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 eucaryotic 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 said receptors for the identification of functional ligands is also encompassed.

1 .) A method for the identification of modulators of GPCRs, comprising the steps of:
 a. transforming a eucaryotic host cell with genetic sequences coding for one or more than one GPCR(s),   b. cultivation of the transformed host cells under conditions sufficient for the functional expression of said one or more GPCRs,   c. contacting the cultivated host cell expressing the one or more GPCRs in a functional manner with a potential modulator of the selected one or more GPCRs,   d. measuring an selected cellular response of the transformed host cell on exposure to the potential modulator, and   e. selecting an identificate, which induces a specific response.   
     
     
         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 II), 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) or eukaryotic non-human cell lines like CHO-K1 (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), Neuro-2a (Mouse Albino neuroblastoma), NIH/3T3 (mouse embryo fibroblast). 
     
     
         3 .) A method according to  claim 1 , wherein the one or more GPCR(s) is a T1R-type GPCR. 
     
     
         4 .) A method according to  claim 1 , wherein two or more GPCRs are expressed in a heterologous co-expression of at least two different T1R-type GPCRs. 
     
     
         5 .) A method according to  claim 1 , wherein the transformation is accomplished with a multicistronic expression vector. 
     
     
         6 .) A method according to  claim 5 , wherein the multicistronic expression vector comprises a multicistronic expression unit comprising downstream from a promoter for the expression in an eucaryotic host and functionally linked thereto, the following cistrons:
 a. GPCR 1      b. GPCR 2  and a   c. selection marker,   wherein the promoter is a strong promoter suitable for use in the selected host cell,   wherein the GPCR, and the GPCR 2  are independently from another being selected from the group consisting of T1R or T2R taste receptors,   wherein the selection marker is being selected from the group consisting of hygromycin r , zeocin r , neomycin r , blasticidin r  or puromycin r ,   wherein both the GPCR1 and the GPCR2 as well as the selection marker are functionally connected by intervening 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 Rbm3 , derived from cold-inducible Rbm3; IRES PV , derived of polioviral origin, IRES RV , derived from rhinovirus, IRESFMDV, derived from food 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 mRNA; IRES MMLV , derived from moloney murine leukemia virus gag mRNA; IRES HIV , derived from human immunodefiency virus env mRNA; IRES PSIV , derived from  Plautia stali  intestine virus; IRES RPV , derived from  Rhopalosiphum padi  virus; IRES KSH , derived from Karposi's sarcoma-associated herpesvirus, preferably the IRES EMCV  being derived from encephalomyocarditis virus (synonym: CITE EMCV ),   and wherein the multicistronic expression unit is terminated by a polyadenylation signal.   
     
     
         7 .) A method according to  claim 5 , wherein the multicistronic cloning vector additionally comprises a cistron coding for a G-protein. 
     
     
         8 .) A method according to  claim 1 , wherein the modulator is selected from the group consisting of small molecules and/or peptides. 
     
     
         9 .) A method according to  claim 1 , wherein the cellular response is measured by measuring the change of intracellular calcium levels relative to intracellular calcium levels without contacting the cell to the modulator. 
     
     
         10 .) A method according to  claim 9 , wherein the change is an increase. 
     
     
         11 .) A method according to  claim 9 , wherein the change is a decrease relative to intracellular calcium levels when the cell is contacted to a sweet compound instead of the modulator. 
     
     
         12 .) Multiparameter optimized nucleic acid molecule coding for a GPCR. 
     
     
         13 .) Protein consisting of the amino acids sequences according to SEQ ID NO: 1 and/or SEQ ID NO: 2 or functionally equivalent proteins. 
     
     
         14 .) Nucleic acid molecules according to  claim 12 , as depicted in one of SEQ ID NO 3 or SEQ ID NO: 4. 
     
     
         15 .) Nucleic acid molecules functionally equivalent to the molecules according to  claim 14 . 
     
     
         16 .) Multicistronic expression vector comprising more than one cistron coding for a GPCR. 
     
     
         17 .) Multicistronic expression vector comprising downstream from a promoter for the expression in an eucaryotic host and functionally linked thereto, the following cistrons:
 d. GPCR 1      e. GPCR 2  and a   f. selection marker,   wherein the promoter preferably is a strong promoter suitable for use in the selected host cell,   wherein the GPCR 1  and the GPCR 2  are independently from another selected from the group consisting of T1R or T2R taste receptors,   wherein the selection marker is selected from the group consisting of hygromycin r , zeocin r , neomycin r , blasticidin r  puromycin r ,   wherein both the GPCR1 and the GPCR2 as well as the selection marker are functionally connected by intervening 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 Rbm3 , derived from cold-inducible Rbm3; IRES PV , derived of polioviral origin, IRES RV , derived from rhinovirus, IRESFMDV, derived from food 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 mRNA; IRES MMLV , derived from moloney murine leukemia virus gag mRNA; IRES HIV , derived from human immunodefiency virus env mRNA; IRES PSIV , derived from  Plautia stali  intestine virus; IRES RPV , derived from  Rhopalosiphum padi  virus; IRES KSH , derived from Karposi's sarcoma-associated herpesvirus, preferably the IRES EMCV  being derived from encephalomyocarditis virus (synonym: CITE EMCV ),   and wherein the multicistronic expression unit is terminated by a polyadenylation signal.   
     
     
         18 .) A vector according to  claim 17 , wherein the multicistronic cloning vector additionally comprises a cistron coding for a g-protein or an equivalent thereof. 
     
     
         19 .) Cell lines stably transfected with a nucleic acid molecule according to  claim 14 . 
     
     
         20 .) A method of identifying a GPCR modulator comprising a method according to  claim 1 . 
     
     
         21 .) A method according to  claim 1 , wherein the host cell is HEK293 (human embryo kidney), Hela (Human Negroid cervix epitheloid carcinoma), CHO-K1 (Hamster Chinese ovary) or Neuro-2a (Mouse Albino neuroblastoma). 
     
     
         22 .) A method according to  claim 1 , wherein the one or more GPCR(s) is T1R1, T1R2 and/or T1R3. 
     
     
         23 .) A method according to  claim 1 , wherein two or more GPCRs are expressed in a heterologous co-expression of T1R1/T1R3. 
     
     
         24 .) A method according to  claim 1 , wherein two or more GPCRs are expressed in a heterologous co-expression of T1R2/T1R3. 
     
     
         25 .) A method according to  claim 1 , wherein the transformation is accomplished with a tricistronic or a tetracistronic cloning vector. 
     
     
         26 .) A method according to  claim 5 ,
 wherein the promoter is selected from the group consisting of: cytomegalovirus promoter (P-CMV), human elongation factor 1 alpha promoter (P-E1α), human ubiquitin promoter (P-ubi), simian virus promoter (P-SV40), and Rous sarcoma virus long terminal repeat promoter (P—RSV-LTR).   
     
     
         27 .) A method according to  claim 5 , wherein the GPCR 1  and the GPCR 2  are independently from another selected from the group of T1R receptors. 
     
     
         28 .) A method according to  claim 5 , wherein the GPCR 1  and the GPCR 2  are independently from another selected from a combination of T1R1-T1R3 or T1R2-T1R3. 
     
     
         29 .) A method according to  claim 5  wherein the multicistronic cloning vector additionally comprises a cistron coding for G-alpha 15. 
     
     
         30 .) A method according to  claim 7 , wherein the g-protein is located between the last GPCR and the selection marker and is functionally connected to both via an IRES. 
     
     
         31 .) A method according to  claim 11 , wherein the sweet compound is selected from the group consisting of glucose, fructose, saccharose, acesulfam K, saccharin, cyclamat, aspartam, xylitol, stevioside, sucralose, thaumatin, monellin, brazzein, perillartine, glycyrrhizin, sucronic acid, P-4000, SC45647, NC174, neohesperidin and sweet tasting amino acids. 
     
     
         32 .) Multiparameter optimized nucleic acid molecule according to  claim 12 , wherein the GPCR, is a T1R-type GPCR. 
     
     
         33 .) Multiparameter optimized nucleic acid molecule according to  claim 28 , wherein the T1R-type GPCR, is a T1R1, T1R2 or T1R3 or a functionally equivalent receptor protein. 
     
     
         34 .) Multicistronic expression vector according to  claim 17 , wherein the promoter is selected host cell, more preferably being selected from the group consisting of: cytomegalovirus promoter (P-CMV), human elongation factor 1 alpha promoter (P-E1α), human ubiquitin promoter (P-ubi), simian virus promoter (P-SV40), Rous sarcoma virus long terminal repeat promoter (P—RSV-LTR). 
     
     
         35 .) Multicistronic expression vector according to  claim 17 , wherein the GPCR 1  and the GPCR 2  are independently from another selected from the group consisting of T1R receptors. 
     
     
         36 .) Multicistronic expression vector according to  claim 31 , wherein the GPCR 1  and GPCR 2  are a combination of T1R1-T1R3 or T1R2-T1R3. 
     
     
         37 .) A vector according to  claim 17 , wherein the multicistronic cloning vector additionally comprises a cistron coding for G-alpha 15 or an equivalent thereof, the g-protein preferably being fused in-frame to above GPCR 1  and/or GPCR 2 . 
     
     
         38 .) Cell lines stably transfected with a vector according to  claim 16 . 
     
     
         39 .) A vector according to  claim 17 , wherein the g-protein is fused in-frame to the GPCR 1  and/or GPCR 2