Methods for identifying compounds for regulating muscle mass or function using corticotropin releasing factor receptors
View Patent ↗Screening methods for identifying compounds that bind to or activate corticotropin releasing factor 2 receptors (CRF 2 R) and regulate or potentially regulate skeletal muscle mass or function in vivo are disclosed. Also disclosed are screening methods for identifying compounds that prolong or augment the activation of CRF 2 Rs or of CRF 2 R signal transduction pathways, increase CRF 2 R or increase CRF expression are provided. Pharmaceutical compositions comprising CRF 2 R agonists, antibodies to CRF 2 R and methods for increasing skeletal muscle mass or function or for the treatment of skeletal muscle atrophy using CRF 2 R as the target for intervention and methods for treatment of muscular dystrophies are described.
1. A method for identifying candidate compounds for regulating skeletal muscle mass or function, comprising:
a. contacting a test compound with a vertebrate CRF 2 R;
b. determining whether the test compound binds to or activates the CRF 2 R;
c. selecting those compounds that bind or activate CRF 2 R, and further determining whether the test compound regulates muscle mass or function in a skeletal muscle atrophy model system; and
d. identifying those test compounds that regulate muscle mass or function as candidate compounds for regulating skeletal muscle mass or function.
2. The method for identifying candidate compounds according to claim 1 , in which the CRF 2 R is expressed on a eukaryotic cell.
3. The method for identifying candidate compounds according to claim 1 wherein the CRF 2 R has the amino acid sequence corresponding to the amino acid sequence of SEQ ID NO: 32.
4. The method for identifying candidate compounds according to claim 2 , in which determining whether the test compound activates the CRF 2 R involves measuring the cellular cAMP level.
5. The method for identifying candidate compounds according to claim 4 , in which the cell further comprises a reporter gene operatively associated with a cAMP responsive element and measuring the cellular cAMP level involves measuring expression of the reporter gene.
6. The method for identifying candidate compounds according to claim 5 , in which the reporter gene is alkaline phosphatase, chloramphenicol acetyltransferase, luciferase, glucuronide synthetase, growth hormone, placental alkaline phosphatase, or Green Fluorescent Protein.
7. A method for identifying candidate compounds for regulating skeletal muscle mass or function comprising:
a. contacting a test compound with a cell expressing a functional vertebrate CRF 2 R, and determining level of activation of CRF 2 R resulting from the test compound;
b. contacting said test compound with a cell expressing a functional vertebrate CRF 1 R, and determining level of activation of CRF 1 R resulting from the test compound;
c. comparing the level of CRF 2 R activation and the level of CRF 1 R activation;
d. selecting those test compounds that selectively activate CRF 2 R and further determining whether said test compound regulates muscle mass or function in a skeletal muscle atrophy model system; and
e. identifying those test compounds that regulate muscle mass or function as candidate compounds for regulating skeletal muscle mass or function.
8. The method according to claim 7 wherein the candidate compound exhibits a 100-fold or greater selectivity for CRF 2 R.
9. The method according to claim 7 wherein the candidate compound exhibits a 1000-fold or greater selectivity for CRF 2 R.
10. The method according to claim 7 wherein the candidate compound exhibits between 1-fold and 100-fold selectivity for CRF 2 R.