Methods for the classification and diagnosis of scoliosis through the use of gi protein receptor

Abstract

Methods for diagnosing a predisposition to developing a scoliosis (e.g., adolescent idiopathic scoliosis (AIS)) and identifying compounds for treating scoliosis based on the modulation of Gi protein-coupled receptor activity are described. Specific embodiments of the methods involve measuring a change in impedance signals of cells expressing a receptor coupled to a Gi protein with a ligand. To identify compounds useful in treatment, the cell is contacted with a test compound and a ligand. A higher impedance in the presence relative to the absence of said test compound is indicative that the test compound is useful for treating scoliosis.

1 . A method for determining whether a test compound is useful for treating a scoliosis, said method comprising:
 (a) contacting a cell expressing a receptor coupled to an inhibitory guanine nucleotide-binding (G i ) protein with a ligand to said receptor in the presence or absence of said test compound;   (b) determining the magnitude of the signal induced by said ligand in said cell;   wherein a higher signal in the presence relative to the absence of said test compound is indicative that said test compound is useful for treating a scoliosis.   
     
     
         2 . The method of  claim 1 , wherein said scoliosis is an idiopathic scoliosis. 
     
     
         3 . the method of  claim 1 , wherein said idiopathic scoliosis is adolescent idiopathic scoliosis (AIS). 
     
     
         4 . The method of  claim 1 , wherein said cell is obtained from a subject suffering from AIS. 
     
     
         5 . The method of  claim 1 , wherein said signal is the change in impedance of said cell. 
     
     
         6 . The method of  claim 5 , wherein said change in impedance is measured by cellular dielectric spectroscopy (CDS). 
     
     
         7 . The method of  claim 1 , wherein said cell is an osteoblast, a myoblast or a peripheral blood mononuclear cell (PBMC). 
     
     
         8 . The method of  claim 1 , wherein said cell is a PBMC. 
     
     
         9 . The method of  claim 8 , wherein said cell is a lymphocyte. 
     
     
         10 . The method of  claim 1 , wherein said receptor is a serotonin receptor, an α-adrenergic receptor, an adenosine receptor or a cannabinoid receptor. 
     
     
         11 . The method of  claim 10 , wherein (i) said serotonin receptor is 5-HT1A; (ii) said α-adrenergic receptor is α2-AD; (iii) said adenosine receptor is A 3 ; or (iv) said cannabinoid receptor is CB2. 
     
     
         12 - 14 . (canceled) 
     
     
         15 . The method of  claim 1 , wherein said ligand is an agonist. 
     
     
         16 . The method of  claim 11 , wherein (i) said serotonin receptor is 5-HT 1A and said ligand is 1-[3-(3,4-Methylenedioxyphenoxy)propyl]-4-phenyl-piperazine maleate (BP554 maleate); (ii) said α-adrenergic receptor is α2-AD and said ligand is 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK14304); (iii) said adenosine receptor is A 3  and said ligand is 1-Deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-β-D-ribofuranuronamide (IB-MECA); or (iv) said cannabinoid receptor is CB2 and said ligand is N-Cyclohexyl-7-chloro-1-[2-(4-morpholinyl)ethyl]quinolin-4(1H)-one-3-carboxamide (CB65). 
     
     
         17 - 19 . (canceled) 
     
     
         20 . The method of  claim 1 , comprising determining the magnitude of the signal induced by at least two different ligands. 
     
     
         21 . The method of  claim 1 , comprising determining the magnitude of the signal for at least two different receptors coupled to a G i  protein. 
     
     
         22 . A method for diagnosing a predisposition to developing a scoliosis in a subject comprising:
 (a) contacting a cell expressing a receptor coupled to an inhibitory guanine nucleotide-binding (G i ) protein from said subject with a ligand to said receptor;   (b) determining the magnitude of the signal induced by said ligand in said cell;   (c) comparing said signal to a corresponding reference signal; and   (d) determining said predisposition based on said comparison.   
     
     
         23 . The method of  claim 22 , wherein said scoliosis is an idiopathic scoliosis. 
     
     
         24 . The method of  claim 23 , wherein said idiopathic scoliosis is adolescent idiopathic scoliosis (AIS). 
     
     
         25 . The method of  claim 22 , wherein said signal is the change in impedance of said cell. 
     
     
         26 . The method of  claim 25 , wherein said change in impedance is measured by cellular dielectric spectroscopy (CDS). 
     
     
         27 . The method of  claim 22 , wherein said cell is an osteoblast, a myoblast or a peripheral blood mononuclear Cell (PBMC). 
     
     
         28 . The method of  claim 22 , wherein said cell is a PBMC. 
     
     
         29 . The method of  claim 28 , wherein said cell is a lymphocyte. 
     
     
         30 . The method of  claim 22 , wherein said receptor is a serotonin receptor, an α-adrenergic receptor, an adenosine receptor or a cannabinoid receptor. 
     
     
         31 . The method of  claim 30 , wherein (i) said serotonin receptor is 5-HT1A; (ii) said α-adrenergic receptor is α2-AD; (iii) said adenosine receptor is A 3 ; or (iv) said cannabinoid receptor is CB2. 
     
     
         32 - 34 . (canceled) 
     
     
         35 . The method of  claim 22 , wherein said ligand is an agonist. 
     
     
         36 . The method of  claim 31 , wherein (i) said serotonin receptor is 5-HT1A and said ligand is 1-[3-(3,4-Methylenedioxyphenoxy)propyl]-4-phenyl-piperazine maleate (BP554 maleate); (ii) said α-adrenergic receptor is α2-AD and said ligand is 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK14304); (iii) said adenosine receptor is A 3  and said ligand is 1-Deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-β-D-ribofuranuronamide (IB-MECA); or (iv) said cannabinoid receptor is CB2 and said ligand is N-Cyclohexyl-7-chloro-1-[2-(4-morpholinyl)ethyl]quinolin-4(1H)-one-3-carboxamide (CB65). 
     
     
         37 - 39 . (canceled) 
     
     
         40 . The method of  claim 22 , comprising determining the magnitude of the signal induced by at least two different ligands. 
     
     
         41 . The method of  claim 22 , comprising determining the magnitude of the signal for at least two different receptors coupled to a G i  protein. 
     
     
         42 . The method of  claim 24 , wherein the subject is a likely candidate for developing adolescent idiopathic scoliosis. 
     
     
         43 . A method of classifying a human subject having idiopathic scoliosis comprising:
 (a) contacting a cell expressing a receptor coupled to an inhibitory guanine nucleotide-binding (G i ) protein from said subject with a ligand to said receptor; and   (b) determining the magnitude of the signal induced by said ligand in said cell, whereby the results of the determining step enables the classification of the subject having idiopathic scoliosis in one subgroup.   
     
     
         44 . The method of  claim 43 , wherein said idiopathic scoliosis is adolescent idiopathic scoliosis (AIS). 
     
     
         45 . The method of  claim 43 , further comprising selecting a treatment for the subject based on the results of the determining step. 
     
     
         46 . The method of  claim 43 , wherein said signal is the change in impedance of said cell. 
     
     
         47 . The method of  claim 46 , wherein said change in impedance is measured by cellular dielectric spectroscopy (CDS). 
     
     
         48 . The method of  claim 43 , wherein said cell is an osteoblast, a myoblast or a peripheral blood mononuclear Cell (PBMC). 
     
     
         49 . The method of  claim 43 , wherein said receptor is a serotonin receptor, an α-adrenergic receptor, an adenosine receptor or a cannabinoid receptor. 
     
     
         50 . The method of  claim 49 , wherein (i) said serotonin receptor is 5-HT1A; (ii) said α-adrenergic receptor is α2-AD; (iii) said adenosine receptor is A 3 ; or (iv) said cannabinoid receptor is CB2. 
     
     
         51 - 53 . (canceled) 
     
     
         54 . The method of  claim 43 , wherein said ligand is an agonist. 
     
     
         55 . The method of  claim 50 , wherein (i) said serotonin receptor is 5-HT1A and said ligand is 1-[3-(3,4-Methylenedioxyphenoxy)propyl]-4-phenyl-piperazine maleate (BP554 maleate); (ii) said α-adrenergic receptor is α2-AD and said ligand is 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK14304); (iii) said adenosine receptor is A 3  and said ligand is 1 -Deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-β-D-ribofuranuronamide (IB-MECA); or (iv) said cannabinoid receptor is CB2 and said ligand is N-Cyclohexyl-7-chloro-1-[2-(4-morpholinyl)ethyl]quinolin-4(1H)-one-3-carboxamide (CB65). 
     
     
         56 - 58 . (canceled) 
     
     
         59 . The method of  claim 43 , comprising determining the magnitude of the signal induced by at least two different ligands. 
     
     
         60 . The method of  claim 43 , comprising determining the magnitude of the signal for at least two different receptors coupled to a G i  protein.