Field of Invention
The present invention describes establislunent of a cell based functional assay for GPCRs, by determining the level of intracellular cAMP levels utilizing reporter gene driven cell based assay. The novel assay described in the current invention, combines features of binding and functional mode in a single type of experimental set up which offers both binding affinity as well as mode of action of compounds interacting with any GPCR in a single set. The novel assay of the invention is useful in identification of compounds acting through GPCRs and their further categorization based on the mode of action. The assay may find high utility in screening of new chemical entities for identification of novel drugs acting through GPCRs. Moreover, the novel assay can also be utilized in exploring the mode of action of established and known drugs and assignment of their pharmacological properties. Background of the Invention
GPCRs also known as seven-transmembrane domain receptors comprise a large protein family of transmembrane receptors that sense molecules outside the cell and activate inside signal transduction pathways and ultimately cellular responses. GPCRs are involved in various physiological functions as well as many diseases, and are also the target of around half of all modem medicinal drugs. There are two principal signal transduction pathways involving the GPCRs: cAMP signal pathway and Phosphatidylinositol signal pathway.
The cytoplasmic tail region of GPCRs interacts to one of the three main classes of G proteins. G proteins are composed of a common Oy subunit and a specific a subunit. The role of the a subunit is well known for translating the extracellular cues to intracellular responses. G proteins are mainly classified into three categories based on the nature of their subunits. G proteins containing Gas or Gai subunits enhance or reduce the cAMP level respectively upon receptor stimulation through adenylate cyclase enzyme. In contrast, G proteins comprising Gaq subunit mobilize intracellular calcium ions upon activation of the receptor through a membrane bound phospholipase C enzyme. In recent years, GBy subunits turned out to be more than a silent partner. They too transmit the message through activation of ERK-MAPK pathways.
The molecular diversity of GPCR-mediated signal transduction pathway complicates the configuration of a common functional assay. The development of high through-put functional assays for GPCRs would greatly enhance the ability to discover and develop novel agonists and antagonists to this important superfamily of pharmaceutical targets. One approach for developing a high through-put functional GPCR assay is the use of reporter gene assays. Reporter gene constructs couple transcriptional enhancers that are regulated by various intracellular second messengers with appropriate promoter and reporter gene elements to produce a surrogate signal transduction system responsive to signaling pathways activated by various hormone receptors (Deschamps, Science, 1985 230:1174-7; Montminy, Proc. Nail. Acad Sci USA, 1986 83:6682-6686; Angel, Cell, 1987, 49:729-39; Fisch, Mol. Cell Biol,

1989 9:1327-31). With the appropriate choice of transcriptional enhancers, promoters, and reporter genes, non-radiometric functional assays have been configured for Gas coupled GPCRs (Konig, Mol. Cell. Neurosciences, 1991, 2:331-337; Chen, Anal. Biochemistry, 1995, 226: 349-354) and Gaq coupled GPCRs (Weyer, Receptor and Channels, 1993 1:193-200) that are amenable to high through-put screening technology.
Earlier, some assays have been developed for the identification of GPCRs. However, some disadvantages are associated with the traditional radioligand binding assay and FLIPR based functional assays. In traditional binding assay a) hazardous radioactive ligands are used; b) the mode of action of a molecule needs to be further investigated using a separate radioactive or cell based functional assay. In the FLIPR based functional assay (European patent application: EP1310800), fluorescent counts are measured within 5 seconds of compound injection which may not be sufficient to bring the compound in equilibrium with the receptor. As a resuh of shorter incubation of compounds in FLIPR assay, important compounds may be missed out or artefactual values may be generated.
In order to overcome the above mentioned disadvant^es, we have developed novel functional assay for GPCRs based on measurement of intracellular cAMP levels by measuring reporter gene activity:
a) which eliminates the usage of hazardous radioactive ligands. Thus an environmental friendly approach can be implemented to identify novel compounds.
b) mode of action (agonist or antagonist) as well as binding affinity of the ligand (pKb or pEC50) are derived from the same experiment.
c) compounds are incubated for few hours to bring them in equilibrium with the receptor. Longer incubation period in reporter gene format is required to have a sustained activation of the receptor leading to the synthesis of c AMP and induction of the reporter gene.
Thus, the reporter gene based assay may be more in line with the physiological conditions wherein the drug is allowed to interact with the target receptor in the body. Summary of Invention
In one aspect, the present invention provides a novel functional assay method for identification of compounds acting through GPCRs, which comprises:
a) Chinese hamster ovary (CHO) cells were transfected with specific GPCR constructs along with excess of reporter gene using Lipofectamine and cultured in suitable medium containing selection antibiotics;
b) individual colonies exhibiting maximum luciferase activity with both forskolin as well as the agonist were picked up and the expression of said GPCRs was analyzed;
c) recombinant CHO cells were plated in a microtitre plate and cultured;
d) increasing concentrations of agonist or new compounds were incubated with the recombinant cells in CO2 incubators to evaluate agonist properties of compounds;

e) for evaluation of compound in antagonist mode, the above recombinant cells were incubated with increasing concentrations of compounds or a known antagonist and a fixed concentration of a specific agonists for few hours at 25-37 °C in CO2 incubator;
f) additional step of serum free growth is required for evaluation of compounds in various serotonin and acetylcholine receptors before doing steps (d or e);
g) after incubation of the cells was over, medium was removed, cells were washed with buffer and lysed in thelysis buffer;
h) reporter gene activity was measured in individual wells.
In another aspect, total RNA isolated from each cell line was used in the cDNA synthesis.
In further aspect, radioligand binding and competition assays were performed with membranes prepared from the recombinant CHO cell lines.
In yet another aspect, mode of action (agonist or antagonist) as well as binding affinity of the ligand (pKb or pEC50) is derived from the same experiment. Brief Description of Diagrams
Figure 1 shows expression of the RNA in recombinant CHO cells.
Figure 2 shows stimulation of luciferase reporter gene in CHO cells expressing human 5- HT2A, adrenergic Alphaib or Histamine Hi receptors.
Figure 3 shows ligands stimulate the specific receptor in a dose dependent manner.
Figure 4 shows blockade of reporter gene expression by antagonists.
Figure 5 shows dose response graph of serotonin on CHO-5HT2A cells (in the presence of 10,100 and 1000 nM Ketanserin), dose response graph of epinephrine on CHO-aib (in the presence of 10,100 and 1000 nM Ketanserin) and dose response graph of histamine on CHO-Hi cells (in the presence 10, 100 or 1000 nM Cetirizine). The agonist concentration used was same as in figure 3.
Figure 6 shows graphical representation of method for screening of G-protein linked receptor compounds. Detailed Description of Invention
Luciferin, T4 DNA Ligase, high fidelity Taq polymerase, superscript reverse transcriptade,
mammalian vector pcDNA3.1, CRE-Luc reporter gene, cell culture media, sera, radioligands Ketanserin
Hydrochloride [ethylene-^H] 60-90 Ci/mmol, Prazosin [7-methoxy-^H] 70-87 Ci/mmole, Pyrilamine
[pyridinyl 5-^H], (Mepyramine) 20-30 Ci/ mmol, scintillation proximity assay beads. Human 5-HT2A
cDNA clone. Adrenergic alphaib cDNA clone, all other DNA restriction enzymes, modification
enzymes, all other reagents and common chemicals
were purchased from well known suppliers.
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Human 5-HT2A cDNA clone was amplified by polymerase chain reaction (PCR) using gene specific primers. Adrenergic alphaib cDNA clone was amplified by PCR using gene specific primers. Human histamine Hi cDNA was generated by reverse transcription using total RNA isolated from HepG2, 1MR32, HEK293 and CaCo2 cell lines and gene specific reverse primers. The cDNA was amplified by PCR using gene specific primers using high fidelity Taq DNA polymerase. Amplified DNA was cloned in to mammalian expression vector pcDNA 3.1. The authenticity of the cloned genes was determined by restriction analysis and nucleotide sequencing. Example 1: Expression of the RNA in recombinant CHO cells
Total RNA irom recombinant or control CHO cells were isolated using TRI reagent (Sigma) as recommended. The quality of each RNA sample was analyzed by agarose gel electrophoresis. Total RNA from each cell line was used in the cDNA synthesis by reverse transcription using Superscript Reverse Transcriptase and gene specific reverse primers for 5-HT2A, Alphas, Hi and P-actin genes. PCR was performed on each cDNA sample using gene specific forward and reverse primers. Samples were separated on 1% agarose gel and visualized after Ethidium bromide staining.
As evident fi"om Figure 1, human 5-HT2A is exclusively expressed in CHO cells transfected with human 5-HT2A gene (CHO-5HT2A) and not in control and other transfected cells. Similarly human histamine Hi or adrenergic alphas mRNA are exclusively expressed in CHO cells transfected with human Hi or ttib cDNA (CHO-Hi or CHO-oib) respectively. P-actin mRNA was also reverse transcribed in the same set of assays using a gene specific reverse primer. The cDNA from each of the cell line was amplified using P-actin specific forward and reverse primers. Presence of a band corresponding to P-actin fragment in Figure 1 demonstrated the presence and good quality of the RNA in each preparation. Example 2: Stimulation of luciferase reporter gene in CHO cells
Each recombinant CHO cell line was plated in 96 well white with clear bottom plates. The ligands as indicated were added to a final concentration of 10 ^M. The luciferase activity was measured in individual wells using luciferin substrate in Perkin Elmer luminometer. The basal luciferase activity for each cell line was assigned an arbitrary value of 1. Fold stimulation with each ligand was determined in relation to the basal luciferase activity.
The assay was also utilized to investigate the specificity of various ligands and authenticity of the generated cell lines in this assay. As evident from Figure 2, serotonin at a concentration of 10 jiM demonsfrated about nine fold induction of luciferase activity in CHO-5HT2A cells. However, histamine and epinephrine at the same concentration did not show a significant induction of luciferase activity in CHO-5HT2A cells. Similarly, epinephrine at a concentration of 10 fiM induced the expression of luciferase activity to about six fold in CHO-aib cells. As expected, CHO-aib cells did not respond to the treatment with 10 ^.M serotonin or histamine (Figure 2). CHO-Hi cells exhibited about three fold enhanced expression of luciferase enzyme with 10 nM histamine. Epinephrine and serotonin (10 ^M
5

each) did not show any effect on the level of luciferase activity in CHO-H] cells, A strong induction in luciferase activity was observed in all the three recombinant cell lines upon treatment with forskolin. Example 3: Ligands stimulate the specific receptor in a dose dependent manner
To further confirm the single dose effect of specific agonists on the recombinant CHO cell lines, a dose response effect with different agonists was measured. Each recombinant cell line was treated with individual agonists from 0.1 nM to 10,000 nM and luciferase activity was measured by using luciferin substrate in Victor Light Luminometer from Perkin Elmer. The agonist stimulated luciferase activity in the absence of a compound was assigned a value of 100% while basal luciferase activity was assigned a value of 0%. Rest of the luminescent values obtained for compounds at various doses were calculated with reference to stimulated and basal luciferase activities. Data was analyzed using Graphpad software.
Serotonin showed a specific dose response effect on the expression of luciferase activity in CHO-5HT2A cells (Figure 3). A pECso value of 6.6 for serotonin in the assay was determined (Table 1). Epinephrine and histamine did not show the induction of luciferase activity in CHO-5HT2A cells up to the highest dose tested (Figure 3). 5-HT2A receptor is not reported to couple to Gas containing G protein or stimulate cAMP production under any experimental conditions reported so far. S-HTZA receptor is well established to couple to Gaq containing G protein (Raymond JR. Mukhin YV, Gelasco A, Turner I, Collinsworth G, Gettys TW, Grewal JS, Gamovskaya MN: Multiplicity of mechanisms of serotonin receptor signal transduction. Pharmacol Ther 2001,92:179-2 12). Some reports also suggest inhibition of cAMP accumulation upon activation of 5-HT2A receptor (Gamovskaya MN, Nebigil CG, Arthur JM, Spumey RF, Raymond JR: 5- hydroxytryptamine 2A receptors expressed in rat renal mesangial cells inhibit cyclic AMP accumulation. Mol Pharmacol 1995, 48: 230-237). A range of pKj values from 8.4 to 6.0 are reported for serotonin in 5-HT2A radioligand binding assays in RJPHAR database. The pECso value determined in the current investigations falls in the middle of the reported range.
CHO-ttib cells showed a robust dose response with increasing concentrations of epinephrine from 0.1 to 10000 nM as evident from the level of luciferase activity observed (Figure 3). The treatment of recombinant cells with epinephrine provided a pECso value of 7.3 (Table 1). pECso values of 7.3 and 6.6 for calcium mobilization and cAMP accumulation respectively are reported for CHO cells expressing human adrenergic alphalb receptor upon treatment with norepinephrine (Horie K, Itoh H, Tsugimoto G: Hamster aib-adrenergic receptor directly activates Gs in the transfected chmese hamster ovary cells. Mol Pharmacol 1995, 48: 392-400), (Gallego M, Setien R, Puebla L, Boyano-Adanez MdC, Arilla E, Casis O: tti-adrenoceptors stimulate a Gas protein and reduce the transient outward K^ current via a cAMP/PKA-mediated pathway in the rat heart. Am J Physiol Cell Physiol 2004, 288: C577-C585). CHO-aib cells did not respond to histamine or serotonin treatment as evident from basal level of luciferase activity observed at the highest concentration of the ligand tested (Figure 3).
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Following on the same pattern, CHO-Hi cells were treated with increasing concentrations (1 to 100,000 nM) of histamine. A dose proportionate increase in luciferase activity was observed in CHO-Hi cells treated with histamine but not with serotonin or epinephrine. Histamine showed a pECso value of 6.0 in this assay. Moniri et. al. reported a pECso value of 5.8 in a cAMP accumulation assay in CHO cells expressing human histamine Hi receptor upon treatment with histamine (Moniri NH, Covington-Strachan D, Booth RG: Ligand-directed functional heterogeneity of histamine HI receptors: novel dual function ligands selectively activate and block HI-mediated phospholipase C and adenylyl cyclase signaling. J Pharmacol Exp Ther 2004; 311: 274-281) Overall, the values reported in the present assay are well in agreement with the published values generated using different strategies for three receptors under investigation. Table 1: pECjo values
Compounds I CHO-SHTIA \ CHO-a,b I CHO-Hi
Serotonin 6.6 ±0.1 <5 <5
Epinephrine <5 7.0 ± 0.3 <5
Histamine <5 <5 6.0 ±0.1
Example 4: Blockade of reporter gene expression by antagonists
Once we observed an induction of reporter gene activity in all recombinant cell lines evaluated in a agonist and dose dependent manner, it was of interest to determine whether the activity was blocked by various known antagonists. A number of compounds already demonstrated to antagonize some or all of the receptors under investigation were selected for the current study. Vehicle or selected compounds (10 ^M concentrations) were incubated along with 10 ^M of specific agonist with the cells and luciferase activity was measured by Victor Light Luminometer from Perkin Elmer. A detailed evaluation of various compounds in specific cell lines is presented in Figure 4. Example 5: Dose response study
Ketanserin, mianserin, olanzapine, clozapine and chlorpromazine (each at 10 pM concentrations) fully antagonized the serotonin induced and 5-HT2A mediated induction of luciferase activity in CHO-5HT2A cells. While haloperidol exhibited a limited antagonism, cetirizine did not show any impact on serotonin induced luciferase activity in CHO-5HT2A cells. Risperidone demonstrated an inverse agonism on 5-HT2A receptor and brought down the luciferase activity to less than vehicle control. A treatment of CHO-5HT2A cells with increasing concentrations of serotonin (0.1 to 10,000 nM) in the presence of 0,10, 100 and 1000 nM ketanserin resulted in a significant right shift in the graph (Figure 5). The rightward shift in the graph in Figure 5 was dependent on the concentration of ketanserin used, with maximum shift observed with 1000 nM dose. To better understand the effect of these compounds on serotonin induced luciferase activity in CHO-5HT2A cells, a dose response study was performed. The cells were incubated
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with the increasing concentrations of above compounds (0.1 to 10,000 nM) along with 10 ^M serotonin and luciferase activity was measured. Table 2 provides a pICso as well as pKb value for each compound tested in all three cell lines. As evident from Table 2, majority of the compounds demonstrated a potent antagonism to serotonin induced luciferase activity in CHO-5HT2A cells with a pICso value of close to or less than 7.0 with the exception of cetu-izine and haloperidol. Cetirizine and haloperidol exhibited none or partial antagonism to serotonin induced luciferase activity in CH0-2A cells. Serotonin completely displaced ketanserin in CHO-5HT2A cells. Similarly, ketanserin fully displaced serotonin when assayed in the same cell line. All these observations support a competitive and reversible binding of ligands to CHO-5HT2A cells.
The effect of same set of compounds, tested in CHO-5HT2A cells, was investigated in CHO-aib cells. Haloperidol, clozapine, chlorpromazine and risperidone (at a concentration of 10 ^M) completely blocked the epinephrine induced luciferase activity in the recombinant cells (Figure 4). In comparison to the above compounds, ketanserin, olanzapine and mianserin showed a limited efficacy, cetirizine did not show any effect on the epinephrine induced luciferase activity in CHO-Oib cells. Chlorpromazine and risperidone demonstrated a potent antagonism to epinephrine induced luciferase activity in CHO-aib with pICso values of 6.9 and 7.1 respectively (Figure 4, Table 2). Rest of the compounds showed a pICso value of close to or less than 6.0. As expected, cetirizine did not show any antagonism of epinephrine induced luciferase activity. We studied the effect of various concentrations of ketanserin on the level of luciferase activity in CHO-aib cells upon treatment with increasing concentrations of epinephrine. Selection of ketanserin for this evaluation was guided by the observation that it had limited impact on blocking the epinephrine induced luciferase activity in CHO-aib cells. As evident from Figure 5, ketanserin did induce a rightward shift in the graph as compared to control epinephrine treated samples. The extent of rightward shift in curves was dependent on the concentration of ketanserin used with the maximum shift at 1000 nM dose. However, the effect of ketanserin on CHO-Oib cells was milder than on CHO-5HT2A cells which are well in agreement with the fact that it has a higher affinity for human 5-HT2A receptor as compared to alpha lb receptor. The assay supported a reversible mode of interaction of epinephrine as well as kentanserin to CHO-aib cells.
CHO-Hi cells were evaluated for the blockade of histamine induced luciferase activity by a defined set of compounds. As evident from Figures 2 and 4, CHO-Hi cells exhibited about four fold stimulation of luciferase activity with 10 ^M histamine which correlated well with a direct cAMP measurement in CHO cells expressing human histamine Hi receptor reported. Histamine induced luciferase activity was completely blocked by Hi specific antagonist cetirizine (Figure 4). In addition, all the tested compounds with the exception of haloperidol (at a concentration of 10 fiM) blocked the histamine induced luciferase activity to the basal level. Effect of different concentrations of cetirizine on the histamine induced luciferase activity in CHO-Hi cells was investigated. Cetirizine treatment resulted
8

in a rightward shift in the curves plotted with an increasing concentration of histamine (Figure 5). Cetirizine also demonstrated an inverse agonist property as evident fh)m Figure 4. Most of the compounds evaluated showed a potent antagonism of histamine induced luciferase activity in CHO-Hi cells in a dose dependent manner. Mianserin demonstrated to be the most potent compound in the assay with a pIC50 value of 8.1. Cetirizine showed a pICso value of 6.3.
The assay apart from demonstrating various mode of action of a compound to GPCRs can also be utilized to characterize orthosteric or allosteric mode of action of a compound. Furthermore, the reporter gene based functional assay can identify whether a compound interacts with the receptor in a reversible or irreversible manner.
Example 6: Once the reporter gene assay was established and detailed pICso and pKb values were determined for a number of compounds, it was of interest to measure their binding affinity for the same set of receptors. Although the binding parameters for majority of these compounds are already reported, it was important to evaluate them in parallel with the reporter gene based functional assays. Table 2: plCjoand pKb values obtained from functional assays
I CHO-SHTiA \ CHO-Oib I CHO-Hj
Compounds pICso pKb pICso pKb pICjo pKb
Ketanserin 7.1 ±0.2 9.0 ± 0.2 5.4 ± 0.2 7.7 ± 0.2 5.9 ±0.1 7.4 ±0.2
Mianserin 7.1 ±0.2 8.9 ± 0.04 5.0 ± 0.3 7.4 ± 0.3 8.1 ±0.04 10.1±0.01
Cetirizine <5 <5 <5 <5 6.3 ± 0.2 8.4 ± 0.2
Olanzapine 7.5 ± 0.3 9.3 ±0.1 5.7 ±0.1 7.9 ± 0.2 7.99 ± 0.3 10 ±0.3
Haloperidol 5.8 ±0.4 7.5 ±0.1 6.2 ±0.1 8.6 ±0.1 5.4 ± 0.2 7.4 ± 0.4
Clozapine 6.5 ± 0.4 8.3 ± 0.2 6.0 ±0.2 8.3 ± 0.2 7.8 ±0.3 9.8 ±0.3
Chlorpromazine 6.9 ± 0.3 8.6 ±0.1 6.9 ± 0.05 9.3 ±0.05 6.75 ±0.06 8.8 ±0.2
Risperidone 8.5 ± 0.3 10.3 ±0.2 7.1 ±0.1 9.5 ±0.1 5.9 ±0.06 7.9 ±0.1
Radioligand binding and competition assays were performed with membranes prepared from the recombinant CHO cell lines as described in following three references:
1. Nelson DL, Lucaites VL, Wainscott DB, Glennon RA: Comparison of hallucinogenic phenylisopropylamine binding affinities at cloned human 5-HT2A, 5-HT2B and 5-HT2c receptors. Naunyn-Schmiedeberg's Arch Pharmacol 1999,359: 1-6.
2. Yoshio R, Taniguchi T, Itoh H, Muramatsu I: Affinity of serotonin receptor antagonists and agonists to recombinant and native a 1 -adrenoreceptor subtypes. JpnJPharmacol 2001, 86: 189-195.
3. Ratnala VRP, Swarts HOP, VanOostrum J, Leurs R, DeGroot HJM, Bakker RA, DeGrip WJ: Large scale overproduction, functional purification and ligand affinities of his-tagged human histamine Hi receptor. Eur J Biochem 2004,271:263 6-2646.
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All the assays were converted to scintillation proximity assay (SPA) based format. For competition binding assays, 0.1 to 10,000 nM of each compound was incubated with a fixed concentration of the specific radioligands, membrane and SPA beads. For adrenergic alphaib receptor, 4.0 nM of Prazosin [7-methoxy-^H] was incubated with recombinant membrane and Polylysine (PLL) coated Yitrium silicate (Ysi) SPA beads for three hours at ambient temperature. For, histamine Hi binding assay, 1 nM of Pyrilamine [pyridinyl 5-^H] was incubated with the membrane and Wheat Germ Agglutinin (WGA) coated Polyvinyl Toluene (Pvt) SPA beads for two hours at the ambient temperature. For 5-HT2A binding assay, 3.1 nM Ketanserin Hydrochloride [ethylene-^H] was incubated with the membrane and WGA coated Ysi SPA beads in dark for four hours. After incubation was over, radioactivity was measured in Microbeta plate reader (Perkin Elmer). Total binding was determined in the absence of any ligands whereas non-specific binding was determined to be counts obtained in the presence of excess amount of specific ligands. Specific activity was calculated from the differences between total and non¬specific counts.
Radioligand binding assay using SPA beads were performed to determine the pKj values for specific compounds. The binding experiment was performed thrice and average values are presented in Table 3. As evident, all the compounds showed a pKj value which well correlates with the already reported values. These compounds also demonstrated target selectivity as reported earlier (http://www.iuphar-db.org/GPCR/ReceptorFarmiliesForward). Serotonin exhibited a pKi value of 6.8 to 5-HT2A receptor but did not show any binding to alpha ib and Hi receptors. Similarly, epinephrine showed strong binding to alpha lb receptor with a pKj value of 6.4 but a weak affinity for 5-HT2A and no binding to Hi receptors. The pKi value determined is well in agreement with the published value. Histamine did not show any binding to either 5-HT2A or alpha ib receptors while giving a pKj value of 5.9 with Hi receptor which is again well in agreement with previously reported values. Ceterizine demonstrated a selective binding to human Hi receptor with a pKb value of 7.7 and no affinity to 5-HT2A or Hi receptors. Clozapine, mianserin, ketanserin and olanzapine showed varying degree of binding to all the above receptors (Table 3).
Table 3: pKi values determined fi'om radioligand binding assays
Compounds/Targets S-HTIA Oib Hi
Serotonin 6.8 ±0.02 <5 <5
Epinephrine 5.1 ±0.1 6.4 ±0.1 <5
Histamine <5 <5 5.9 ±0.01
Clozapine 7.8 ±0.1 7.2 ± 0.2 8.8 ±0.1
Mianserin 8.2 ±0.2 6.9 ±0.1 9.7 ±0.1
Ketanserin 8.9 ±0.1 7.2 ±0.1 7.7 ± 0.03
1 ' t
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Olanzapine i 7.5 ±0.1 \ 6.5 ±0.1 I 8.6 ±0.1
Cetirizine <5 <5 7.7 ±0.1
Example 7: pKj and pKb are derived parameters from pICso value which are determined by binding assays and functional assays respectively. As the pICso value for the same ligand and receptor combination may vary depending on the amount of radioligand used in the binding assay or amount of agonist used in the functional assay, derived pKj and pKb values demonstrate a constant parameter for a specific compound. Thus, we compared the pKj and pKb values for specific compounds derived from radioligand binding assay or cell based fiinction assay. For an agonist, pECso value derived from the functional assay is compared with the pKj value determined from the binding assay. As evident from Table 4, majority of the compounds showed a good correlation between pKb or pECso values derived from the functional assay and pKi values generated from the radioligand binding assay.
Table 4: pECso, pKj and pKb value comparison
5-HT2A Olb Hi
Compounds/ pECso or pKj pECso or pKj pECso or pKj
Targets pKb pKb pKb
Serotonin 6.6 ±0.1 6.8 ± 0.02 <5 <5 <5 <5
Epinephrine <5 5.1±0.1 7.0 ± 0.3 6.4 ±0.1 <5 <5
Histamine <5 <5 <5 <5 6.0 ±0.1 5.9 ±0.01
Clozapine 8.3 ± 0.2 7.8 ±0.1 8.3 ± 0.2 7.2 ± 0.2 9.8 ± 0.3 8.8 ±0.1
Mianserin 8.9 ± 0.04 8.2 ± 0.2 7.4 ± 0.3 6.9 ±0.1 10.1± 9.7 ±0.1
0.01
Ketanserin 9.0 ± 0.2 8.9 ±0.1 7.7 ± 0.2 7.2 ±0.1 7.4 ± 0.2 7.7 ± 0.03
Olanzapine 9.3 ±0.1 7.5 ±0.1 7.9 ± 0.2 6.5 ±0.1 10 ±0.3 8.6 ±0.1
Cetirizine <5 <5 <5 <5 8.4 ± 0.2 7.7 ±0.1
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1. An assay method for screening the compounds of G protein linked receptor (GPCRs) using reporter
gene based assay in a microtitre plate comprising:
a) transfecting Chinese hamster ovary (CHO) cells with specific GPCR constructs along with excess of reporter gene using Lipofectamine and cultured in suitable medium containing selection antibiotics;
b) picking up individual colonies exhibiting maximum luciferase activity with both forskolin as well as the agonist and analysing the expression of said GPCRs;
c) plating recombinant CHO cells in a microtitre plate and culturing them;
d) incubating increasing concentrations of agonist or new compounds with the recombinant cells in CO2 incubators to evaluate agonist properties of compounds;
e) incubating said recombinant cells for evaluation of compound in antagonist mode, with increasing concentrations of compounds or a known antagonist and a fixed concentration of a specific agonists for few hours at 25-37 °C in CO2 incubator;
f) evaluating compounds in various serotonin and acetylcholine receptors before doing steps (d or e) by an additional step of serum free growth;
g) after incubation of the cells was over, removing the medium and and washing the cells with
buffer
and lysed in the lysis buffer; h) measuring reporter gene activity in individual wells.
2. The method as claimed in claim 1, wherein said GPCRs comprises serotonin receptors, muscuramic receptors, histamine receptors and adrenergic receptors.
3. The method as claimed in claims 1 and 2, wherein said GPCRs are selected from the group consisting of 5-HT2A, histamine HI, adrenergic alpha lb, S-HTe and 5-HT4.
4. The method as claimed in any preceding claim, wherein said luciferase reporter gene constructs was three fold in excess.
5. The method as claimed in any preceding claim, wherein said reporter gene is CRE-Luc reporter gene.
6. The method as claimed in any preceding claim, wherein said microtitre plate is used for high throughput screening.

7. The method as claimed in any preceding claim, wherein said reporter system is selected from group
consisting of luciferin substrate and fluorescent proteins.
8. The method as claimed in any preceding claim, wherein said incubating steps (d) and (e) occurs for
from about 4 hours to 5 hours.