Method of use of sonic hedgehog protein as a ligand for patched
AssigneeThe Johns Hopkins University School of Medicine
InventorPhilip A. Beachy
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Abstract
The invention relates to hedgehog proteins that are involved in signaling developmental processes. The proteins of the invention modulate differentiation of neural plate. Also provided are methods for identifying compounds that modulate early events in development and more particularly to methods to identify compounds that modulate differentiation of neural plate using hedgehog proteins as ligands for the patched receptor.
Claims46
An isolated protein comprising the amino acid sequence set forth in SEQ ID NO:1 wherein alanine is substituted for the amino acid residue at a position selected from 51, 52, 56, 75, 90, 76, 81, 105, 116, 132, 135, 138, 168, 177, 189 and 195, and any combination thereof.
An isolated protein comprising the amino acid sequence set forth in a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8 and SEQ ID NO:12.
A method for identifying a compound that modulates a cellular response mediated by a hedgehog protein comprising:
- (a) incubating the compound with a cell expressing a hedgehog protein and a patched receptor under conditions sufficient to permit the compound to interact with the patched receptor or the hedgehog protein;
- and
- (b) comparing a cellular response mediated by a hedgehog protein in the cell incubated with the compound with the response of a cell not incubated with the compound,
- wherein a difference in response between the cell incubated with the compound and the cell not incubated with the compound is indicative of a compound that modulates a cellular response mediated by the hedgehog protein.
A method for identifying a compound that modulates a cellular response mediated by a patched receptor comprising:
- (a) incubating the compound with a cell expressing the patched receptor and a hedgehog protein under conditions sufficient to permit the compound to interact with the cell;
- and
- (b) comparing a cellular response in a cell incubated with the compound with the cellular response of a cell not incubated with the compound,
- wherein a difference in response is indicative of a compound that modulates a cellular response mediated by the patched receptor.
A method for identifying a compound that modulates differentiation of neural plate cells, comprising:
- (a) incubating the compound and a hedgehog protein with neural cells expressing a patched receptor under conditions sufficient to permit the compound to interact with the neural plate cells;
- and
- (b) comparing the differentiation of neural plate cells incubated with the compound with the differentiation of neural plate cells not incubated with the compound;
- wherein a difference in differentiation of neural plate cells incubated with the compound compared to differentiation of neural plate cells incubated without the compound is indicative of a compound that modulates differentiation of neural plate cells.
A method of increasing the stability of a hedgehog protein, comprising:
- (a) introducing into the amino acid sequence of a hedgehog protein a thiol-containing amino acid at a position corresponding to position 51, 52, 56, 75, 90, 76, 81, 105, 116, 132, 135, 138, 168, 177, 189, 195, or combinations thereof of mouse sonic hedgehog;
- and
- (b) modifying the introduced amino acid by attaching to a maleimide-linked moiety;
- thereby increasing the stability of a hedgehog protein.
A method for modifying the pharmacokinetic properties of a hedgehog protein, comprising:
- (a) introducing into the amino acid sequence of a hedgehog protein a thiol-containing amino acid at position 51, 52, 56, 75, 90, 76, 81, 105, 116, 132, 135, 138, 168, 177, 189, 195, or combinations thereof;
- and
- (b) modifying the introduced amino acid by attaching a maleimide-linked moiety;
- thereby modifying the pharmacokinetic properties of a hedgehog protein.
A method of identifying a surface amino acid of a hedgehog protein involved in hedgehog signaling, comprising:
- (a) incubating a hedgehog protein having one or more substituted amino acid residues and a cell expressing a patched receptor under conditions that allow the hedgehog protein to interact with the cell;
- and
- (b) assaying hedgehog-mediated signaling;
- wherein a difference in hedgehog mediated signaling in the cell incubated with the hedgehog protein as compared to hedgehog mediated signaling in a cell not incubated with the hedgehog protein is indicative of a hedgehog protein involved in hedgehog signaling.
Dependencies are inferred from the claim text.