Screening assays for hedgehog agonists and antagonists
First Claim
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1. An assay for identifying compounds able to potentiate or inhibit biding of a hedgehog polypeptide to a naturally occurring patched receptor, comprising:
- (a) forming a reaction mixture including;
(i) a vertebrate hedgehog polypeptide;
(ii) a naturally occurring patched receptor; and
(iii) a test compound; and
(b) detecting binding of the hedgehog polypeptide and the patched receptor;
wherein a change in the binding of the hedgehog polypeptide and the patched receptor in the presence of the test compound, relative to the binding in the absence of the test compound, indicates that said test, compound potentiates or inhibits binding of said hedgehog polypeptide to said patched receptor.
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Abstract
The present invention concerns the discovery that proteins encoded by a family of vertebrate genes, termed here hedgehog-related genes, comprise morphogenic signals produced by embryonic patterning centers, and are involved in the formation of ordered spatial arrangements of differentiated tissues in vertebrates. The present invention makes available compositions and methods that can be utilized, for example to generate and/or maintain an array of different vertebrate tissue both in vitro and in vivo.
67 Citations
27 Claims
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1. An assay for identifying compounds able to potentiate or inhibit biding of a hedgehog polypeptide to a naturally occurring patched receptor, comprising:
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(a) forming a reaction mixture including;
(i) a vertebrate hedgehog polypeptide;
(ii) a naturally occurring patched receptor; and
(iii) a test compound; and
(b) detecting binding of the hedgehog polypeptide and the patched receptor;
wherein a change in the binding of the hedgehog polypeptide and the patched receptor in the presence of the test compound, relative to the binding in the absence of the test compound, indicates that said test, compound potentiates or inhibits binding of said hedgehog polypeptide to said patched receptor.
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2. An assay for identifying a test compound which inhibits or potentiates binding of a hedgehog polypeptide to a naturally occurring patched receptors comprising:
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(a) forming a reaction mixture including;
(i) a hedgehog polypeptide, which polypeptide binds a naturally occurring patched receptor and comprises an amino acid sequence encoded by a nucleic acid sequence which hybrdizes under stringent conditions, including a wash step of 0.5×
SSC at 60°
C. conditions with a nucleic acid sequence selected from the group consisting of SEQ ID No;
1, SEQ ID No;
2, SEQ ID No;
3, SEQ ID No;
4, SEQ ID No;
5, SEQ ID No;
6 or SEQ ID No;
7;
(ii) a naturally occurring patched receptor; and
(iii) a test compound; and
(b) detecting binding of said hedgehog polypeptide to said patched receptor;
wherein a change in the binding of said hedgehog polypeptide to said patched receptor in the presence of the test compound, relative to binding in the absence of the test compound, indicates that said test compound potentiates or inhibits binding of said hedgehog polypeptide to said patched receptor. - View Dependent Claims (3, 4, 5, 6, 7, 8, 9)
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10. An assay for screening a test compound which potentiates or inhibits the binding of a hedgehog polypeptide to a naturally occurring patched receptor, comprising:
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i. providing a cell expressing a naturally occurring patched receptor;
ii. contacting the cell with a hedgehog polypeptide having an amino acid sequence encoded by a nucleic acid sequence which hybridizes under stringent conditions including a wash step of 0.5×
SSC at 60°
C., with a nucleic acid sequence selected from the group consisting of SEQ ID No;
1, SEQ ID No;
2, SEQ ID No;
3, SEQ ID No;
4, SEQ ID No5, SEQ ID No;
6 or SEQ ID No;
7, and a test compound; and
iii. detecting binding of the hedgehog polypeptide to said patched receptor, wherein a change in the binding of said hedgehog polypeptide and said patched receptor is indicative of a compound that potentiates or inhibits the binding of said hedgehog polypeptides to said patched receptor. - View Dependent Claims (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27)
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Specification