WO2005085867A2

WO2005085867A2 - Screening method for emulators of neural activity and digestive system using gpr35

Info

Publication number: WO2005085867A2
Application number: PCT/IB2005/000382
Authority: WO
Inventors: Yasuhito Taniguchi; Hiroko Kachi; Kimihiko Tomotoshi; Katsuhiro Shinjo; Tohru Komada
Original assignee: Pfizer Japan, Inc.; Pfizer Inc.
Priority date: 2004-02-27
Filing date: 2005-02-14
Publication date: 2005-09-15
Also published as: WO2005085867A8; CA2558721A1; EP1720530A2; WO2005085867A3

Abstract

The present invention primarily relates to function analysis of GPR35, an orphan GPCR (G protein coupled receptor), a screening method to identify modulators (agonists/antagonists) of the receptor, cells stably expressing the receptor, and the discovery of modulators of the receptor. The present invention also relates to novel rat GPR35.

Description

GPR35

Tbchnical ?Pield of the Invention The present invention primarily relates to function analysis of GPR35, an orphan GPCR (G protein coupled receptor), a screening method to identify modulators (agonists/antagonists) of the receptor, cells stably expressing the receptor, and the discovery of modulators of the receptor. The present invention also relates to novel rat GPR35.

Background Art of the Invention G protein coupled receptors are a large superfamily of integral membrane proteins, involved in a broad range of signaling pathways. Most G protein-coupled receptors are characterised by 7 transmembrane-spanning helices, and are therefore also called 7- transmembrane receptors (7TMs). There are at least several hundred members of this family, which include receptors responding to a wide range of different stimuli, including peptides, biogenic amines, Hpids, neurotransmitters, hormones, nucleotides, sugar-nucleotides, cytokines, etc. Structurally, the receptors of tins family consist of an extracellular amino terminal domain, seven membrane-spanning hydrophobic regions, six loop regions, three of which are extracellular, the other three being intracellular, and an intracellular carboxy terminal domain. The hydrophobic regions show considerable homology between the different members of this family, whereas the loop regions, as well as the amino and carboxy terminal domains, are quite diverse, showing high homology only amongst closely related receptor subtypes. G proteins are heterotrimeric complexes, containing an α, a β, a γ-subunits in which α is bigger than β and β is bigger than γ. At least 20 human genes are ?known to encode the GTP-binding orsubunits. Furthermore, there are at least 5 genes encoding the β -subunits and at least 11 genes encoding ysubunits. The α-subunit family is divided into four subfamilies on the basis of their homology: (l) The Gs family is typically able to stimulate adenylate cyclase upon agonist binding to the receptor coupling to it, and therefore lead to an increase in intracellular cyclic AMP. (2) The Gq family, which includes Gqα, Gil α, G14 α, G16 α, lead to activation of phospholipase C.

(3) G12 and G13 appear to regulate classes of small molecular weight G proteins and Na⁺Η⁺ exchange. (4) The Gi family typically mediate inhibition of adenylate cyclase. More than half of the orsubunits are members of the Gi family and include the ubiquitously expressed and nearly identical Gil α, Gi2 α and Gi3 α, as well as several with a limited expression, such as Gz α, Goα and transducin. Initiation of signal txansduction cascades involving G proteins requires the binding of a hgand to a G protein-coupled receptor (GPCR). This results in the stabilisation of conformations of the GPCR that increases the rate of dissociation of the bound GDP from the nucleotide binding pocket of G α subunit of a G protein. Binding of GTP is thus allowed. With GTP bound, the G protein dissociates into GTP-G α and Gβγ moieties and can hence regulate directly or otherwise the activity of several enzymes, w?hich generate intracellular signalling molecules (e.g. cyclic AMP), or the probability of opening a range of ion channels (Gil an, A.G. (1987) Annu Rev. Biochem. 56, 614-649). Following G protein activation, hydrolysis of bound GTP by intrinsic GTPase activity terminates its ability to regulate its effectors and leads to crGDP reassociation with βy. The G protein therefore serves a dual role, as a relay transmitting the signal from the receptor to the effector, and as a clock that controls the duration of the signal. Therefore, depending on which GPCR couples to which G protein, very different intracellular effects can result, e.g. stimulation or inhibition of adenylate cyclase, leading to increased or decreased cyclic AMP concentrations in the cells, opening of ion channels, etc. GPCRs are very important targets for pharmaceutical intervention. Many agonists and antagonists of the GPCRs of lcnown functions are important for the treatment of diseases. Examples include but are not limited to, βl-adrenergic receptor antagonists used for treating hypertension, angina and heart failure, dopamine D2 antagonists used for the treatment of schizophrenia, and β2-adrenergic receptor agonists used for the treatment of asthma. Over the past 15 years, nearly 350 therapeutic agents targeting GPCRs have been introduced onto the market, and it is estimated that around 30% of clinically prescribed drugs function as either agonists or antagonists at GPCRs. Therefore, it is likely that amongst the orphan receptors, there are further therapeutic targets which can play a key role in treating or preventing diseases or dysfunctions, including, but not limited to, pain, cancers, obesity, eating disorders, hypertension, hypotension, heart failure, incontinence, asthma, chronic bronchitis, angina, ulcers, psychotic and neurological disorders, viral infections including HIV-1 or HIV-2, other infections, inflammatory conditions, sexual dysfunction, urogenital disorders, and many other disorders, diseases or dysfunctions. It is therefore clearly highly desirable to find the physiological function for each GPCR. However, it is not at all easy to find the physiological function for each GPCR as discussed below. The conserved structure and hydrophobicity profile of this receptor family has allowed the identification and cloning of some members of this family, both through cloning experiments, e.g. degenerate polymerase chain reaction or cDNA library screening with probes derived from GPCR sequences using low stringency, and through bioinformatic mining of sequence databases. For many of these receptors, it is yet xinclear what their functions are. These receptors are referred to as "orphan receptors". Through detailed analysis of their sequences and comparison with the sequences of GPCRs with lαiown ligands, hypotheses can be generated as to the class of ligands that the orphan receptor may respond to. However, these predictions are often tenuous, and it is still difficult to identify the

Hgand that such an orphan receptor may respond to. This is mainly because stimulation of GPCRs can lead to a multitude of different intraceHular effects, which are mediated through the coupling to different G proteins. The G proteins in turn act on a whole range of signalling proteins, including phosphoHpase C, adenylate cyclase and ion channels, and thereby initiate a cascade of events in the cells. It is difficult to predict from the sequence of the GPCR which G protein it will couple to, and in some cases the resulting intraceHular effects are difficult to measure. Furthermore, the usual host cells used for heterologous expression of cloned GPCRs only express certain G proteins. Therefore, if the G protein that the orphan GPCR couples to is not expressed in these cells, the binding of an agonist to this receptor will not have a measurable effect, making it unlikely that one could find the agonist(s) for the GPCR in such a system. It is therefore necessary to engineer specialized host ceH lines, expressing various heterologous G proteins, to enable the estabfishment of host eel systems that can lead to measurable responses for many of the different GPCRs. With regard to GPR35, there are several articles that report nucleotide and/or amino acid sequence of human and/or mouse GPR35. However, none of them reveals specific physiological function of GPR35. All that has been previously disclosed merely presumus that GPR35 is a GPCR. "Discovery of three novel G-proteύrcoupled receptor genes (O'Dowd BF et al. Genomics 47 (2), 310-313 (1998))" discloses cDNA and amino acid sequences of human GPR35. However, this report merely assumes the GPR35 is a GPCR. Needless to say, it does not mention any physiological function of the GPR35. Northern blotting was employed to determine tissue distribution. However, in rats, GPR35 expression was detected in the intestine but not in the heart, spleen, Hver, lung, ovary, kidney, nor whole brain. In humans, the expression was not detected in the caudate-putamen, thalamus, frontal cortex, midbrain, lung, nor adrenal. The expression in the DRG, dorsal horn, or spinal cord which is closely related to a neural activity, was therefore not detected. "Genetic variation in the gene encoding calpain 10 is associated with type 2 diabetes melHtus (Horikawa Y et al. Nat. Genet. 26:163-175 (2000))" discloses the cloning of mouse GPR35 gene. In this article, however, genes and SNPs located in the ISHDDMl region on chromosome 2 (which is suspected of relating to NIDDlVl), were examined at random to verify the linkage (association) between the disease and SlNEPs, in order to identify genetic elements related to non-insulin dependent diabetes melHtus (NIDD?1VI) also ?known as type 2 diabetes. Although this article provides the findings that the GPR35 gene is located in the ISflDDMl region and that there are a number of polymorphisms of human GPR35, it did not mention specific physiological functions of GPR35 nor lead to a positive conclusion concerning its relationship with ?NIDDM. Since the sequences of human GPR35 and mouse GPR35 are known, some biotechnology companies may prepare antibodies or lαiockout animals according to the consignment and, in general, the nondisclosure agreement. These can be employed only as research tools, but does not clarify the functions by themselves. The inventors of the present invention overcame these difficulties and disclosed specific physiological function of GPR35. Brief Disclosure of the Invention The present invention encompasses the following non-Hmiting aspects. One aspect of the present invention is; a method of screening for a compound that modulates a GPR35 protein, comprising the step o£ (a) contacting a GPR35 protein or a partial polypeptide thereof with a test compound. The GPR35 protein or the partial polypeptide thereof used in the screening method of the present invention can exist in ceHs or a membrane fraction. The screening method of the present invention can be used to screen compounds that modulates neural activity and/or digestive system. In the screening method of the present invention, test compound can consist of a plurality of compounds. In this case, what is determined is whether these compounds can, as a combination, modulate a GPR35 protein. In the screening method of the present invention, a GPR protein or a partial polypeptide thereof of the foHowing can be used,' (a) a polypeptide having the amino acid sequence of sequence ID. 2, 4, or 6,

(b) a polypeptide having the same amino acid sequence as the polypeptide of (a) except that one or more amino acids are deleted, substituted, or added,

(c) a polypeptide having an amino acid sequence that has at least 80% identity to the the polypeptide of (a), (d) a polypeptide having an amino acid sequence that has at least 90% identity to the the polypeptide of (a),

(e) a polypeptide having an amino acid sequence that has at least 98% identity to the the polypeptide of (a), and

(0 a human GPR35 protein, a mouse GPR35 protein, or a rat GPR35 protein. Another aspect of the present invention isJ a method of screening for a compound that inhibits the binding of a Hgand to a GPR35 protein, comprising the steps o£

(a) contacting a GPR35 protein or a partial polypeptide thereof with the Hgand,

(b) contacting a GPR35 protein or a partial polypeptide thereof prepared in the substantially same manner as the one used in the step (a) with the Hgand prepared in the substantiaHy same manner as the one used in the step (a), in the presence of a test compound, and

(c) comparing the results of the step (a) and the step (b) to determine whether the binding of the Hgand is affected by the presence of the test compound. In this screening method of the binding assay, a detectable label can be bound to the Hgand, and the step (c) can comprise the step of comparing the detected amount of label in the step (a) and the detected amoimt of label in the step (b). In this screening method of the binding assay, the Hgand can be selected from zaprinast, an analogue thereof, or a mimetic thereof. Another aspect of the present invention is a screening method for agonists or antagonists of a GPR35 protein by functional assay. In the screening method for agonists or antagonists of a GPR35 protein by functional assay, the functional response to be observed can be, for example, the increase of intraceHular calcium concentration. One aspect of agonist screening by functional assay is; a method of screening for a compound that is an agonist of a GPR35 protein, comprising the steps o£

(a) adding a test compound to ceUs expressing a GPR35 protein or a partial polypeptide thereof or to a membrane fraction from the ceUs, and

(b) determining whether a functional response is observed. One aspect of antagonist screening by functional assay is,' a method of screening for a compound that is an antagonist of GPR35 protein, comprising the steps ofi

(a) adding an agonist to cells ejφressing GPR35 protein or a partial polypeptide thereof or to a membrane fraction from the cells, (b) adding an agonist prepared in the substantiaHy same manner as the one used in the step (a) and a test compound to the ceUs or the membrane prepared in the substantiaHy same manner as the one used in the step (a), and

(c) comparing a functional response in the step (a) and one in the step (b) to determine whether the functional response is reduced by the test compound. In this screening method of the binding assay, the agonist can be selected from zaprinast, an analogue thereof, or a mimetic thereof. Another aspect of the present invention is use of zaprinast, an analogue thereof, or a mimetic thereof as a modulator of a GPR35 protein. Another aspect of the present invention is an isolated and/or purified polypeptide of one of the foHowing,

(a) a polypeptide having the amino acid sequence of sequence ID. 2,

(b) a polypeptide having the same amino acid sequence and the same kind of activity as the polypeptide of (a) except that one or more amino acids are deleted, substituted, or added,

(c) a polypeptide having an amino acid sequence that has at least 86% identity to the polypeptide of (a),

(d) a polypeptide having an amino acid sequence that has at least 90% identity to the polypeptide of (a), and

(e) a polypeptide having an amino acid sequence that has at least 98% identity to the polypeptide of (a). Another aspect of the present invention is a transformed ceUs that express the above mentioned polypeptide and an antibody immunospecific for the above mentioned polypeptide. Another aspect of the present invention is an isolated and/or purified polynucleotide that encodes the above mentioned polypeptide. Another aspect of the present invention is a an isolated and/or purified polynucleotide of one of the foHowing,

(a) apolynucleoti.de having the nucleotide sequence of sequence ID. 1,

(b) a polynucleotide that is capable of hybridizing under stringent conditions to a polynucleodide having a nucleotide sequence complementary to the nucleotide sequence of the polynucleotide of (a) and that encodes a polypeptide having the same kind of activity as the polypeptide encoded by the polynucleotide of (a),

(c) a polynucleotide having a nucleotide sequence that has at least 90% identity to the polynucleotide of (a),

(d) a polynucleotide having a nucleotide sequence that has at least 95% identity to the polynucleotide of (a),

(e) a polynucleotide having a nucleotide sequence that has at least 98% identity to the polynucleotide of (a), and

(0 a polynucleotide that encodes any of polypeptides (A) " (E) below; (A) a polypeptide having the amino acid sequence of sequence ID. 2, (B) a polypeptide having the same amino acid sequence and the same kind of activity as the polypeptide of (A) except that one or more amino acids are deleted, substituted, or added,

(E) a polypeptide having an amino acid sequence that has at least 98% identity to the polypeptide of (A). Another aspect of the present invention is an expression vector containing the above mentioned polynucleotide. Another aspect of the present invention is transformed cells that stably express any of foHowing polypeptides;

(a) a polypeptide having the amino acid sequence of sequence ID. 2, 4, or 6,

(b) a polypeptide having the same amino acid sequence and the same ?kind of activity as the polypeptide of (a) except that one or more amino acids are deleted, substituted, or added, (c) a polypeptide having an amino acid sequence that has at least 80% identity to the polypeptide of (a),

(e) a polypeptide having an amino acid sequence that has at least 98% identity to the polypeptide of (a). Another aspect of the present is a novel compounds that can be identified by any of the above mentioned screening method. Another aspect of the present is a medicament for use to modulate GPR35 activity comprising a compound that can be identified by any of the above mentioned screening method. Non-Hmiting examples of the modulation of GPR35 activity include modulation of neural activity and modulation of digestive system. In particular, such a compound can be selected from the group consisting of the foHowing compounds,'

2-methyl-5-phenyl-pyrazolo[l,5-a]pyrimidin-7(4H)-one,

3-(4,7-(Hhy(fro-l-methyl-7-oxo-3-propyl-lH-p5τaz»lo[4,3-d]pyriι (Hn-5-y])-4-ethoxy-benzenep ropanoic acid,

3-[3-(4,5-(Hhy(fro-3-methyl-4-oxo-l-propyl-lH-pyrazolo[3,4-d]pyrimidin-6-y?D-4-propoxypheny

]]-2-propenoic acid,

2,4-dihy<fro-2-methyl-5-[2-(2-methylpropoxy)-5-(lH-tetiazol-5-y?D-3-pyridinyl]-3-propyl-7H-P yrazolo[4,3-cUpyrimidin-7-one, 2,4-dihydro-2-methyl-5-[2-(2-methylpropo^)-5-(lH-l,2,3-triazol-4-y?D-3-pyridiny?l]-3-propyl-7

H-?Pyrazolo[4, 3-d]pyrimidin-7-one,

5-(2-ethoxyphenyB-l,4-dihy<fro-7H-l,2,3-Triazolo[4,5-d]pyrimidin-7-one,

3-(4,7-(Hhy< o-7-oxo-lH-l,2,3-triaz»lo[4,5-cUpyώ cliloride, 3-(4,7-ch ιycfro-7-oxo-lH-l,2,3-iria2»lo[4,5-(^

5-Nitro-2-(3-phenylpropylamino)benzoic acid,

2-Cyano-4-hydroxyindole,

2-(2-Propoxypheny?0-8-trifluoromethylpxmn-6-one, and

6-phenyl-l-(phenyHnethy?0-lH-Bis[l,2,3]tiia2»lo[l,5-a:4',5'-e]pyrinήdin-4(5H)-one. Another aspect of the present invention is use of a compound that is identifiable by any of the above mentioned screening method to modulate GPR35 activity. Non-Hmiting examples of the modulation of GPR35 activity include modulation of neural activity and modulation of digestive system. Another aspect of the present invention is a method of modulating GPR35 activity by administering a compound that can be identified by any of the above mentioned screening method. Non-Hmiting examples of the modulation of GPR35 activity include modulation of neural activity and modulation of digestive system.

Definitions The terms "homologue" as used herein with regard to polypeptide refers to a polypeptide having a certain homology with a reference polypeptide. Such a homologue can have a homology of preferably, 80% or 85%, more preferably, 90% or 95%, even more preferably 98%. Such a homologue includes a polypeptide having otherwise the same amino acid sequence as a reference polypeptide except that one or more (preferably, several) amino acids are added/substituted/deleted. Such a homologue includes a fragment of a polypeptide. The terms "homologue" as used herein with regard to polynucleotides refers to a polynucleotide having a certain homology with a reference polynucleotide. Such a homologue can have a homology of preferably, 80% or 85%, more preferably, 90% or 95%, even more preferably 98%. Such a homologue includes a polynucleotide that encodes a homologue of a polypeptide that a reference polynucleotide encodes. Such a homologue also includes a polypeptide that is complementary to a polynucleotide capable of hybridizing under stringent conditions (e.g. 65°C and O.lxSSC {lxSSC=0.15M NaCl, 0.015M Na₃ citrate pH7.0}) to a reference polynucleotide. Such a homologue also includes a polynucleotide fragment or partial polypeptide. Such sequence homology (identity) can be easily assessed by pubHcly or commerciaUy avaHable bioinformatics software, such as Blast2 (Altschul, S.F. et al (1997) Nucl. Acids Res.25, 3389-3402), or programs included in the GCG software package (Devereux et al (1984) Nucl. Acids Res. 12, 387,' Wisconsin Package Version 10, Genetics Computer Group (GCG, Madison, Wisconsin), such as Bestfit or Gap. The term "analogue" as used herein relates to any substance which is similar in structure to a reference agent (e.g. Zaprinast). As used herein, the term "mimetic" relates to any substance which has the same Irind of activity or effect as a reference agent (e.g.Zaprinast). The term "modulator of a receptor" as used herein refers to any substance which has an direct or indirect effect on the receptor. The term "agonist of a receptor" refers to a substance which can stimiilate the receptor. The term "antagonist of a receptor" refers to a substance which can inhibit the stimulation of the receptor by an agonist. The term "functional response" as used herein refers to the reaction that, for example, stimulation of a receptor leads to in ceUs. In the case of G-protein coupled receptors, this can include, for example, a change in the concentration of cychc AMP, a transient rise in intraceHular calcium concentration, or an opening of an ion channel. The term "compound" as used herein refers to any chemical entity, including but not limited to a smaH organic molecule, a peptide, a protein, a modified protein such as a glycoprotein or a Hpoprotein, antibodies or fragments thereof, a nucleic acid such as DNA or RNA or modified nucleic acids, such as oHgonucleotides with a modified backbone. The term "isolated and/or purified" polynucleotide/polypeptide as used herein refers to any polynucleotide/polypeptide that is not in a natural condition. Therefore, "isolated and/or purified" polynucleotide includes the case in which only a promoter or an enhancer of an endogenous polynucleotide is modified keeping the polynucleotide intact.

Sequence listing Sequence IDs. 1 and 2 respectively show a cDNA nucleotide sequence and an amino acid sequence of rat GPR35. Sequence IDs. 3 and 4 respectively show a cDNA nucleotide sequence and an amino acid sequence of human GPR35. Sequence IDs. 5 and 6 respectively show a cDNA nucleotide sequence and an amino acid sequence of rat GPR35. Sequence IDs. 7-23 show nucleotide sequences of primers used in examples.

DETAILED DESCRIPTION OF THE INVENTION The present invention is described in further detail below.

Screening method Gener aHy, the screening method of the present invention is! - a method of screening for a compound that modulates a GPR35 protein, comprising the step o£ (a) contacting a GPR35 protein or a partial polypeptide thereof with a test compound. The screening method can be, but is not limited to, a screening method by binding assay or functional assay. Typical but non-Hmiting example of a modulating compound is an agonist or an antagonist. The GPR35 protein or the partial polypeptide thereof used in the screening method of the present invention can exist in ceHs or a membrane fraction. Thus, a sample of GPR35 may comprise (l) transformed cells prepared as described below, or (2) cells naturaUy expressing GPR35, or (3) membrane preparations prepared from any ceUs expressing GPR35, or (4) GPR35 protein enriched or purified from such ceUs or membranes. The sldHed person will be weH aware of methods that can be used to enrich or purify GPR35, which include affinity chromatography, size exclusion chromatography, ion exchange chromatography and other methods suitable for the separation of protein from complex mixtures. Tbst compounds that are employed in the screening method of the present invention include, for example, peptides, proteins, non-peptide compounds, synthetic compounds, microbial fermentation products, marine organism extracts, plant extracts, ceH extracts, and animal tissue extracts. These test compounds may be either novel or known. Compounds that may be used for screening include, but are not limited to, peptides such as, for example, soluble peptides, including but not limited to members of random peptide Hbraries; (see, e.g., Lam et al. (1991) Nature 354, 82-84; Houghten et al. (1991) Nature 354, 84-86) , and combinatorial chemistry-derived molecular Hbrary made of D- and/or L-configuration amino acids, phosphopeptides (including, but not Hmited to, members of random or partiaHy degenerate, directed phosphopeptide Hbraries! see, e.g., Songyang et al. (1993) CeU 72, 767-778), antibodies (including, but not Hmited to, polyclonal, monoclonal, humanized, anti-idiotypic, chimerie or single chain antibodies, and Fab, F(ab')2 and Fab expression Hbrary fragments, and epitope-binding fragments thereof), and smaH organic or inorganic molecules. Peptide Hbraries may be used as a source of test compounds that can be used to screen in the methods of the invention. Diversity Hbraries, such as random or combinatorial peptide or nonpeptide Hbraries can be screened for molecules that specificaHy bind to a GPR35 protein. Many Hbraries are known in the art that can be used, e.g., chemicaUy synthesized Hbraries, recombinant (e.g. phage display Hbraries), and in vitro translation-based Hbraries. Examples of chemicaUy synthesized Hbraries are described in Fodor et al. (1991)

Science 251, 767-773; Houghten et al. (1991), Nature 354, 84-86; Lam et al. (1991), Nature 354, 82-84; Medynski (1994) Bio/Technology 12, 709-710; GaUop et al. (1994), J. Medicinal Chemistry 37, 1233-1251; Ohlmeyer et al. (1993), Proc. Natl Acad. Sci. USA 90, 10922-10926; Erb et al. (1994), Proc. Natl. Acad. Sci. USA 91, 11422-11426; Houghten et al. (1992) Biotechniques 13, 412; Jayawickreme et al. (1994), Proc. Natl. Acad. Sci. USA 91, 1614-1618; Salmon et al. (1993) Proc. Natl. Acad. Sci. USA 90, 11708-11712; PCTPubHcation No. WO 93/20242; and Brenner and Lerner (1992) Proc. Nati. Acad. Sci. USA 89, 5381-5383. Examples of phage display Hbraries are described in Scott & Smith (1990) Science 249'-386-390; Devlin et al. (1990) Science 249, 404-406; Christian, et al. (1992), J. Mol. Biol. 227, 711-718; Lenstra (1992) J. Immunol. Meth. 152, 149-157; Kay et al. (1993) Gene 128, 59-65; and PCT PubHcation No. WO 94/18318 dated August 18, 1994. By way of example of nonpeptide Hbraries, a benzodiazepine Hbrary (see e.g. Birnin et al. (1994), Proc. Natl. Acad. Sci. USA 91, 4708-4712) can be adapted for use. Peptoid Hbraries (Simon et al. (1992) Proc. Natl. Acad. Sci. USA 89, 9367-9371) can also be used. Another example of a Hbrary that can be used, in which the amide functionaHties in peptides have been permethylated to generate a chemicaUy transformed combinatorial Hbrary, is described by Ostresh et al. (1994) Proc. Natl. Acad. Sci. USA 91, 11138-11142). Screening the Hbraries can be accompHshed by any of a variety of commonly known methods. See, e.g., the foHowing references, which disclose screening of peptide Hbraries: Parmley & Smith (1989) Adv. Exp. Med. Biol. 251, 215-218; Scott & Smith (1990) Science 249, 386-390; Fowlkes et al. (1992) BioTechniques 13, 422-427; Oldenburg et al. (1992) Proc. Nati. Acad. Sci. USA 89, 5393-5397; Yu et al. (1994) CeU 76, 933- 945; Staudt et al. (1988) Science 241, 577-580; Bock et al. (1992) Nature 355, 564-566,^' Tuerk et al. (1992) Proc. Natl. Acad. Sci. USA 89, 6988- 6992; Ellington et al. (1992) Nature 355, 850-852; U.S. Patent No. 5,096, 815, US. Patent No. 5,223,409, and U.S. Patent No. 5, 198,346, aU to Ladner et al.; Itebar & Pabo (1993) Science 263, 671-673; and PCT PubHcation No. WO 94/18318. Compounds that can be tested and identified using the methods described herein can include, but are not Hmited to, compounds obtained from any commercial source, including Aldrich (1001 West St. Paul Ave., Milwaukee, WI 53233), Sigma Chemical (P.O. Box 14508, St. Louis, MO 63178), Fluka Chemie AG (Industriestrasse 25, CH- 9471 Buchs, Switzerland (Fluka Chemical Corp. 980 South 2nd Street, Ronkonkoma, NY 11779)), Eastman Chemical Company, Fine Chemicals (P.O Box 431, Ifingsport, TN 37662), Boehringer Mannheim GmbH (Sandhofer Strasse 116, D-68298 Mannheim), Takasago (4 Volvo Drive, Rockleigh, NJ 07647), SST Corporation (635 Brighton Road, Clifton, NJ 07012), Ferro (ill West Irene Road, Zachary, LA 70791), Riedel- deHaen AktiengeseUschaft (P.O. Box D-30918, Seelze, Germany), PPG Industries Inc., Fine Chemicals (One PPG Place, 34th Floor, Pittsburgh, PA 15272). Further any lrind of natural products may be screened using the methods of the invention, including microbial, fungal, plant or animal extracts. Furthermore, diversity Hbraries of test compounds, including small molecule test compounds, may be utiHzed. For example, Hbraries may be commercially obtained from Specs and BioSpecs B.V. (Rijswijk, The Netherlands), Chembridge Corporation (San Diego, CA), Contract Service Company (Dolgoprudny, Moscow Region, Russia), ComgenexUSA Inc. (Princeton, NJ), Maybridge Chemicals Ltd. (CornwaUPL34 OHW, United Kingdom), and Asinex (Moscow, Russia). Still furthermore, combinatorial Hbrary methods ?known in the art, can be utilized, including, but not Hmited to: biological Hbraries; spatiaHy addressable paraUel soHd phase or solution phase Hbraries,' synthetic Hbrary methods requiring deconvolution; the "one-bead one-compound" Hbrary method; and synthetic Hbrary methods using affinity chromatography selection. The biological Hbrary approach is Hmited to peptide Hbraries, while the other four approaches are appHcable to peptide, non-peptide oHgomer or smaU molecule Hbraries of compounds (Lam (1997) Anticancer Drug Des.12, 145). Combinatorial Hbraries of test compounds, including smaH molecule test compounds, can be utiHzed, and may, for example, be generated as disclosed in Eichler & Houghten (1995) Mol. Med. Today 1, 174-180; DoUe (1997) Mol. Divers. 2, 223-236; and Lam (1997) Anticancer Drug Des. 12, 145-167. Examples of methods for the synthesis of molecular Hbraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90, 6909; Erb et al. (1994)

Proc. Natl. Acad. Sci. USA 91, 11422; Zuckermann et al. (1994) J. Med. Chem. 37, 2678; Cho et al. (1993) Science 261, 1303; CarreU et al. (1994) Angew. Chem. Int. Ed. Engl. 33, 2059;

CareU et al. (1994) Angew. Chem. Int. Ed. Engl. 33, 2061; and GaHop et al. (1994) J. Med. Chem. 37, 1233. Libraries of compounds may be presented in solution (e.g. Houghten (1992)

Bio/Techniques 13, 412-421), or on beads (Lam (1991) Nature 354:82-84), chips CFodor (1993)

Nature 364, 555-556), bacteria (U.S. Patent No. 5,223,409), spores (Patent Nos. US 5,571,698;

US 5,403,484; and US 5,223,409), plasmids (CuU et al. (1992) Proc. Natl. Acad. Sci. USA 89, 1865- 1869) or phage (Scott and Smith (1990) Science 249, 386-390; Devlin (1990) Science 249,

404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87, 6378-6382; and FeHci (1991) J.

Mol. Biol. 222, 301-310). Screening the Hbraries can be accomphshed by any of a variety of commonly known methods. See for example the foUowing references, which disclose screening of peptide Hbraries: Parmley & Smith (1989) Adv. Exp. Med. Biol. 251, 215-218; Scott & Smith (1990)

Science 249, 386-390; Fowlkes et al. (1992) BioTechniques 13, 422- 427; Oldenburg et al.

(1992) Proc. Natl. Acad. Sci. USA 89, 5393-5397; Yu et al. (1994) CeU 76, 933-945; Staudt et al.

(1988) Science 241, 577-580; Bock et al. (1992) Nature 355, 564-566; Tuerk et al. (1992) Proc.

Natl. Acad. Sci. USA 89, 6988-6992; Ellington et al. (1992) Nature 355, 850-852; U.S. Patent No. 5,096,815, U.S. Patent No. 5,223,409, and U.S. Patent No. 5, 198,346, aU to Ladner et al.,

Rebar & Pabo (1993) Science 263, 671- 673; and PCT PubHcation No. WO 94/18318. Another aspect of the present invention is; a method of screening for a compound that inhibits the binding of a Hgand to a GPR35 protein, comprising the steps o£ (a) contacting a GPR35 protein or a partial polypeptide thereof with the Hgand,

(b) contacting a GPR35 protein or a partial polypeptide thereof prepared in the substantiaHy same manner as the one used in the step (a) with the Hgand prepared in the substantiaHy same manner as the one used in the step (a), in the presence of a test compound, and

(c) comparing the results of the step (a) and the step (b) to determine whether the binding of the Hgand is affected by the presence of the test compound. In this screening method of the binding assay, a detectable label can be bound to the Hgand, and the step (c) can comprise the step of comparing the detected amount of label in the step (a) and the detected amount of label in the step (b). For example, Hgands labeled with ¹²⁵I, ³⁵S, ³H, ¹⁴C, Hgands labeled with fluorescent compounds such as fluorescein, or Hgands labeled with enzymes such as horseradish peroxidase can be used as a labeled Hgand. Alternatively, these labeled Hgands can be prepared according to perse known methods. Another aspect of the screening method of the present invention is a screening method for agonists or antagonists of GPR35 by functional assay. One aspect of the agonist screening of the present invention is; a method of screening for a compound that is an agonist of a GPR35 protein, comprising the steps o£

(b) determining whether a functional response is observed. One aspect of the antagonist screening of the present invention is; a method of screening for a compound that is an antagonist of GPR35 protein, comprising the steps ofi

(a) adding an agonist to ceUs expressing GPR35 protein or a partial polypeptide thereof or to a membrane fraction from the ceUs, (b) adding an agonist prepared in the substantially same manner as the one used in the step

(a) and a test compound to the cells or the membrane prepared in the substantiaHy same manner as the one used in the step (a), and

(c) comparing a functional response in the step (a) and one in the step (b) to determining whether the functional response is reduced by the test compound. With regard to the agonist/antagonist screening method described above, in a preferred aspect of the invention, the functional response can be a transient rise in intraceUular calcium concentration, measured by using fluorescent dyes such as ?Fluo-3 or Indo-1 or by other means ?known to the person skiUed in the art; in another preferred aspect of the invention, the functional response is an increase in the rate of respiration of the cells as measured by an increase in the rate of acidification of the medium surroiuiding the ceUs as measured by microphysiometry. In yet another preferred aspect of the invention, the functional response is an increase or decrease in the cycHc AMP concentration or increase in the calcium concentration in the ceUs, as measured e.g. by increased activity of a reporter gene product wherein the coding region of the reporter gene is functionaUy linked to a promoter comprising at least one cycHc AMP response element or one element that can respond to the signal transmission that is associated with the increase of the intraceHular calcium concentration. Other methods to measure changes in cycHc AMP concentration or calcium concentration in ceUs are weH ?known to the person slriUed in the art. In a further preferred aspect of the invention, cells are transfected with a plasmid or plasmids leading to co-expression of GPR35 and GFP-β-arrestin complex, and an agonist of the receptor is identified by observing clustering of fluorescence, i.e. GFP-β-arrestin complex, on the ceH surface. Suitable ceUs expressing GPR35 can be, for example, selected from ceUs naturaUy expressing GPR35, ceUs where the expression of GPR35 has been upregulated, or cells that has been transfected to express GPR35.

Transfection The transfectants of the present invention can be prepared by the foHowing non-Hmiting methods. AcDNAor genomic DNA that encodes the GPR35 protein can be used to construct an expression vector that comprises a DNA encoding the GPR 35 protein. A DNA that encodes a partial polypeptide having the substantiaHy same lrind of Hgand-binding activity as the GPR35 protein can also be used. A cDNA or genomic DNA that encodes a known or novel GPR35 protein can be used as weU as a synthetic DNA. SpecificaHy for example, a DNA that encodes the GPR35 protein having the amino acid sequence represented by SEQ ID No. 2, 4, or 6 can be used. An example of such a DNA is a DNA having a base sequence represented by SEQ ID No.l, 3, or 5. These DNAs can also be prepared by lαiown gene engineering techniques. As an expression vector, for example, pcDNA3.1, pAKKO-111, pAKKO-lllH, pXTl, pRC/CMV pRC RSV etc. can be used. As a promoter, any one which functions efficiently in a host ceU can be used, for example, SV40 promoter, CMV promoter, HSVTK promoter, SRα promoter, RSV promoter, etc. can be used. In addition, it is preferable to use the expression vector further containing an enhancer, spHcing signal, poly A addition signal, selective marker, etc. The selective marker includes dihydrofolate reductase genes, neomycin resistance genes (G418 resistance), and the Hke. The preferred expression vector holding a DNA encoding the GPR35 protein of the present invention is a vector that is inserted with an above-mentioned promoters (in particular, SRα promoter, CMV promoter, RSV promoter, etc.) upstream of the DNA encoding the GPR35 protein, a poly A addition signal downstream of the DNA, a selective marker such as neomycin resistance gene or DHFR gene downstream of the poly A addition signal, and an ampiciHin resistance gene downstream of the selective marker. More specifically, an expression vector that comprises a CMV promoter upstream of a DNA that encodes human, rat or mouse GPR35 protein, a poly A addition signal downstream of the DNA, a neomycin resistance gene downstream of the poly A addition signal, and an ampiciHin resistance gene downstream of the selective marker is preferable. By the introduction of thus obtained expression vector holding a DNA encoding the GPR35 protein to a host ceU, a ceH that highly expresses the GPR35 can be obtained. As to host cells, human embryonic kidney 293 (HEK293) ceUs (Exp Physiol, 75(3): 309-319 (1990)) and CHO cells (J. Exp. Med., 108: 945 (1995)) are preferred. The HEK293 ceUs and CHO ceUs can not only highly express the GPR35 protein but also remarkably stably express it. Preferable combination of an expression vector and host cells can be optimaUy chosen. For example, the combination of the expression vector represented by hGPR35/pcDNA3.1 and the CHO ceUs is preferable. For the introduction of an expression vector to host ceUs, any ?known method such as calcium phosphate txansfection ("Virology, 52: 456-467 (1973)), electroporation (EMBO J., V- 841-845 (1982)), commerciaUy available transfection reagents (for example, FuGE?NE6 Transfection Reagent by Roche), and the Hke can be used. The ceUs highly expressing the human GPR35 receptor protein may be prepared by first selecting a transformant from the cells transfected with the above-mentioned expression vector using a selective marker as an indicator, foUowed by clonal selection. When a neomycin resistant gene is used, the resistant ceUs may be selected by continuous incubation with addition of G418 to multiply the transgene in the ceUs, thereby yielding much more expressing ceUs. In the incubation of the ceUs (host ceUs and transfected ceUs (regardless of transient or stable expression)) used in the present invention, 0.5-20% fetal calf serum EMEM, DMEM, RPMI1640, MEM-o , and the like may be used. Particularly, when an expression vector containing a neomycin gene as a selective marker is used, it is preferable to use a D?MEM medium containing G418/geneticin neomycin and fetal calf serum. The preferred pH is at approximately 6 to 8. The incubation is usuaHy carried out at about 30-40 °C for a period of about 15 to 200 hours, if required with aeration, agitation, and exchange of the culture medium. Despite considerable difficulties, the Inventors finaUy constructed animal ceUs that continuously and stably express GPR35 after aU their commitment and effort. Such ceUs are indispensable research tool for high through put and large scale screening (for example, HTS). Preparation of such a transfectant is explained in detail in ?EXAMP?LES.

Antibody The present invention further provides antibodies immunospecific for the rat GPR35 protein. The antibodies may be obtained by administering the polypeptides or epitope-bearing fragments, analogs or cells to an animal, preferably a non-human animal, according to known techniques. For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous ceU line cultures can be used. Examples include the hybridoma technique (Kohler, G. and IVHlstein, G, Nature (1975) 256'495-497), the trioma technique, the human B-cell hybridoma technique (Kozboret al., Immunology Today (1983) 4.72) and the EBVhybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy 77-96, Alan R. Liss, Inc., 1985). Tbchniques for the production of single chain antibodies, such as those described in U.S. Pat. No. 4,946,778, can also be adapted to produce single chain antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms, including other mammals, may be used to express humanized antibodies. The above-described antibodies may be employed to isolate or to identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography. Antibodies against polypeptides of the present invention may also be employed to treat the diseases, amongst others.

Medical Indication Compounds of the present invention (namely, compounds that modulated GPR35 protein (GPR modulator)) can be used to modulate GPR35 protein and hence modulate neurotransmission in the nervous systems (or spinal cord) so as to treat a variety of neurological disorders (for example, CNS disorders). Non limiting examples of such conditions are pain, anxiety, convulsions, cognition disorders, obesity, schizophrenia, neurodegeneration, depression, attention deficit hyperactivity disorder, mania, memory deficit, eating disorders, Parkinson's disease, Huntington's disease, Alzheimer's disease, amyotropliic lateral sclerosis, drug addiction, bipolar disorders, circadian rhythm disorders, migraine, sexual dysfunctions, sleep disorders and eating disorders, urinary diseases, etc. Further, compounds of the present invention (namely, compounds that modulated GPR35 protein (GPR modulator)) can be used to modulate GPR35 protein and hence modulate digestive system so as to treat a variety of disease conditions. Non limiting examples of such conditions that can be treated by the agonism of GPR35 are functional dyspepsia, Irritable Bowel Syndrome (IBS), diarrhea, eating disorders, emesis. Non limiting examples of such conditions that can be treated by the antagonism of GPR35 are gastro-esophageal reflux disease, Barrett's esophagus, esophageal achalasia, functional dyspepsia, gastroparesis, postoperative Ueus, Irritable Bowel Syndrome (IBS), constipation, eating disorders, non-cardiac chest pain (NCCP), bronchial asthma (BA), bronchitis, and chronic cough (for BA, bronchitis, and chronic cough, those caused by reflux of stomach content are particularly assumed). Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in ceU cultures or experimental animals, e.g. for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeuticaUy effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a dehvery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects. The data obtained from the ceU culture assays and animal studies can be used in formulating a range of dosages for use in humans. The dosage of such compounds Hes preferably within a range of circulating concentrations that include the ED 50 with Httle or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utiHzed. For any compound used in the method of the invention, the therapeuticaUy effective dose can be estimated initially from ceH culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC5θ(i. e. the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in ceU culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance Hquid chromatography. Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologicaUy acceptable carriers or excipients. Thus, the compounds and their physiologicaUy acceptable salts and solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration. For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceuticaUy acceptable excipients such as binding agents (e.g. pregelatinised maize starch, polyvinylpyrroHdone or hydroxypropyl methylceUulose); fillers (e.g. lactose, microcrystalline ceUulose or calcium hydrogen phosphate); lubricants (e.g. magnesium stearate, talc or siHca); disintegrants (e.g. potato starch or sodium starch glycolate); or wetting agents (e.g. sodium lauryl sulphate). The tablets may be coated by methods weH ?known in the art. liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such Hquid preparations may be prepared by conventional means with pharmaceuticaUy acceptable additives such as suspending agents (e.g. sorbitol syrup, ceUulose derivatives or hydrogenated edible fats); emulsifying agents (e.g. lecithin or acacia); non-aqueous vehicles (e.g. almond on, oUy esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g. methyl or propybp- hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated to give controUed release of the active compound. For buccal administration the compositions may take the form of tablets or lozenges formulated in conventional manner. For administration by inhalation, the compounds for use according to the present invention are conveniently deHvered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propeHant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dic?hlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deHver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The compounds may be formulated for parenteral administration by injection, e. g. by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g. in ampules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in ofly or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g. sterile pyrogen-free water, before use. The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g. containing conventional suppository bases such as cocoa butter or other glycerides. In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foU, such as a bHster pack. The pack or dispenser device may be accompanied by instructions for adniinistration. EXAMPLES The present invention is explained more specificaHy by examples below, which do not limit the scope of the invention.

Example l: Original Cloning of human GPR35 A fuU-length cDNA of human GPR35 was isolated from human Standard ?Mjχed

DRG cDNA Hbrary (LIFE TECHNOLOGIES; 11649-019, Lot No: 80160b) based upon the ?known sequence of human GPR35 protein using very weU-?known technology. Compared with the pubHshed sequence of GPR35 (accession number,' AF027957), the clone had three missense mutations (T108M, R174A, and R294S) that seemed to be S?NPs (the substitutions were registered in databases independently. See also Nat. Genet., 26:163-175 (2000)). The nucleotide sequence of the clone is shown in sequence ID. 3 and the amino acid sequence of the polypeptide encoded by the clone is shown in sequence ID. 4. This was used as template DNA for foHowing experiments to construct expression vectors for human GPR35.

Example % Construction of an Expression Vector of ?FTAG-tagged human GPR35 A cDNA of FLAG-tagged human GPR35 was generated by PCR using two primers of sequence ID. 7 and sequence ID. 8. The former primer contains EcoRI site and the latter primer Xbal site. The PCR was performed using Advantage-HF2 PCR Kit

(CLONTECH; K1914-1) with the foHowing program: 94 °C for 4min,' 30 cycles of 94 °C for 20 sec, 50 °C for 30 sec, 72 °C for 90secJ 72 °C for 4 min. The PCR product was purified by High Pure™ PCR Product Purification Kit (Roche 1732 676). The purified PCR product was digested with 20 U of Eco I (NEB; R0101S) and 20 U of Xbal (NEB; R0145S) in lxSuRE Cut Buffer H (Roche) at 37 °C for 2.5 hr. A mammalian expression vector, pcDNA3.l(+) (Invitrogen; V790-20) was also digested in the same manner. The samples were electrophoresed with 1% agarose gel, and desired fragments were gel-purified using DNA PREP (IATRON; AGC-001K). Digested pcDNA3.l(+) was treated with "Alkaline Phosphatase, shrimp" (Roche,- 1 758 250 ) at 37 °C for 1 hr. The enzyme was then inactivated at 65 °C for lδmin. The treated pcDNA3.l(+) and human FLAG-GPR35 fragments were Hgated using Rapid DNA Iigation Kit (Roche,' 1 635 379). Then, One Shot TOP10 Chemically Competent E. coH (C4040-03) was transformed using 4 μl of the reaction mixture. About 200 transformants were obtained, and 16 colonies were picked up to check by PCR using universal primers, T7 and BGH reverse. Four clones were checked by DNA sequencing. The DNA sequencing was performed by TOYOBO Gene Analysis using T7 and BGH reverse primer, and the data were analyzed by GE?NETYX-WIN version 4. Finally, two of the properly constructed clones were obtained and used for further experiments. The plasmid map of this vector is shown in Figure 1.

Example 3: Construction of an Expression Vector of Native human and mouse GPR35 A pcDNA3.1 (+) expression vector containing a cDNA of native (namely, not-tagged) human GPR35 was constructed by the substantiaHy same method as those described above. Primers used for PCR were the primer of sequence ID. 9 (5' primer) and the primer of sequence ID. 8 (3' primer). The former primer contains EcόRI site and the latter primer Xbal site. The plasmid map of the vector is shown in Figure 2. SimUarly, a pcDNA3.1 (+) expression vector containing a cDNA of native mouse GPR35 was constructed. Primers used for PCR were the primer of sequence ID. 10 and the primer of sequence ID. 11. Mouse bone marrow total RNA was used for cloning.

?Eχaτnρle 4: Cloning of rat GPR35 and Sequencing thereof Tb identify and clone rat GPR35 that has not been pubHshed, RTPCR experiments were performed, lb generate first-strand cDNA templates for RTPCR, 5 μg of Rat Colon Total RNA (CLONTECH; #64066-1) was reverse-transcribed by SUPERSCRIPT Preamplification Systems (GIBCO BRL/Invitrogen) according to the attached protocol. Tb amplify rat GPR35 by RTPCR, a 5' primer of Sequence ID. 12 and a 3' primer of Sequence ID. 13 was used. These primers were designed according to nucleotide sequences of mouse GPR35 (accession number; BC027429). Mixtures for PCR were prepared using LA Taq with GC Buffer II (TaKaRa,^' RR02AG) according to 'general reaction mixture for PCR' described in the attached protocol. PCR was performed under the foHowing condition: 94 °C for 4min; 35 cycles of 94 °C for 20 sec, 50 °C for 30 sec, 72 °C for 1 min,' 72 °C for 4 min. After PCR, 10 μl of the samples were separated by electrophoresis. Although non-specific amplifications were also observed, PCR product with expected size was obtained. The fragment with expected size was gel-purified using DNA Prep (IATRON; AGC-OOHO and TA-cloned using TOPO TA Cloning Kit (InvitrogenJ K4500-01). Plasmids from the several independent dones were purified using QIAprep Spin ?Miniprep ?Kit (QIAGEN; #27106), and they were sequenced by TOYOBO Gene Analysis using T7 primer and M13 reverse primer. Determined sequences were analyzed by GE?NETYX-SV R version 5.2 (GENETYX) and B?LAST Search. The clones (plasmids) contained 921-bp ORE The nucleotide sequence of the clone is shown in sequence ID. 1 and the amino add sequence of the polypeptide encoded by the clone is shown in sequence ID. 2. Analysis by BLAST Search revealed that it encodes polypeptide that is most simUar to the mouse and human GPR35. Homology of rat GPR35 was 72% to human GPR35 and 85% to mouse GPR35 in amino add sequence. Homology of rat GPR35 was 78% to human GPR35 and 89% to mouse GPR35 in nucleic add sequence. Therefore, we concluded that the 921-bp ORF was rat GPR35 gene, lb determine error-free rat GPR35 gene, several independent experiments were performed.

Example 5: Construction of an Expression Vector of rat GPR35 To prepare insert for mammaHan expression vector for rat GPR35, PCR was performed using Advantage HF2 PCR Kit (CLONTECH; K1909-1). TA-cloned error-free rat GPR35 was used as a template for the PCR. Two primers used for the PCR were 5' primer of Sequence ID. 14 and 3' primer of Sequence ID. 15. Conditions for the PCR were as foUows: 94 °C for 4 min,^' 30 cydes of 94 °C for 20 sec, 50 °C for 30 sec, 72°C for 1 min; 72°C for 4 min. The fragments in the PCR products were separated by electrophoresis and extracted by DNA Prep. The gel-purified PCR fragments were digested by 20 U of EcoΕH and 20 U of Xhol in lxSuRECut Buffer H (Roche), and Hgated into EcoRl-Xhol sites of pcDNA3.l(+). Several single clones were checked by DNA sequencing (TOYOBO Gene Analysis). For large-scale preparation of the plasmid, EndoFree Plasmid Maxi Kit (QIAGEN; 12362) was used.

Example 6: Transient Transfection HEK293 ceUs were maintained in D?MEM (Invitrogen; 11965-084) supplemented with 10% FBS and penicillin/streptomycin. Expression vectors of FLAG-tagged human GPR35 (FLAG-hGPR35), native human GPR35, and rat GPR35 were prepared as described above. These expression vectors as weU as expression vectors (cloned in pcDNA3 (Invitrogen)) of chimerie G proteins (Gqs5, Gqi5 and Gqo5) and Gαl6. were used for transfection of HEK293 ceUs. Transfection of HEK293 ceUs was performed using FuGElNTE 6 Transfection Reagent (Roche,^' 814443) according to the attached protocol.

Example T- Stable Transfection CommerciaHy available CHO-Gαl5-?NFAT βTactamase ceUs were maintained in DMEM (Invitrogen 11995-065) supplemented with 10% FBS (Biowhittaker 14-502F), 0.1 mM NEAA (Invitrogen 11140-050), 1.0 mM Na-pyruvate (Invitrogen 11360-070), 25 mM HEPES (Invitrogen 15630-080), 3 μg/ml blasticidin HC1 (Invitrogen R210-01), lOO μg/ml zeocin (Invitrogen R250-01) and penidUin/streptomycin (Invitrogen 15140-122). About 6 x 10⁵ cells were transfected with 4.5 μg of human GPR35-expression vector using 15μl of Iipofectamine 2000 (Invitrogen 11668-019). One-day after transfection, the cells were harvested and re-suspended in a medium prepared by further supplementing D?MEM above with lmg/ml G418 (Invitrogen 11811-031). The ceUs were seeded into T75 flask (Corning 430641) at a density of 1 x 10⁶ ceUs/flask and incubated for 11 days in CO2 incubator. The stable cells were stimulated with 10 μM of zaprinast (Sigma Z0878) overnight, and selection of zaprinast-sensitive ceUs was performed on Huorescence Activated CeU Sorter (FACSVantage SE; Becton Dickinson). For sorting, a substrate of β-lactamase was prepared by mixing CCF4 (Invitrogen K1028) with pluronic F127 (Invitrogen K1026), PBS Θ (Invitrogen 14190-144) and probenecid (Sigma P8761) just before use. The response to zaprinast on each sorted ceU was confirmed by βTactamase assay and Ca assay. Thus, stably-expressing human GPR35 ceU line was established.

Two days after transfection, the cells were washed twice with Hanks' Balanced Salt Solution (GIBCO BRL; 14175-095), and loaded with 5 μM Fura 2-AM (DOJINDO; 343-05401) in Hanks' Balanced Salt Solution containing Pluronic F-127 (0.05 %v/v, SIGMA' P-2443) for 1 hour in CO2 incubator. After the incubation, the ceUs were harvested by centrifugation and suspended in Hanks' Balanced Salt Solution (GIBCO BRL; 14025-092) (this solution contains calcium chloride, magnesium chloride, and magnesium sulfate) at a density of l~3xl0⁵ ceUs/ml. CeU suspension (90 μl) was dispensed to 96-weU plates (COSTER; 3904). Addition of 10 μl of the test compounds and measurement of intraceUular calcium mobiHzation were performed with FDSS6000 (Hamamatsu Photonics).

Example 9: Western Blotting lb confirm expression of FLAG-hGPR35, 1.5 x 10⁶ cells were dissolved in 150 μl of Tris-SDS-BME Sample Loading Buffer (Owl; ER33) and analyzed by western blotting analysis. 20 μl of the samples were loaded to READY GELS J (BIO-RAD; 161J391) and separated by electrophoresis in Tris/Glycine/SDS Buffer (BIO-RAD; 161-0732) at 200 volts for 30 min. Proteins were blotted to PVDF membrane (MLIIIPORE; IPVH304FO) at 15 V for 30min, and the blotted membrane was blocked by TBS buffer (150 mM NaCl, 20 mM Tris-HCl; pH7.5) containing 1.2 % bovine albumin (SIGMA; A-7030) for 30min. The membranes were incubated in 1,000-fold diluted ANTI-FLAG M2 Monoclonal Antibody (SIGMA F-3165) for 1 hour and washed with TTBS buffer (TBS buffer containing 0.1% Tween20) three times. Membranes were incubated in 500-fold cHluted anti-mouse IgG-HRP (IMMUNOTECH; 0817) as secondary antibody and washed with TTBS buffer three times. FLAG-GPR35 was visualized by ECL Western blotting detection reagents (Amersham pharmacia biotech; RPN 2209), and the signals were detected by IMAGE READER LAS- 1000 Pro version 2.0 (FUJI PHOTO FILM).

?Example 1Q: Screening for Agonists for human GPR35 To screen agonists for GPR35, we prepared HEK293 ceUs transiently co-expressing FLAG-tagged human GPR35 (FLAG"hGPR35), chdmeric G proteins (Gqs5, Gqi5 and Gqoδ) and a promiscuous G protein (Gαl6). After the ceUs were loaded with Fura 2-AM (calcium indicator), intraceUular Ca²⁺ mobilization evoked by addition of 10 μM of compounds was detected with FDSS6000 by the method described above. About 1,000 compounds were screened, and we discovered that zaprinast induced intraceHular Ca²⁺ mobilization in the co-transfectant. It had no such effect observed in parental HEK293 ceUs. As a result of the screening of various compounds, besides Zaprinast, the foUowing compounds were identified to induce intraceHular Ca²⁺ mobilization in the co-transfectant.

Table 1 : identified compounds

To confirm the agonistic activity of zaprinast, we tested dose dependencies of the responses in the co-transfectants by the same method. In the co-transfectant, intraceHular Ca²⁺ mobilization was induced by the compound dose-dependentiy, whUe ?HEK293 cells co-expressing only the chimeric G proteins and Gαl6 (mock) showed no response (Figure 3) to the compounds. The expression of FLAGTιGPR35 in the co-transfectant was confirmed by western blotting (data not shown). We also confirmed the agonism of zaprinast for native hGPR35 (Figure 3).

Example 11: Confirmation of the Coupling of GPR35 We also examined which G protein(s) was/were coupled to GPR35 using zaprinast. When FIAG-hGPR35 was co-expressed with Gqi5, Gqo5 (these are commerciaUy avaUable) or Gαl6 in HEK293 ceUs, intraceUular calcium mobiHzation was induced by zaprinast (Figin-e 4). On the other hand, the response was not observed in either co-transfectant of Gqs5 and FLAG-hGPR35 or transfectant of FLAGTιGPR35 alone (Figure 2). The expression of FLAGTιGPR35 in the co-transfectant was confirmed by western blotting (data not shown). These data suggest that human GPR35 couples to God, Gαo, and Gαl6. Example 12: IntraceUular Caldum Mobilization Assay using rat GPR35 lb confirm agonist activity of zaprinast for rat GPR35, HEK293 ceUs transiently co-expressing rat GPR35 and Gqi5 (chimeric G protein) were prepared, and chaUenged with zaprinast by a method described above (Figure 5). Zaprinast showed a moderate agonistic activity for human GPR35 (mean EC50 value = 1136 nM) but showed surprisingly high agonistic activity for rat GPR35 (mean EC50 value = 21 nM,). Transfectant expressing Gqi5 alone (mock) did not respond to zaprinast (Figure 5). Iransfectant expressing GPR35 alone also did not respond to zaprinast (data not shown).

Example 13: Comparative Experiment to Confirm Mechanism Zaprinast is a weU-known PDE5 inhibitor (BIOORG MED CHEM LETT (1996) 6 (15), 1819-1824 and EUR J PHARMACOL (2001) 411, MO). In the experiments above, Zaprinast showed its activity only when a GPR35 protein and an appropriate G protein are co-expressed. Therefore, it is understood that the above activity of Zaprinast is mediated by GPR35. However, just for further clarification, self-developed compound (hereinafter referred to as compound A) that is a strong PDE5 inhibitor (its chemical formula is 3-ethyl-5-{5-[(4-ethylpiperazmo)sulphonyl]-2-propoxyphenyl}-2-(2-pyridylmethy?D-6,7-dihydro -2H-pyra2»lo[4,3-d]pyrimicfin-7-one. IC50 value against PDE5 is 540nM for zaprinast and only 0.8nM for the compound A. Compound A is disclosed in Xenobiotica (2001), 31(8-9), 651-664 and 665-676) was used for the caldum mobilization assay. As a result, the compound A, unlike Zaprinast did not show an agonistic activity (Figure 6). This confirms that Zaprinast showed the activity via GPR35 protein. This was also confirmed by a comparative experiment that compared an activity of Zaprinast and the compound A using spinal cord as described below.

?Example 14: Electrophysiological Experiment Spinal cord preparation Transversely shced spinal cord preparation was obtained from an adult rat by a method already described in an article (Nakatsuka, et al. Alteration in synaptic inputs through C-afferent fibers to substantia gelatinosa neurons of rat spinal dorsal horn during postnatal development Neuroscience, 99, 549-556, 2000). Briefly, male adult Sprague-Dawley rats (200-350 g,^' 7-8 weeks old, SLC) were anesthetized with urethane (1.5 g kg, i.p.). After laminectomy, lumbosacral spinal cord was removed and transferred into ice cold Krebs solution (l^~3°C) pre-equiHbrated with 95% O2 / 5% CO2. The composition of the Krebs solution was ( mM): 117 NaCl, 3.6 KC1, 2.5 CaCl2 , 1.2 MgCk , 1.2 NaH₂ P0₄ , 25 NaHC0₃ and 11 glucose. The pia was removed after cutting aU the ventral and dorsal roots near the root entry zone. The spinal cord was mounted on a microsHcer (DTK-3000W, Dosaka EM Co. Ltd., Kyoto, Japan) and then a transverse sHce (500 μm thick) was cut. The shce was placed on a nylon mesh in the recording chamber of about 0.5 ml volume. The cut shce was continuously superfused at a flow rate of 18-20ml/min with the Krebs solution in 95% O2 / 5% CO2 , pH 7.2, for pre-incubation (longer than lhr, 36+1 °C). The institutional animal care and use committee approved the experimental procedure. Patch clamp recordings Substantia gelatinosa (SG) neurons were identified by their location in the preparation by a method in previously-reported article (Yoslnmura and, Nishi Blind patch-clamp recordings from substantia gelatinosa in adult rat spinal cord sHces^: pharmacological properties of synaptic currents, Neuroscience, 53, 519-526, 1993). Briefly, under a binocular stereomicroscope and with transmitted iHumination, the SG neurons were clearly discernable as a translucent band. Blind whole-ceU recordings were performed from SG neurons with patch pipettes which had tip resistance of 8^~10 MΩ when the pipettes were filled with the solution containing (in mM): 135 K-gluconate, 5 KC1, 2 MgCla, 0.5 CaC , 5 EGTA, 5 HEPES, and 5 ATP. Each recording was performed on a ceU in a fresh sHce without prior appHcation of any drugs, except some experiments. Signals were gained with a patch-clamp amplifier (Axopatch 200B, Axon Instruments). Data were low-pass filtered at 2 ?kHz, digitized at 333 kHz with an A/D converter, stored and analyzed with a personal computer using the software of pCLAMP (Version 9.0, Axon Instruments), Axograph (Version 4.6.2, Axon Instruments) and Origin (Version 6.1, ?Microcal). AH experiments were performed to isolate exdtatory postsynaptic currents (EPSCs) at the holding potential (VH) of —70 mN at which GABA and glycine-mediated synaptic currents were neghgible. ?M?iniature EPSCs (mEPSCs) were recorded in the presence of 2 μM tetrodotoxin (TTX). AppHcation of drugs AH tested compounds were apphed through the bath solution by exchanging the perfusion solution at a flow rate of 14-18 ml/min. Zaprinast was purchased from Sigma (St. Louis, MO, USA) and TTX was purchased from WAKO (Japan). The above mentioned self developed compound A was provided by medicinal chemists at Pfizer Sandwich Laboratories. Data analysis and statistics Data analyses were performed with the help of detection program of synaptic currents in the Axograph software. The events that posses the shape of a rapid rise foUowed by a slow decay and the peak exceeded -3 or -4 pA were identified as mEPSCs to be analyzed. Each selected synaptic current was visuaUy inspected to eliminate detection errors. Changes of the inter-event interval and ampHtude of mEPSCs were evidenced with the help of cumulative histograms of these parameters, and ascertained with the Kolmogorov-Smirnov test. For this test, P < 0.01 was taken to be statisticaUy significant. Data were presented as mean + SEM Result: Effect of zaprinast on mEPSCs Regarding to the effects of zaprinast on mEPSCs in SG neurons of the spinal cord, zaprinast (10 μM) inhibited the frequency of the mEPSCs (Fig. 7). Figs. 7B and 7C demonstrate the effects of zaprinast (10 μJVJD on cumulative distribution of the inter-event interval and the ampHtude of the mEPSC. Zaprinast significantly inhibited the frequency (P < 0.001) of the mEPSCs (7B), whfle the ampHtude of mEPSCs (p > 0.01) was not (7C). The Irinetics of mEPSCs was not affected by zaprinast (7D). Of 5 cells examined, 4 cells showed significant suppressions of mEPSC frequency in each cells (P < 0.01, ?KS"test). Membrane current was not induced by zaprinast up to 100 μM (n = 2, Data not shown). These data suggest that zaprinast inhibited the excitatory transmission presynapticaHy. Result: Mechanism of the inhibition of mEPSC frequency by zaprinast In order to study whether this inhibition of the mEPSC induced by zaprinast was GPR35 agonism or PDE-5 inhibition, we examined the effect of the compound A above, a selective PDE-5 inhibitor, on the mEPSCs. The frequency and ampHtude of mEPSCs were not affected by the compound A of its concentration from 20 nM up to lOμM (Fig. 8). Since this no inhibitory effect of the compound A was also confirmed in the SG neurons whose frequency of mEPSCs was reduced by the appHcation of zaprinast of lOμM (n=2, Data not shown), the compound A and zaprinast are quite different from their mode of action on mEPSCs. These results indicate that zaprinast would exert its inhibitory effect on mEPSCs via GPR35 agonism but not PDE5 inhibition. Result As described above, it has been demonstrated that electrophysiological evidence that activation of GPR35 produced suppression in the release of excitatory neurotransmitters from the presynaptic terminals of excitatory interneuron. These presynaptic GPR35 may play an important role in the inhibitory controls of nociceptive transmission at the spinal cord level.

?Example 15: Tissue Distribution of human GPR35 Total RNA samples used to synthesize templates are Hsted in Table 2. ?Reverse transcription was performed using SUPERSCRIPT PreampHfication Systems (CLOISPTECH) according to the attached protocol. To amplify transcripts of human GPR35, two nucleotide primers, the primer of Sequence. ID. 16 and the primer of Sequence ID 17 were designed. Premix Taq Ex Taq Version (TaKaRa,' RR003A) was used for PCR. PCR was performed in a volume of 20 μl under the foHowing program: 94 °C for 4min,' 32 cycles of 94 °C for 20 sec, 65 °C for 30 sec, 72 °C for 60 sec^' 72 °C for 4 min. The samples were analyzed by electrophoresis using 1% agarose gel. For a positive control, 1 pg of expression vector of FLAG-tagged GPR35 was used. For an internal control, GAPDH was amplified with the same procedure except for annealing temperature (50 °C), number of cycles (25 cycles) and primers (Sequence IDs 18 and 19). To exclude the possibility of contamination of genomic DNA to the templates, experiments without reverse transcription (in absence of SuperScri.pt II reverse transcriptase) were performed, and no amplifications were observed except for positive control.

Tissue Maker Catalogue # Conc.(μg/μl)

Trachea CLONTECH #K4000-1 (Human Panel I) 1.00

Heart CLONTECH #K4001-1 (Human Panel II) 1.00

Kidney GIBCO BRL 11410-016 2.50

Adrenal grand CLONTECH 64016-1 1.00

Stomach CLONTECH #K4000-1 (Human Panel I) 1.00 Small intestine CLONTECH #K4001-1 (Human Panel II) 1.00

Colon CLONTECH #K4001-1 (Human Panel II) 1.00

Brain CLONTECH #K4003-1 (Human Panel IV) 1.00

Cerebellum CLONTECH #K4003-1 (Human Panel IV) 1.00

Spinal cord CLONTECH #K4003-1 (Human Panel IV) 1.00

Dorsal horn ABS custom-made RNA 0.95

DRG ABS custom-made RNA 0.76

Table 2. List of total RNA used for RT-PCR.

Tissue distribution of GPR35 in human was determined by RTPCR (Figure 9). Striking signals of GPR35 were observed in spinal cord (whole), (spinal) dorsal horn, DRG, brain, cerebeUum, smaH intestine and colon. On the other hand, Httle or no signal was observed in trachea, heart, kidney, adrenal gland, and stomach. The internal control (GAPDH) was amplified equaUy with each template. Furthermore, it was confirmed that no amplified products were obtained by experiment without the reverse-transcription reaction (data not shown). These data show that GPR35 is mainly expressed in the nervous system, smaH intestine and colon.

Example 16: Tissue Distribution of rat GP?R35 Rat DRG and spinal cord were extracted from a 10-week-old pregnant female Sprague-Dawley rat sacrificed by CO2 (all animal care and sample coUection were approved by the Research Ethics Committee). The tissues were extracted in 1 ml of ISOGEN (Nippon Gene), and total RNAs were purified according to attached protocol. Other total RNAs were purchased from BD Clontech or UNITECH. They are summarized in Table 3.

Tissue Maker Catalogue #

Brain CLONTECH (BD) 64075-1

Cerebellum UNITECH 203

Cerebrum UNITECH 202

Skeletal muscle UNITECH 215 Uterus UNITECH 213

Heart UNITECH 205

Lung UNITECH 206

Liver UNITECH 208

Bladder CLONTECH (BD) 64077-1

Stomach UNITECH 207

Small intestine UNITECH 211

Colon CLONTECH (BD) 64066-1

DRG Prepared in this study

Spinal cord Prepared in this study

Table 3. List of total RNAs of rat used in this study

To generate first-strand cDNA templates for RTPCR, 5 μg of total RNA Oisted in Table l) was reverse-transcribed by SUPERSCRIPT PreampHfication Systems (GIBCO BRL Invitrogen) according to the attached protocol. Tb exclude the possibility of contamination of genomic DNA to the templates, a series of samples without SuperScri.pt II RT (-RT) were prepared in parallel as negative controls. Twentieth of the each reverse-transcribed sample was used as template for subsequent PCR. lb amplify rat GPR35 by RTPCR, two primers (Sequence Ids 20 and 21) were designed according to nucleotide sequences of rat GPR35. ?Mixtures for PCR were prepared using LA Taq with GC Buffer (TaKaRa; RR02AG) according to 'general reaction mixture for PCR' described in the attached protocol. PCR for rat GPR35 was performed under the foHowing condition: 94 °C for 4 min,^' 35 cycles of 94 °C for 20 sec, 58 °C for 30 sec, 72 °C for 1 min; 72 °C for 4 min. For internal controls of reverse transcription, GAPDH was amplified with the same procedure except for cycle number (30 cycles) and primers (Sequence IDs 22 and 23). After PCR, 10 μl of each sample was separated by electrophoresis, and DNAs were visualized by etbidium bromide staining. lb determine tissue distribution of rat GPR35, RTPCR analysis was performed. Predominant expression in digestive system (colon, smaH intestine, stomach) and DRG were observed (Figure 10). This result is consistent with a previous study in human GPR35. Example 17: Iigand Binding Assay Transiently or stably transfected ceUs expressing GPR35 are harvested by scraping (24-72 hours after the transfection in the case of transient transfection) and resuspended in 50 ml of ice-cold HEPES buffer (pH7.4) (containing 0.32 M Sucrose, lOmM EDTA, lOmM MgCla). The resulting suspension is centrifuged at 48,000 g, 4°C for 30 minutes. The resulting peUet is resuspended in the same buffer and centrifugation described above is repeated. The resulting peUet (membrane fraction) is suspended in an appropriate volume of an assay buffer and preserved at -80 °C. The protein concentration is determined via Bradford's assay (Biorad), according to the manufacturer's recommendations. The membrane fraction containing 50 μg protein is then incubated with various potential Hgands, radiolabeled to high specific activity, for about 30-120 minutes at room temperature (the optimal conditions, ion concentrations, incubation time and temperature can be determined for each Hgand with our undue difficulty according to teclmical lαiowledge weU ?known to those slriHed in the art). Reaction solution is filtrated by suction filtration using SKATRON ceUs harvester when the reaction ends. Right after filtration, the filter is washed by the assay buffer three times. WaUac GF/B filter is immersed in 0.3% (v/v) PEI (Polyethyleneimine; Sigma) for 1 hour before use. The GF/B filter is dried after washing and bound radioactivity on FUtermats is determined using a Hquid scintiUation counter. The specific binding is defined as the difference between total radioactivity bound minus the radioactivity measured in the presence of an excess of unlabeHed Hgand. Mock-transfected ceUs are also measured to assess whether the host ceUs express receptors for the Hgands used endogenously.

Example 18: Gastric emptying rate lb evaluate the involvement of GPR35 in gastric emptying rate, male Crj:SD(IGS) rats (Charles River Japan, Inc., 200-270 g) were used after fasted overnight. Drugs were administered to rats as summarized in Table 4 below, and after the administration of the drugs, a semrsoHd meal (methylceHulose: 7 g, glucose: 7.5 g, cornstarch: 9 g, casein: 15 g in 275 ml of water,^' hereinafter "test meal") was given oraUy by 3 mLhead.

Table 4. Application of Drug

Thirty minutes after test meal was given, the rats were sacrificed with CO2. The stomach was removed and weighed ("Weight A"). The stomach was opened and rinsed with saline, and weighed again ("Weight B"). "Weight of the stomach content" was calculated as the difference between them ("Weight A"-"Weight B") "Gastric emptying rate" was calculated by the foHowing formula

"Gastric emptying rate" (%) =100 x ("Weight of given test meal "-"Weight of the stomach content") / (Weight of given test meal)

Zaprinast was purchased from Sigma (St. ?Louis, MO, USA). Cisapride, a positive control, and Compound A (see Example 13), a selective PDE5 inhibitor were synthesized at Pfizer. Drugs were suspended with 0.1% methylceUulose solution and administered oraHy.

In the table above, 'Vehicle" means 0.1 % methylceUulose solution. Dose of drugs or vehicle was 5mL kg for the Experiment 18-1 and 18-2, and 2mL kg for the Experiment 18-3. Data were presented as mean ± SEM. The comparison between the control group and the drug-treated group was carried out by Dunnettfs test or Student's t-test. P values of less than 0.01 were regarded as significant. Figure 11 shows the result of Experiment 18-1 and 18-2. Zaprinast, a GPR35 agonist, delayed rat gastric emptying abiHty in a dose dependent manner when oraHy administered by 1-lOOmg/kg. This delaying effect was statistically significant. In the figure, it can be deemed that negative "gastric emptying rate" is due to that gastric emptying abUity was inliibited and that gastric juices and the Hke secreted remained in stomach resulting in stomach content overweighing test meal. Figure 12 shows the result of Experiment 18-3. This experiment was conducted to observe the effect of PDE 5 inhibitor, compound Aon rat gastric emptying abiHty in order to confirm that rat gastric emptying abUity is reaHzed by agonism of GPR35, because Zaprinast is an agonist of GPR35 as weU as an antagonist of PDE5. Compound A enhanced rat gastric emptying abiHty and this enhancement was statisticaUy significant. Therefore, it can be concluded that inhibition of gastric emptying abUity caused by Zaprinast is reaHzed by agonism of GPR35. As discussed above, agonism of GPR35 wiH inhibit rat gastric emptying abiHty.

Claims

1. A method of screening for a compound that modulates a GPR35 protein, comprising the step o£

(a) contacting a GPR35 protein or a partial polypeptide thereof with a test compound.

2. A method of screening for a compound that modulates neural activity, comprising the steps ofi (a) contacting a GPR35 protein or a partial polypeptide thereof with a test compound.

3. A method of screening for a compound that modulates digestive system, comprising the steps ofi (a) contacting a GPR35 protein or a partial polypeptide thereof with a test compound.

4. A method of screening for a compound that inhibits the binding of a Hgand to a GPR35 protein, comprising the steps ofi

(a) contacting a GPR35 protein or a partial polypeptide thereof with the Hgand,

(b) contacting a GPR35 protein or a partial polypeptide thereof prepared in the substantiaHy same manner as the one used in the step (a) with the Hgand prepared in the substantially same manner as the one used in the step (a), in the presence of a test compound, and

(c) comparing the results of the step (a) and the step (b) to determine whether the binding of the Hgand is affected by the presence of the test compound.

5. The method according to claim 4, wherein a detectable label is bound to said Hgand, and said step (c) comprises the step of comparing the detected amoimt of label in the step (a) and the detected amount of label in the step (b).

6. A method of screening for a compound that is an agonist of a GPR35 protein, comprising the steps ofi (a) adding a test compound to ceUs expressing a GPR35 protein or a partial polypeptide thereof or to a membrane fraction from the ceUs, and (b) determining whether a functional response is observed.

7. A method of screening for a compound that is an antagonist of GPR35 protein, comprising the steps ofi (a) adding an agonist to ceUs expressing GPR35 protein or a partial polypeptide thereof or to a membrane fraction from the ceUs,

(b) adding an agonist prepared in the substantiaHy same manner as the one used in the step (a) and a test compound to the ceUs or the membrane prepared in the substantially same manner as the one used in the step (a), and (c) comparing a functional response in the step (a) and one in the step (b) to determine whether the functional response is reduced by the test compound.

8. A method according to claim 6 or 7, wherein said functional response is a transient rise in intraceHular caldum concentration.

9. The method according to claim 4 or 7, wherein said Hgand or said agonist is selected from zaprinast, an analogue thereof, or a mimetic thereof.

10. The method according to any one of claims 1-4, wherein the GPR35 protein or the partial polypeptide thereof in the step (a) exists in ceUs or a membrane fraction.

11. The method according to any one of claims 1-4, 6, and 7 to screen a compound for use in the treatment or prevention of any of the foUowing conditions: pain, anxiety, convulsions, cognition disorders, obesity, schizophrenia, neurodegeneration, depression, attention deficit hyperactivity disorder, mania, memory deficit, eating disorders, Parkinson's disease, Huntington's disease, Alzheimer's disease, amyotrophic lateral sclerosis, drug addiction, bipolar disorders, Orcadian rhythm disorders, migraine, sexual dysfunctions, sleep disorders, functional dyspepsia, Irritable Bowel Syndrome (IBS), diarrhea, emesis, gastro-esophageal reflux disease, Barrett's esophagus, esophageal achalasia, gastroparesis, postoperative Ueus, constipation, non-cardiac chest pain (NCCP), bronchial asthma(BA), bronchitis, and chronic cough.

12. The method according to any one of claims 1-4, 6, and 7, wherein said test compound consists of a pluraHty of compounds.

13. A method according to any one of claims 1-4, 6, and 7, wherein said GPR35 protein is selected from the foHowing,'

(a) a polypeptide having the amino acid sequence of sequence ID. 2, 4, or 6,

(b) a polypeptide having the same amino acid sequence as the polypeptide of (a) except that one or more amino acids are deleted, substituted, or added, (c) a polypeptide having an amino acid sequence that has at least 80% identity to the the polypeptide of (a),

(d) a polypeptide having an amino acid sequence that has at least 90% identity to the the polypeptide of (a),

(f) a human GPR35 protein, a mouse GPR35 protein, or a rat GPR35 protein.

14. Use of zaprinast, an analogue thereof, or a mimetic thereof as a modulator of a GPR35 protein.

15. An isolated and/or purified polypeptide of one of the foHowing,^' (a) a polypeptide having the amino acid sequence of sequence ID. 2,

(b) a polypeptide having the same amino add sequence and the same Irind of activity as the polypeptide of (a) except that one or more amino acids are deleted, substituted, or added,

(c) a polypeptide having an amino acid sequence that has at least 86% identity to the polypeptide of (a), (d) a polypeptide having an amino add sequence that has at least 90% identity to the polypeptide of (a), and

(e) a polypeptide having an amino acid sequence that has at least 98% identity to the polypeptide of (a).

16. An isolated and/or purified polynucleotide of one of the foHowing; (a) a polynudeoti.de having the nudeotide sequence of sequence ID. 1,

(b) a polynucleotide that is capable of hybridizing under stringent conditions to a polynudeodide having a nucleotide sequence complementary to the nucleotide sequence of the polynudeoti.de of (a) and that encodes a polypeptide having the same kind of activity as the polypeptide encoded by the polynucleotide of (a), (c) a polynucleotide having a nucleotide sequence that has at least 90% identity to the tpolynucleoti.de of (a),

(e) a polynucleotide having a nucleotide sequence that has at least 98% identity to the polynucleotide of (a), and © a polynucleotide that encodes the polypeptide according to the claim 15.

17. An expression vector containing the polynucleotide according to claim 16.

18. Transformed ceUs that express the polypeptide according to claim 15.

19. An antibody immunospecific for the polypeptide according to claim 15.

20. Transformed ceUs that stably express any of foHowing polypeptides,"

(a) a polypeptide having the amino acid sequence of sequence ID. 2, 4, or 6,

(b) a polypeptide having the same amino acid sequence and the same ?kind of activity as the polypeptide of (a) except that one or more amino acids are deleted, substituted, or added,

(c) a polypeptide having an amino acid sequence that has at least 80% identity to the polypeptide of (a),

(d) a polypeptide having an amino add sequence that has at least 90% identity to the polypeptide of (a), and

21. A novel compound that is identifiable by the method according to any one of claims 1-4, 6, and 7.

22. A medicament for use to modulate GPR35 activity comprising a compound that is identifiable by the method according to any of claimsl-3, 5, and 6.

23. The medicament according to claim 21 comprising a compound selected from the group consisting of the foHowing compounds,^'

2-methyl-5-phenyl-pyrazolo[l,5-a]pyrimidin-7(4H)-one,

3-(4, 7-dihydro- l-methyl-7-oxo-3-propyl- lH-pyrazolo[4,3-d]pyrimidin-5-y])-4-ethoxy-benzenep ropanoic acid,

3-[3-(4,5-dU ydro-3-methyl-4-oxo-l-propyl-lH-pyrazolo[3,4-d]pyrimidin-6-yl)-4-propoxypheny l]-2-propenoic acid,

2,4-(Hhydro-2-methyl-5-[2-(2-methylpropoxy)-5-(lH-teteazol-5-y?D-3-pyridiny?l]-3-propyl-7H-P yrazolo[4,3-cGpyriιmdin-7-one,

2,4-(Hhy(fro-2-methyl-5-[2-(2-methylpropoxy)-5-(lH-l,2,3-triazol-4-y?0-3-pyridiny?l]-3-propyl-7

H-?Pyrazolo[4, 3-d]pyrimidin-7-one, 5-(2-ethoxypheny?0-l,4-dihydro-7H-l,2,3-l iazolo[4,5-d]pyιinrύdin-7-one, 3-(4, 7-dΛy(fro-7-oxo-lH- 1,2, 3-triazolo[4,5-α0pyr^ chloride,

3-(4,7-(Hhydro-7-oxo-lH-l,2,3-tiiazolo[4,5-d]pyrimidm-5-y?D-4-propoxy-benzenesuUonaιm 5-Nitro-2-(3-phenylpropylamino)benzoic acid, 2-Cyano-4-hydroxyindole,

2-(2-Propoxypheny?D-8-trifluoromethylpurin-6-one, and

6-phenyl-l-(phenylmethy?D-lH-Bis[l,2,3]triazolo[l,5-a:4',5'-e]pyrimidin-4(5H)-one.

24. Use of a compound that is identifiable by the method according to any one of claimsl-4, 6, and 7 to modulate GPR35 activity.

25. Use of a compound that is identifiable by the method according to any one of claims 1-4, 6, and 7 for the manufacture of a medicament to modulate GPR35 activity.

26. A method of modulating GPR35 activity by administering a compound that is identifiable by the method according to any one of claimsl-4, 6, and 7.