EP1720530A2 - Recepteur gpr35 - Google Patents
Recepteur gpr35Info
- Publication number
- EP1720530A2 EP1720530A2 EP05702505A EP05702505A EP1720530A2 EP 1720530 A2 EP1720530 A2 EP 1720530A2 EP 05702505 A EP05702505 A EP 05702505A EP 05702505 A EP05702505 A EP 05702505A EP 1720530 A2 EP1720530 A2 EP 1720530A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- polypeptide
- gpr35
- compound
- protein
- sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
- G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
Definitions
- 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.
- 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.
- 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 ⁇ , a ⁇ , a ⁇ -subunits in which ⁇ is bigger than ⁇ and ⁇ is bigger than ⁇ . At least 20 human genes are ?known to encode the GTP-binding orsubunits.
- the ⁇ -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 Gq ⁇ , Gil ⁇ , G14 ⁇ , G16 ⁇ , lead to activation of phospholipase C.
- G12 and G13 appear to regulate classes of small molecular weight G proteins and Na + ⁇ + exchange.
- 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 ⁇ , Gi2 ⁇ and Gi3 ⁇ , as well as several with a limited expression, such as Gz ⁇ , Go ⁇ and transducin. Initiation of signal txansduction cascades involving G proteins requires the binding of a hgand to a G protein-coupled receptor (GPCR).
- GPCR G protein-coupled receptor
- G protein 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, ⁇ l-adrenergic receptor antagonists used for treating hypertension, angina and heart failure, dopamine D2 antagonists used for the treatment of schizophrenia, and ⁇ 2-adrenergic receptor agonists used for the treatment of asthma.
- ⁇ l-adrenergic receptor antagonists used for treating hypertension, angina and heart failure
- dopamine D2 antagonists used for the treatment of schizophrenia
- ⁇ 2-adrenergic receptor agonists used for the treatment of asthma.
- 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 l ⁇ iown 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
- 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.
- the usual host cells used for heterologous expression of cloned GPCRs only express certain G proteins.
- 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.
- 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.
- 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.
- 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,
- 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 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).
- 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.
- 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£
- 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
- 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,
- 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,
- 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),
- (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;
- 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.
- such a compound can be selected from the group consisting of the foHowing compounds,'
- 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.
- homologue 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.
- homologue 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%.
- a homologue includes a polynucleotide that encodes a homologue of a polypeptide that a reference polynucleotide encodes.
- a homologue also includes a polypeptide that is complementary to a polynucleotide capable of hybridizing under stringent conditions (e.g.
- a homologue also includes a polynucleotide fragment or partial polypeptide.
- sequence homology 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.
- analogue as used herein relates to any substance which is similar in structure to a reference agent (e.g. Zaprinast).
- mimetic relates to any substance which has the same Irind of activity or effect as a reference agent (e.g.Zaprinast).
- modulator of a receptor refers to any substance which has an direct or indirect effect on the receptor.
- agonist of a receptor refers to a substance which can stimiilate the receptor.
- antagonist of a receptor refers to a substance which can inhibit the stimulation of the receptor by an agonist.
- functional response 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.
- isolated and/or purified polynucleotide/polypeptide 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.
- 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.
- 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.
- 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.
- 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)
- Hbrary 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.
- 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.
- any lrind of natural products may be screened using the methods of the invention, including microbial, fungal, plant or animal extracts.
- diversity Hbraries of test compounds may be utiHzed.
- 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).
- combinatorial Hbrary methods 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 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)
- 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,
- 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).
- Hgands labeled with 125 I, 35 S, 3 H, 14 C, Hgands labeled with fluorescent compounds such as fluorescein, or Hgands labeled with enzymes such as horseradish peroxidase can be used as a labeled Hgand.
- 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£
- 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
- 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.
- 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.
- 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.
- cells are transfected with a plasmid or plasmids leading to co-expression of GPR35 and GFP- ⁇ -arrestin complex, and an agonist of the receptor is identified by observing clustering of fluorescence, i.e. GFP- ⁇ -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.
- 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.
- These DNAs can also be prepared by l ⁇ iown gene engineering techniques.
- an expression vector for example, pcDNA3.1, pAKKO-111, pAKKO-lllH, pXTl, pRC/CMV pRC RSV etc. can be used.
- a promoter any one which functions efficiently in a host ceU can be used, for example, SV40 promoter, CMV promoter, HSVTK promoter, SR ⁇ promoter, RSV promoter, etc. can be used.
- 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, SR ⁇ 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.
- promoters in particular, SR ⁇ promoter, CMV promoter, RSV promoter, etc.
- 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.
- a ceH that highly expresses the GPR35 can be obtained.
- 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.
- the combination of the expression vector represented by hGPR35/pcDNA3.1 and the CHO ceUs is preferable.
- 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.
- 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.
- 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.
- any technique which provides antibodies produced by continuous ceU line cultures can be used. Examples include the hybridoma technique (Kohler, G.
- 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.
- transgenic mice, or other organisms, including other mammals may be used to express humanized antibodies.
- 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.
- 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).
- 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.
- compounds of the present invention can be used to modulate GPR35 protein and hence modulate digestive system so as to treat a variety of disease conditions.
- GPR35 modulator namely, compounds that modulated GPR35 protein (GPR modulator)
- GPR35 protein 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.
- IBS Irritable Bowel Syndrome
- 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.
- 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.
- 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 IC5 ⁇ (i. e. the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in ceU culture.
- IC5 ⁇ i. e. the concentration of the test compound which achieves a half-maximal inhibition of symptoms
- levels in plasma may be measured, for example, by high performance Hquid chromatography.
- compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologicaUy acceptable carriers or excipients.
- 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.
- 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.
- 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.
- compositions may take the form of tablets or lozenges formulated in conventional manner.
- 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.
- a suitable propeHant e.g., dichlorodifluoromethane, trichlorofluoromethane, dic?hlorotetrafluoroethane, carbon dioxide or other suitable gas.
- the dosage unit may be determined by providing a valve to deHver a metered amount.
- 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.
- the active ingredient may be in powder form for constitution with a suitable vehicle, e.g.
- 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.
- 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.
- 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.
- 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.
- Example l Original Cloning of human GPR35 A fuU-length cDNA of human GPR35 was isolated from human Standard ?Mj ⁇ ed
- DRG cDNA Hbrary (LIFE TECHNOLOGIES; 11649-019, Lot No: 80160b) based upon the ?known sequence of human GPR35 protein using very weU-?known technology.
- 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
- the PCR product was purified by High PureTM 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 l ⁇ min. The treated pcDNA3.l(+) and human FLAG-GPR35 fragments were Hgated using Rapid DNA Iigation Kit (Roche,' 1 635 379).
- 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 Ec ⁇ RI site and the latter primer Xbal site.
- the plasmid map of the vector is shown in Figure 2.
- 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.
- 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 ⁇ l 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.
- Example 5 Construction of an Expression Vector of 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.
- 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 G ⁇ l6. 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-G ⁇ l5-?NFAT ⁇ Tactamase 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 ⁇ g/ml blasticidin HC1 (Invitrogen R210-01), lOO ⁇ g/ml zeocin (Invitrogen R250-01) and penidUin/streptomycin (Invitrogen 15140-122).
- the stable cells were stimulated with 10 ⁇ M of zaprinast (Sigma Z0878) overnight, and selection of zaprinast-sensitive ceUs was performed on Huorescence Activated CeU Sorter (FACSVantage SE; Becton Dickinson).
- Huorescence Activated CeU Sorter FACSVantage SE; Becton Dickinson.
- a substrate of ⁇ -lactamase was prepared by mixing CCF4 (Invitrogen K1028) with pluronic F127 (Invitrogen K1026), PBS ⁇ (Invitrogen 14190-144) and probenecid (Sigma P8761) just before use.
- the response to zaprinast on each sorted ceU was confirmed by ⁇ Tactamase assay and Ca assay.
- stably-expressing human GPR35 ceU line was established.
- CeU suspension (90 ⁇ l) was dispensed to 96-weU plates (COSTER; 3904). Addition of 10 ⁇ l 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 10 6 cells were dissolved in 150 ⁇ l of Tris-SDS-BME Sample Loading Buffer (Owl; ER33) and analyzed by western blotting analysis. 20 ⁇ l 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.
- Tris-SDS-BME Sample Loading Buffer Owl; ER33
- 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.
- TBS buffer 150 mM NaCl, 20 mM Tris-HCl; pH7.5
- SIGMA bovine albumin
- 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
- FLAG FLAG-tagged human GPR35
- chdmeric G proteins Gqs5, Gqi5 and Gqo ⁇
- G ⁇ l6 chdmeric G proteins
- G ⁇ l6 promiscuous G protein
- Example 11 Confirmation of the Coupling of GPR35
- G protein(s) was/were coupled to GPR35 using zaprinast.
- Gqi5 these are commerciaUy avaUable
- G ⁇ l6 in HEK293 ceUs
- intraceUular calcium mobiHzation was induced by zaprinast (Figin-e 4).
- the response was not observed in either co-transfectant of Gqs5 and FLAG-hGPR35 or transfectant of FLAGT ⁇ GPR35 alone ( Figure 2).
- 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).
- 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).
- Zaprinast is a weU-known PDE5 inhibitor (BIOORG MED CHEM LETT (1996) 6 (15), 1819-1824 and EUR J PHARMACOL (2001) 411, MO).
- 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.
- compound A 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.
- 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.).
- 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 NaH 2 P0 4 , 25 NaHC0 3 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 ⁇ m 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.
- 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.
- 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.
- CLOISPTECH SUPERSCRIPT PreampHfication Systems
- Premix Taq Ex Taq Version (TaKaRa,' RR003A) was used for PCR.
- PCR was performed in a volume of 20 ⁇ l 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.
- 1 pg of expression vector of FLAG-tagged GPR35 was used.
- 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).
- experiments without reverse transcription in absence of SuperScri.pt II reverse transcriptase were performed, and no amplifications were observed except for positive control.
- 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.
- 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.
- 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.
- GAPDH was amplified with the same procedure except for cycle number (30 cycles) and primers (Sequence IDs 22 and 23). After PCR, 10 ⁇ l of each sample was separated by electrophoresis, and DNAs were visualized by etbidium bromide staining.
- 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 ⁇ g 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 l ⁇ iowledge 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.
- test meal methylceHulose: 7 g, glucose: 7.5 g, cornstarch: 9 g, casein: 15 g in 275 ml of water, ' hereinafter "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.
- '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.
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US7491699B2 (en) | 2002-12-09 | 2009-02-17 | Ramot At Tel Aviv University Ltd. | Peptide nanostructures and methods of generating and using the same |
US8568637B2 (en) | 2004-08-02 | 2013-10-29 | Ramot At Tel-Aviv University Ltd. | Method of forming a fiber made of peptide nanostructures |
WO2006018850A2 (fr) * | 2004-08-19 | 2006-02-23 | Tel Aviv University Future Technology Development L.P. | Compositions et leurs methodes d'utilisation dans le traitement de maladies associees aux amyloides |
WO2007043048A2 (fr) | 2005-10-11 | 2007-04-19 | Ramot At Tel Aviv University Ltd. | Hydrogels auto-assembles et leurs procedes d'elaboration et d'utilisation |
US9040583B2 (en) | 2009-07-22 | 2015-05-26 | Temple University-Of The Commonwealth System Of Higher Education | Treatment of disorders associated with G protein-coupled receptor 35 (GPR35) |
US20120022116A1 (en) * | 2010-07-20 | 2012-01-26 | Huayun Deng | Compositions and methods for the treatment of pathological condition(s) related to gpr35 and/or gpr35-herg complex |
KR20210107035A (ko) * | 2018-12-21 | 2021-08-31 | 글리콤 에이/에스 | 장관벽 치유를 촉진하기 위한 조성물 및 방법 |
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WO2000008155A2 (fr) * | 1998-08-07 | 2000-02-17 | Incyte Pharmaceuticals, Inc. | Proteines associees a un recepteur humain |
EP1363655A4 (fr) * | 2000-12-18 | 2005-04-06 | Merck & Co Inc | Molecules d'acides nucleiques isolees codant un recepteur humain et murin couple a la proteine g - gpr54 -, proteines codees, cellules transformees par ces molecules et utilisations correspondantes |
WO2002061087A2 (fr) * | 2000-12-19 | 2002-08-08 | Lifespan Biosciences, Inc. | Peptides antigeniques destines a des recepteurs couples a la proteine g (gpcr), anticorps s'y rapportant, et systeme d'identification desdits peptides antigeniques |
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