Human G protein-coupled receptors and modulators thereof for the treatment of metabolic-related disorders

Abstract

The present invention relates to methods of identifying whether a candidate compound is a modulator of a G protein-coupled receptor (GPCR). In preferred embodiments, the GPCR is human. In other preferred embodiments, the GPCR is coupled to Gi and lowers the level of intracellular cAMP. In other preferred embodiments, the GPCR is expressed endogenously by adipocytes. In further preferred embodiments, the GPCR inhibits intracellular lipolysis. In other further preferred embodiments, the GPCR is a nicotinic acid receptor. The present invention also relates to methods of using a modulator of said GPCR. Preferred modulator is agonist. Agonists of the invention are useful as therapeutic agents for the prevention or treatment of metabolic-related disorders, including dyslipidemia, atherosclerosis, coronary heart disease, stroke, insulin resistance, and type 2 diabetes.

1 . A method of identifying whether a candidate compound is a modulator of a nicotinic acid GPCR, said receptor comprising an amino acid sequence selected from the group consisting of: 
 (a) SEQ. ID. NO.:36 (hRUP25);    (b) SEQ. ID. NO.:137 (mRUP25); and    (c) SEQ. ID. NO.:139 (rRUP25);    or an allelic variant, a biologically active mutant, or a biologically active fragment of said amino acid sequence;    comprising the steps of:    (a′) contacting the candidate compound with the receptor; and    (b′) determining whether the receptor functionality is modulated;    wherein a change in receptor functionality is indicative of the candidate compound being a modulator of a nicotinic acid GPCR.    
     
     
         2 . A modulator of a nicotinic acid GPCR (RUP25) identified according to the method of  claim 1 , provided that the modulator is not identical to a compound selected from the group consisting of:  
       
         
           
           
               
               
           
         
         wherein: 
 R 1  is selected from the group consisting of halogen, hydroxyl, acetylamino, amino, alkoxy, carboalkoxy, alkylthio, monoalkylamino, dialkylamino, N-alkylcarbamyl, N,N-dialkylcarbamyl, alkylsulfonyl, said alkyl groups containing from 1 to 4 carbons, trifluoromethyl, trifluoromethoxy, trifluoromethylthio, methoxymethyl, carboxy, carbamyl, alkanoyloxy containing up to 4 carbon atoms, phenyl, p-chlorophenyl, p-methylphenyl and p-aminophenyl;  
 R 2  is selected from the group consisting of halogen, alkannoyloxy containing from 14 carbon atoms, carboalkoxy containing from 2 to 5 carbon atoms, carbamyl, N-alkyl carbamyl and N,N-dialkylcarbamyl wherein said alkyl groups contain from 1-4 carbon atoms and trifluoromethyl;  
 n is a whole number from 0 to 4; and  
 N-oxides thereof;  
                     
 R 3  and R 4  are hydrogen, alkyl containing from 1 to 4 carbon atoms or cycloalkyl containing from 3 to 7 carbon atoms;  
 n is a whole number from 0 to 4; and  
 N-oxides thereof.  
                     
 
         wherein: 
 R 5  and R 6  are each selected from the group consisting of H, halogen, hydroxyl, amino, alkyloxy, alkylthio, monoalkylamino, dialkylamino, N-alkylcarbamyl, N,N-dialkylcarbamyl, alkylsulfoxy, alkylsulfony, said alkyl groups containing from 1 to 4 carbons, trifluoromethyl, trifluoromethoxy, trifluoromethylthio, carboxy, carbamyl, alkanoyloxy containing up to 4 carbon atoms, phenyl, p-chlorophenyl, p-methylphenyl and p-aminophenyl;  
 n is a whole number from 0 to 4; and  
 N-oxides thereof;  
 
         d)  
         
           
             
             
                 
                 
             
           
         
         wherein: 
 at least one of R 7 , R 8  and R 9  is C 1-4  alkyl and the others are hydrogen atoms; R 10  is hydroxy or C 1  alkoxy, or a salt of the compounds wherein R 4  is hydoxy with a pharmaceutically acceptable base;  
 
         e)  
         
           
             
             
                 
                 
             
           
         
         wherein: 
 at least one of R 7 , R 8  and R 9  is C 1-4  alkyl and the others are hydrogen atoms; each of R 11  and R 12 , which may be the same or different, is hydrogen or C 1-4  alkyl;  
 
         f)  
         
           
             
             
                 
                 
             
           
         
         wherein: 
 at least one of R 13  represents an alkyl group of 7-11 carbon atoms and R 14  represents H or a lower alkyl group of up to two carbon atoms, and a pharmaceutically acceptable carrier;  
 
         g) pyrazine-2-carboxylic acid amide, 5-chloro-pyrazine-2-carboxylic acid amide, 5-amino-pyrazine-2-carboxylic acid amide, 5-benzyl-pyrazine-2-carboxylic acid amide, 6-chloro-pyrazine-2-carboxylic acid amide, 6-methoxy-pyrazine-2-carboxylic acid amide, 3-chloro-pyrazine-2-carboxylic acid amide, 3-methoxy-pyrazine-2-carboxylic acid amide, pyrazine-2-carboxylic acid ethylamide, morpholin-4-yl-pyrazine-2-ylmethanone, 5-methyl-pyrazine-2-carboxylic acid (6-methyl-pyrazin-2-yl)-amide, 5-methyl-pyrazine-2-carboxylic acid (5-methyl-pyrazin-2-yl)-amide, 5-methyl-pyrazine-2-carboxylic acid (3-methyl-pyrazin-2-yl)-amide, (5-methyl-pyrazin-2-yl)-morpholin-4-yl-methanone, 5-methyl-pyrazine-2-carboxylic acid hydroxyamide, pyrazine-2-carboxylic acid, 5-amino-pyrazine-2-carboxylic acid, 5-benzyl-pyrazine-2-carboxylic acid, 6-chloro-pyrazine-2-carboxylic acid, 6-methoxy-pyrazine-2-carboxylic acid, 3-hydroxy-pyrazine-2-carboxylic acid, 5-methyl-pyrazine-2-carboxylic acid 2-hydroxy-ethyl ester, 5-methyl-pyrazine-2-carboxylic acid allyl ester, 5-methyl-pyrazine-2-carboxylic acid phenyl ester, 5-methyl-pyrazine-2-carboxylic acid ethoxycarbonylmethyl ester, pyrazine-2-carboxylic acid methyl ester or 2-methyl-5-(1H-tetrazol-5-yl)-pyrazine; and 4-N-oxides thereof;  
         h) 5-(3-(5-Methyl)isoxazolyl)tetrazole;  
         i) 5-(5-(3-Methyl)isoxazolyl)tetrazole;  
         i) 5-(3-Quinolyl)tetrazole;  
         k) Nicotinic acid;  
         l) Pyridazine-4-carboxylic acid;  
         m) 3-pyridine acetic acid;  
         n) 5-Methylnicotinic acid;  
         o) 6-Methylnicotinic acid;  
         p) Nicotinic acid-1-oxide;  
         q) 2-Hydroxynicotinic acid;  
         r) Furane-3-carboxylic acid;  
         s) 5-Methylpyrazole-3-carboxylic acid; and  
         t) 3-Methylisoxazole-5-carboxylic acid.  
       
     
     
         3 . A method of modulating the activity of a nicotinic acid GPCR, said receptor comprising an amino acid sequence selected from the group consisting of: 
 (a) SEQ. ID. NO.:36 (hRUP25);    (b) SEQ. ID. NO.:137 (mRUP25); and    (c) SEQ. ID. NO.:139 (rRUP25);    or an allelic variant, a biologically active mutant, or a biologically active fragment of said amino acid sequence;    comprising the step of contacting the receptor with the modulator of  claim 2 .    
     
     
         4 . A method of preventing or treating a disorder of lipid metabolism in an individual comprising contacting a therapeutically effective amount of the modulator of  claim 2  with a nicotinic acid GPCR, said receptor comprising an amino acid sequence selected from the group consisting of: 
 (a) SEQ. ID. NO.:36 (hRUP25);  
 (b) SEQ. ID. NO.:137 (mRUP25); and  
 (c) SEQ. ID. NO.:139 (rRUP25);  
 or an allelic variant or biologically active fragment of said amino acid sequence.  
 
     
     
         5 . A method of preventing or treating a metabolic-related disorder in an individual comprising contacting a therapeutically effective amount of the modulator of  claim 2  with a nicotinic acid GPCR, said receptor comprising an amino acid sequence selected from the group consisting of: 
 (a) SEQ. ID. NO.:36 (hRUP25);  
 (b) SEQ. ID. NO.:137 (mRUP25); and  
 (c) SEQ. ID. NO.:139 (rRUP25);  
 or an allelic variant or biologically active fragment of said amino acid sequence.  
 
     
     
         6 . A method of preparing a composition which comprises identifying a modulator of a nicotinic acid GPCR and then admixing a carrier and the modulator, wherein the modulator is identifiable by the method of  claim 1  and provided that the modulator is not identical to a compound selected from the group consisting of: 
 a)  
                     
 wherein: 
 R 1  is selected from the group consisting of halogen, hydroxyl, acetylamino, amino, alkoxy, carboalkoxy, alkylthio, monoalkylamino, dialkylamino, N-alkylcarbamyl, N,N-dialkylcarbamyl, alkylsulfonyl, said alkyl groups containing from 1 to 4 carbons, trifluoromethyl, trifluoromethoxy, trifluoromethylthio, methoxymethyl, carboxy, carbamyl, alkanoyloxy containing up to 4 carbon atoms, phenyl, p-chlorophenyl, p-methylphenyl and p-aminophenyl;  
 R 2  is selected from the group consisting of halogen, alkannoyloxy containing from 1-4 carbon atoms, carboalkoxy containing from 2 to 5 carbon atoms, carbamyl, N-alkyl carbamyl and N,N-dialkylcarbamyl wherein said alkyl groups contain from 1-4 carbon atoms and trifluoromethyl;  
 n is a whole number from 0 to 4; and  
 N-oxides thereof;  
                     
 R 3  and R 4  are hydrogen, alkyl containing from 1 to 4 carbon atoms or cycloalkyl containing from 3 to 7 carbon atoms;  
 n is a whole number from 0 to 4; and  
 N-oxides thereof.  
                     
 
 wherein: 
 R 5  and R 6  are each selected from the group consisting of H, halogen, hydroxyl, amino, alkyloxy, alkylthio, monoalkylamino, dialkylamino, N-alkylcarbamyl, N,N-dialkylcarbamyl, alkylsulfoxy, alkylsulfony, said alkyl groups containing from 1 to 4 carbons, trifluoromethyl, trifluoromethoxy, trifluoromethylthio, carboxy, carbamyl, alkanoyloxy containing up to 4 carbon atoms, phenyl, p-chlorophenyl, p-methylphenyl and p-aminophenyl;  
 n is a whole number from 0 to 4; and  
 N-oxides thereof;  
                     
 
 wherein: 
 at least one of R 7 , R 8  and R 9  is C 1-4  alkyl and the others are hydrogen atoms; R 10  is hydroxy or C 1-4  alkoxy, or a salt of the compounds wherein R 4  is hydoxy with a pharmaceutically acceptable base;  
                     
 
 wherein: 
 at least one of R 7 , R 8  and R 9  is C 1-6  alkyl and the others are hydrogen atoms; each of R 11  and R 12 , which may be the same or different, is hydrogen or C 1-6  alkyl;  
                     
 
 wherein: 
 at least one of R 13  represents an alkyl group of 7-11 carbon atoms and R 14  represents H or a lower alkyl group of up to two carbon atoms, and a pharmaceutically acceptable carrier;  
 
 g) pyrazine-2-carboxylic acid amide, 5-chloro-pyrazine-2-carboxylic acid amide, 5-amino-pyrazine-2-carboxylic acid amide, 5-benzyl-pyrazine-2-carboxylic acid amide, 6-chloro-pyrazine-2-carboxylic acid amide, 6-methoxy-pyrazine-2-carboxylic acid amide, 3-chloro-pyrazine-2-carboxylic acid amide, 3-methoxy-pyrazine-2-carboxylic acid amide, pyrazine-2-carboxylic acid ethylamide, morpholin-4-yl-pyrazine-2-ylmethanone, 5-methyl-pyrazine-2-carboxylic acid (6-methyl-pyrazin-2-yl)-amide, 5-methyl-pyrazine-2-carboxylic acid (5-methyl-pyrazin-2-yl)-amide, 5-methyl-pyrazine-2-carboxylic acid (3-methyl-pyrazin-2-yl)-amide, (5-methyl-pyrazin-2-yl)-morpholin-4-yl-methanone, 5-methyl-pyrazine-2-carboxylic acid hydroxyamide, pyrazine-2-carboxylic acid, 5-amino-pyrazine-2-carboxylic acid, 5-benzyl-pyrazine-2-carboxylic acid, 6-chloro-pyrazine-2-carboxylic acid, 6-methoxy-pyrazine-2-carboxylic acid, 3-hydroxy-pyrazine-2-carboxylic acid, 5-methyl-pyrazine-2-carboxylic acid 2-hydroxy-ethyl ester, 5-methyl-pyrazine-2-carboxylic acid allyl ester, 5-methyl-pyrazine-2-carboxylic acid phenyl ester, 5-methyl-pyrazine-2-carboxylic acid ethoxycarbonylmethyl ester, pyrazine-2-carboxylic acid methyl ester or 2-methyl-5-(1H-tetrazol-5-yl)-pyrazine; and 4-N-oxides thereof;  
 h) 5-(3-(5-Methyl)isoxazolyl)tetrazole;  
 i) 5-(5-(3-Methyl)isoxazolyl)tetrazole;  
 j) 5-(3-Quinolyl)tetrazole;  
 k) Nicotinic acid;  
 l) Pyridazine-4-carboxylic acid;  
 m) 3-pyridine acetic acid;  
 n) 5-Methylnicotinic acid;  
 o) 6-Methylnicotinic acid;  
 p) Nicotinic acid-1-oxide;  
 q) 2-Hydroxynicotinic acid;  
 r) Furane-3-carboxylic acid;  
 s) 5-Methylpyrazole-3-carboxylic acid; and  
 t) 3-Methylisoxazole-5-carboxylic acid.  
 
     
     
         7 . A pharmaceutical or physiologically acceptable composition comprising, consisting essentially of, or consisting of the modulator of  claim 2 .  
     
     
         8 . A method of changing lipid metabolism comprising providing or administering to an individual in need of said change said pharmaceutical or physiologically acceptable composition of  claim 7 .  
     
     
         9 . A method of preventing or treating a metabolic-related disorder comprising providing or administering to an individual in need of said prevention or treatment said pharmaceutical or physiologically acceptable composition of  claim 7 .  
     
     
         10 . A method of using the modulator of  claim 2  for the preparation of a medicament for the treatment of a disorder in lipid metabolism in an individual.  
     
     
         11 . A method of using the modulator of  claim 2  for the preparation of a medicament for the treatment of a metabolic-related disorder in an individual.  
     
     
         12 . A method of identifying whether a candidate compound binds to a nicotinic acid GPCR, said receptor comprising an amino acid sequence selected from the group consisting of: 
 (a) SEQ. ID. NO.:36 (hRUP25);    (b) SEQ. ID. NO.:137 (mRUP25); and    (c) SEQ. ID. NO.:139 (rRUP25);    or an allelic variant or a biologically active fragment of said amino acid sequence;    comprising the steps of:    (a′) contacting the receptor with a labeled reference compound known to bind to the GPCR in the presence or absence of the candidate compound; and    (b′) determining whether the binding of said labeled reference compound to the receptor is inhibited in the presence of the candidate compound;    wherein said inhibition is indicative of the candidate compound binding to a nicotinic acid GPCR.    
     
     
         13 . A method of making a mouse genetically predisposed to a metabolic-related disorder or a disorder of lipid metabolism comprising the step of knocking out the gene encoding the polypeptide of SEQ. ID. NO.:137 (mRUP25) or the polypeptide of SEQ. ID. NO.:151 (mRUP19).  
     
     
         14 . A knockout mouse according to the method of  claim 13 .  
     
     
         15 . A method of using the knockout mouse of  claim 14  to identify whether a candidate compound has therapeutic efficacy for the prevention or treatment of said metabolic-related disorder or said disorder of lipid metabolism.  
     
     
         16 . A method of making a rat genetically predisposed to a metabolic-related disorder or a disorder of lipid metabolism comprising the step of knocking out the gene encoding the polypeptide of SEQ. ID. NO.:139 (rRUP25) or the polypeptide of SEQ. ID. NO.:157 (rRUP19).  
     
     
         17 . A knockout rat according to the method of  claim 16 .  
     
     
         18 . A method of using the knockout rat of  claim 17  to identify whether a candidate compound has therapeutic efficacy for the prevention or treatment of said metabolic-related disorder or said disorder of lipid metabolism.  
     
     
         19 . An isolated EFA-hRUP25 polynucleotide selected from the group consisting of: 
 (a) a polynucleotide comprising the nucleotide sequence of SEQ. ID. NO.:158;    (b) a polynucleotide having the nucleotide sequence of SEQ. ID. NO.:158;    (c) a polynucleotide comprising a polynucleotide encoding the polypeptide having the amino acid sequence of SEQ. ID. NO.:159 or a biologically active fragment of said polypeptide; and    (d) a polynucleotide encoding the polypeptide having the amino acid sequence of SEQ. ID. NO.:159 or a biologically active fragment of said polypeptide.    
     
     
         20 . An isolated EFA-hRUP25 polypeptide selected from the group consisting of: 
 (a) a polypeptide comprising the amino acid sequence of SEQ. ID. NO.:159, or a biologically active fragment of said polypeptide; and    (b) a polypeptide having the amino acid sequence of SEQ. ID. NO.:159, or a biologically active fragment of said polypeptide.    
     
     
         21 . A recombinant vector comprising the polynucleotide of  claim 19 .  
     
     
         22 . A host cell comprising the recombinant vector of  claim 21.