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US 5,352,660 AGranted
Method for assaying for a substance that affects a SH2-phosphorylated ligand regulatory system
AssigneeMount Sinai Hospital
InventorAnthyony J. Pawson
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AssigneeMount Sinai Hospital
InventorAnthyony J. Pawson
Estimating the term…
A method for assaying a medium for the presence of a substance that affects an SH2-phosphorylated ligand regulatory system. The method employs an SH2-like domain or a subdomain thereof and a phosphorylated ligand. The phosphophorylated ligand is capable of interacting with the SH2-like domain or a subdomain thereof to form an SH2-phosphorylated ligand complex. The SH2-like domain or subdomain and/or the phosphorylated ligand are present in a known concentration. The SH2-like domain or a subdomain thereof and the phosphorylated ligand are incubated with a substance which is suspected of affecting an SH2-phosphorylated ligand regulatory system. The method is carried out under conditions which permit the formation of the SH2-phosphorylated ligand complex. SH2-phosphorylated ligand complex, free SH2-like domain or subdomains thereof, or non-complexed phosphorylated ligand are assayed. The invention also relates to an isolated SH2-phosphorylated ligand complex; a method of using an isolated SH2-like domain or a subdomain thereof to screen for phosphorylated ligands which are active in an SH2-phosphorylated ligand regulatory system; a method of using an isolated SH2-like domain or a subdomain thereof to regulate the interaction of a signalling protein with a related phosphorylated ligand; and a pharmaceutical composition comprising an isolated SH2-like domain or a subdomain thereof for use as an agonist or antagonist of the interaction of a signalling protein with a related phosphorylated ligand.
A method for assaying a medium for the presence of a substance that affects a Src homology region 2-phosphorylated ligand regulatory system comprising providing a Src homology region 2 having the amino acid sequence of the Src homology region 2 of c-Src, c-Yes, Fgr, Fyn, Lck, Lyn, Hck, Blk, c-Abl, Arg, Dabl, c-fps, Fer, PLC-.gamma.1N, PLC-.gamma.2N, PLC-.gamma.1C, PLC-.gamma.2C, GAP-N, GAP-C, p85.alpha.-N, p85.beta.-N, p85.alpha.-C, p85.beta.-C, Nck, Tensin, or Vav as shown in FIG. 2, and a phosphorylated ligand, the Src homology region 2 and the phosphorylated ligand being selected so that they bind to form a Src homology region 2-phosphorylated ligand complex which is capable of activating the Src homology region 2-phosphorylated ligand regulatory system, the Src homology region 2 and/or the phosphorylated ligand being present in a known concentration, and incubating with a test substance which is suspected of affecting the Src homology region 2-phosphorylated ligand regulatory system, under conditions which permit the formation of the SH2-phosphorylated ligand complex, and assaying for the SH2-phosphorylated ligand complex, free SH2-domain, or non-complexed phosphorylated ligand and comparing to a control to determine the effect of the substance.
A method for assaying a medium for the presence of an agonist or antagonist substance of a Src homology region 2-phosphorylated ligand regulatory system comprising providing a Src homology region 2 having the amino acid sequence of the Src homology region 2 of c-Src, c-Yes, Fgr, Fyn, Lck, Lyn, Hck, Blk, c-Abl, Arg, Dabl, c-fps, Fer, PLC-.gamma.1N, PLC-.gamma.2N, PLC-.gamma.1C, PLC-.gamma.2C, GAP-N, GAP-C, p85.alpha.-N, p85.beta.-N, p85.alpha.-C, p85.beta.-C, Nck, Tensin, or Vav as shown in FIG. 2, and a phosphorylated ligand, the Src homology region 2 and the phosphorylated ligand being selected so that they bind to form a Src homology region 2-phosphorylated ligand complex which is capable of activating the Src homology region 2-phosphorylated ligand regulatory system, the Src homology region 2 and/or the phosphorylated ligand being present in a known concentration, and incubating with a suspected agonist or antagonist substance, under conditions which permit the formation of the SH2-phosphorylated ligand complex, and assaying for the Src homology region 2-phosphorylated ligand complex, free Src homology region 2, or non-complexed phosphorylated ligand and comparing to a control to determine the effect of the substance.
A pharmaceutical composition comprising an isolated Src homology region 2 having the amino acid sequence of the Src homology region 2 of c-Src, c-Yes, Fgr, Fyn, Lck, Lyn, Hck, Blk, c-Abl, Arg, Dabl, c-fps, Fer, PLC-.gamma.1N, PLC-.gamma.2N, PLC-.gamma.1C, PLC-.gamma.2C, GAP-N, GAP-C, p85.alpha.-N, p85.beta.-N, p85.alpha.-C, p85.beta.-C, Nck, Tensin, or Vav as shown in FIG. 2 for use as an agonist or antagonist of the interaction of a signalling protein with a related phosphorylated ligand.
Dependencies are inferred from the claim text.