for binding and/or catalytic activity as proteases or hydrolases and includes phosphatases, e.g., tyrosine phosphatases and proteases, e.g. cysteine proteases, including the cathepsins, i.e., Cathepsin K (O2) and the caspases.
Further, use of the mutant enzyme is not limited to the scintillation proximity assay, but can be used in a wide variety of known assays including colorimetric, spectrophotometric, ligand-binding assays, radioimmunoassays and the like.
We have furthermore discovered a new method of amplifying the effect of a binding agent ligand, e.g., radioactive inhibitor, useful in the assay by replacing two or more phosphotyrosine residues with 4-phosphono(difluoromethyl) phenylalanine (F
2
Pmp) moieties. The resulting inhibitor exhibits a greater and more hydrolytically stable binding affinity for the target enzyme and a stronger scintillation signal.
By this invention there is provided a process for determining the binding ability of a ligand to a cysteine-containing wild-type enzyme comprising the steps of:
(a) contacting a complex with the ligand, the complex comprising a mutant form of the wild-type enzyme, in which cysteine, at the active site, is replaced with serine, in the presence of a known binding agent for the mutant enzyme, wherein the binding agent is capable of binding with the mutant enzyme to produce a measurable signal.
Further provided is a process for determining the binding ability of a ligand, preferably a non-radioactive (cold) ligand, to an active site cysteine-containing wild-type tyrosine phosphatase comprising the steps of:
(a) contacting a complex with the ligand, the complex comprising a mutant form of the wild-type enzyme, the mutant enzyme being PTP1B, containing the same amino acid sequence 1-320 as the wild type enzyme, except at position 215, in which cysteine is replaced with serine in the mutant enzyme, in the presence of a known radioligand binding agent for the mutant enzyme, wherein the binding agent is capable of binding with the mutant enzyme to produce a measurable beta radiation-induced scintillation signal.
Also provided is a new class of peptide binding agents selected from the group consisting of:
N-Benzoyl-L-glutamyl-[4-phosphono(difluoromethyl)]-L-phenylalanyl-[4-phosphono(difluoromethyl)]-L-phenylalanineamide (BzN-EJJ-CONH
2
), where E is glutamic acid and J is 4-phosphono(difluoro-methyl)]-L-phenylalanyl;
N-Benzoyl-L-glutamyl-[4-phosphono(difluoromethyl)]-L-phenylalanyl-[4-phosphono(difluoromethyl)]-L-phenylalanine amide;
N-(3,5-Dibromo)benzoyl-L-glutamyl-[4-phosphono(difluoromethyl)]-L-phenylalanyl-[4-phosphono(difluoromethyl)]-L-phenylalanine amide;
N-Acetyl-L-glutamyl-[4-phosphono(difluoromethyl)]-L-phenylalanyl-[4-phosphono(difluoromethyl)]-L-phenylalanine amide;
L-Glutamyl-[4-phosphono(difluoromethyl)]-L-phenylalanyl-[4-phosphono(difluoromethyl)]-L-phenylalanine amide;
L-Lysinyl-[4-phosphono(difluoromethyl)]-L-phenylalanyl-[4-phosphono(difluoromethyl)]-L-phenylalanine amide;
L-Serinyl-[4-phosphono(difluoromethyl)]-L-phenylalanyl-[4-phosphono(difluoromethyl)]-L-phenylalanine amide;
L-Prolinyl-[4-phosphono(difluoromethyl)]-L-phenylalanyl-[4-phosphono-(difluoromethyl)]-L-phenylalanine amide; and
L-Isoleucinyl-[4-phosphono(difluoromethyl)]-L-phenylalanyl-[4-phosphono(difluoromethyl)]-L-phenylalanine amide; and their tritiated and I
125
iodinated derivatives.
Also provided is a class of compounds represented by structure A which are peptide binding agents and inhibitors of the PTP 1B enzyme.
Further provided is a novel tritiated peptide, tritiated BzN-EJJ-CONH
2
, being N-(3,5-Ditritio)benzoyl-L-glutamyl-[4-phosphono(difluoromethyl)]-L-phenylalanyl-[4-phosphono(difluoromethyl)]-L-phenylalanineamide, wher
Desmarais Sylvie
Dufresne Claude
Friesen Richard
LeBlanc Yves
Roy Patrick
Durette Philippe L.
Low Christopher S. F.
Lukton David
Merck Frosst Canada & Co.
Quagliato Carol S.
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