Screening methods for compounds that modulate the activity of g-protein coupled receptors

Abstract

The present invention relates to a screening system for modulators of GPCRs. Further it relates to recombinant vector systems for the heterologous expression of heterodimeric G-protein coupled receptors (GPCRs) in eukaryotic host cells. Preferably the functional expression of engineered GPCRs for the perception of sweet and L-amino acid taste or more preferably the use of these receptors for the identification of functional ligands is also encompassed.

1 . A method for the identification of a compound that potentiates the sweetness of a sweet tasting compound, comprising the steps of:
 a. providing a transformed eukaryotic host cell; wherein said host cell is transformed with genetic sequences coding for at least two different G-protein coupled receptors (GPCRs), wherein the transformation was accomplished with a multicistronic expression vector which comprises a multicistronic expression unit comprising downstream from a promoter for the expression in an eukaryotic host cell and functionally linked thereto, the following cistrons:
 I. GPCR 1    
 II. GPCR 2 , and 
 III. a selection marker, 
 wherein the GPCR 1  and the GPCR 2  are each independently selected from the group consisting of T1R or T2R taste receptors, 
   b. cultivating the transformed host cell under conditions sufficient for the functional expression of said at least two different GPCRs,   c. contacting the cultivated host cell expressing the at least two different GPCRs in a functional manner with a sweet tasting compound and a candidate compound that potentially potentiates the sweetness of a sweet tasting compound,   d. measuring intracellular calcium levels of the transformed host cell on exposure to the sweet tasting compound and the candidate compound, and   e. selecting a compound that increases the intracellular calcium levels in the presence of the sweet compound greater than the sweet tasting compound alone, wherein the greater increase in the intracellular calcium levels is indicative of a compound that potentiates the sweetness of a sweet tasting compound.   
     
     
         2 . A method according to  claim 1 , wherein the host cell is selected from the group consisting of: HEK293 (human embryo kidney); Hela (Human Negroid cervix epitheloid carcinoma); HT29 (Human Caucasian colon adenocarcinoma grade IQ); A431 (human squamous carcinoma); IMR 32 (human caucasian neuroblastoma); K562 (Human Caucasian chronic myelogenous leukaemia); U937 (Human Caucasian histiocytic lymphoma); MDA-MB-231 (Human Caucasian breast adenocarcinoma); SK-N-BE(2) (Human Caucasian neuroblastoma); SH-SY5Y (Human neuroblastoma); HL60 (human promyelocytic leukemia); CHO-KI (Hamster Chinese ovary); COS-7 (Monkey African green kidney, SV40 transformed); S49 (mouse lymphoma); Ltk (Mouse C34/An connective tissue); NG108-15 (Mouse neuroblastoma×Rat glioma hybrid); B50 (Rat nervous tissue neuronal, ECACC); C6 (Rat glial tumour); Jurkat (Human leukaemic T cell lymphoblast); BHK (Hamster Syrian kidney); and Neuro-2a (Mouse Albino neuroblastoma); and NIH/3T3 (mouse embryo fibroblast). 
     
     
         3 . A method according to  claim 1 , wherein the selection marker is selected from the group consisting of hygromycin, zeocin, neomycin, blasticidin r  and puromycin r . 
     
     
         4 . The method of  claim 1 , wherein both the GPCR 1 , the GPCR 2 , and the selection marker are functionally connected by intervening Internal Ribosome Entry Sites (IRES) selected from the group consisting of IRES EMCV , derived from encephalomyocarditis virus (synonym: CITE EMCV ); IRES GTX , derived from the GTX homeodomain mRNA; IRES BM3 , derived from cold-inducible Rbm3; IRES PV , derived of polioviral origin; IRES RV , derived from rhinovirus; IRES FMDV , derived from foot and mouth disease virus; IRE HV , derived from hepatitis C virus; IRES CSFV , derived from classic swine fever virus; IRES BVDV , derived from bovine viral diarrhea virus; IRES FMLV , derived from friend murine leukemia virus gag tnRNA; IRES MMLV , derived from moloney murine leukemia virus gag mRNA; IRESmv, derived from human immunodeficiency virus env mRNA; IRES PSIV , derived from  Plautia stali  intestine virus; TRES RPV , derived from  Rhopalosiphum padi  virus; and IRES KSH , derived from Karposi's sarcoma-associated herpesvirus. 
     
     
         5 . The method of  claim 1 , wherein the multicistronic expression unit is terminated by a polyadenylation signal. 
     
     
         6 . A method according to  claim 1 , wherein the multicistronic expression vector additionally comprises a cistron coding for a G-protein. 
     
     
         7 . A method according to  claim 1 , wherein the candidate compound is selected from the group consisting of small molecules and peptides. 
     
     
         8 . The method of  claim 1 , wherein the sweet tasting compound is selected from the group consisting of glucose, fructose, saccharose, acesulfam K, saccharin, cyclamat, aspartam, xylitol, stevioside, sucralose, thaumatin, moncllin, brazzein, perillartine, glycyrrhizin, sucronic acid, P-4000, SC45647, NC174, neohesperidin, and sweet tasting amino acids. 
     
     
         9 . A method according to  claim 1 , wherein GPCR 1  is human T1R2 and wherein GPCR 2  is human T1R3. 
     
     
         10 . A method according to  claim 1 , wherein the promoter is selected from the group consisting of: cytomegalovirus promoter (P-CMV), human elongation factor 1 alpha promoter (P-EF1alpha), human ubiquitin promoter (P-ubi), simian virus promoter (P-SV40), and Rous sarcoma virus long terminal repeat promoter (P-RSV-LTR). 
     
     
         11 . The method according to  claim 1 , wherein the multicistronic expression vector additionally comprises a cistron coding for a G-protein. 
     
     
         12 . The method of  claim 11 , wherein the G-protein is located between the last GPCR and the selection marker and is functionally connected to both via an Internal Ribosome Entry Site (IRES). 
     
     
         13 . The method of  claim 11 , wherein the cistron codes for G-alpha 15. 
     
     
         14 . The method of  claim 1 , further comprising transforming said eukaryotic host cell with a multicistronic expression vector which comprises a multicistronic expression unit comprising downstream from a promoter for the expression in an eukaryotic host cell and functionally linked thereto, the following cistrons:
 I. GPCR 1      II. GPCR 2 , and   III. a selection marker,   wherein the GPCR 1  and the GPCR 2  are each independently selected from the group consisting of T1R or T2R taste receptors.   
     
     
         15 . The method of  claim 1 , further comprising contacting the cultivated host cell expressing the at least two different GPCRs in a functional manner with a sweet tasting compound alone; measuring intracellular calcium levels of the transformed host cell on exposure to the sweet tasting compound alone; and selecting a compound which induces a greater change in the intracellular calcium levels in the presence of the sweet tasting compound than the sweet tasting compound alone. 
     
     
         16 . The method of  claim 1 , further comprising contacting the cultivated host cell expressing at least two different GPCRs in a functional manner with the candidate compound alone, measuring intracellular calcium levels of the transformed host cell on exposure to the candidate compound alone, and selecting a compound which does not induce a change in the intracellular calcium levels when administered alone.