WO2000063371A1 - Protease activated receptor 2 variants - Google Patents
Protease activated receptor 2 variants Download PDFInfo
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- WO2000063371A1 WO2000063371A1 PCT/GB2000/001455 GB0001455W WO0063371A1 WO 2000063371 A1 WO2000063371 A1 WO 2000063371A1 GB 0001455 W GB0001455 W GB 0001455W WO 0063371 A1 WO0063371 A1 WO 0063371A1
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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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
- A61P11/06—Antiasthmatics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/06—Antipsoriatics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/12—Antihypertensives
Definitions
- the present invention relates to the identification of polymorphic form of protease activated receptor 2 polypeptide having altered properties compared to wild type.
- protease activated receptors (1).
- Proteolytic cleavage of a PAR amino-terminal exodomain unmasks a tethered ligand that binds intramolecularly onto the body of the receptor to initiate signaling.
- PARs four PARs have been cloned of which PAR-1 and PAR-3 are activated by thrombin (2,3), PAR-2 is activated by trypsin and mast cell tryptase (4-6), PAR-4 is activated by both thrombin and trypsin (7).
- SLIGKV-NH synthetic peptides corresponding to the first five amino acids of the receptor's tethered ligand can activate the PARs (1).
- SLIGKV-NH is used to activate human PAR-2, although the mouse sequence SLIGRL-NH 2 and the selective PAR-2 agonist tc-LIGRLO-NH 2 are more potent activators of PAR-2 (8).
- the human PAR-1 agonist SFLLR-NH 2 has proved to be a less reliable agonist of PAR-1 because it activates human PAR-2 (9,10).
- PAR-2 is expressed on a variety of cells including epithelial cells (2,11), endothelial cells (12,13), smooth muscle cells (14), keratinocytes (15,16), neutrophils (17) and some T-cell lines (18).
- epithelial cells 2,11
- endothelial cells (12,13)
- smooth muscle cells 14
- keratinocytes (15,16)
- neutrophils 17
- T-cell lines 18
- the resulting edema occurs via neuronal PAR-2 activation (22).
- Mast cell tryptase a major mediator of inflammation and activator of PAR-2, stimulates cytokine release from human endothelial (23) and epithelial cells (24). and when injected into the skin of guinea pigs induces vascular leakage (25) and the accumulation of eosinophils and neutrophils (26). Because the proteases trypsin and tryptase can initiate an inflammatory response by the selective activation of PAR-2. receptor polymorphisms that can alter receptor activation may have important implications for disease.
- PAR 2 F240S displayed a significant reduction in sensitivity towards trypsin ( ⁇ 3.7-fold), the PAR-activating peptides, SKLIGV-NH 2 ( ⁇ 2.5-fold) and SLIGRL-NH 2 (-2.8 fold), but an increased sensitivity towards the selective PAR-2 agonist, trans-cinnamoyl-LlGRLO- NH 2 (4-fold). Increased sensitivity was also observed towards the selective PAR-1 agonist, TFLLR-NFL ( ⁇ 7 fold), but not to other PAR-1 agonists tested. Although the PAR-4 agonist GYPGKF-NF was without effect, trans-cinnamoyl-YPGKF-NH 2 could selectively activate the PAR 2 F240S receptor but not the wild type receptor.
- TLIGRL-NH 2 is a selective PAR 2 F240S agonist.
- F240S mutation By introducing the F240S mutation into rat-PAR-2, we observed shifts in agonist potencies that mirrored those seen with the human PAR 2 F240S confirming that F240 plays a key role in agonist specificity.
- the dramatically altered pharmacological profile induced by this polymorphism will have important implications for the design of PAR-targeted antagonists and may contribute to. or be predictive of an inflammatory disease.
- the present invention provides a variant protease activated receptor 2
- TLIGRL-NH 2 which polypeptide or fragment thereof comprises an extracellular loop 2 (ECL-2) having at least one amino acid difference from the corresponding ECL-2 amino acid sequence of the wild type polypeptide.
- wild type polypeptide used for comparison purposes is human PAR-2 having the amino acid sequence set out in SEQ ID No.2.
- the at least one amino acid difference includes a modification at residue 240 of the amino acid sequence set out in SEQ ID No.2, or its equivalent, more preferably a substitution at residue 240 of the amino acid sequence set out in SEQ ID No. 2, or its equivalent.
- the present invention provides a PAR-2 polypeptide or fragment thereof which comprises a substitution at residue 240, or its equivalent, such that residue 240 is not phenylalanine.
- amino acid residue 240 is not an aromatic amino acid.
- a polypeptide of the invention comprises a phenylalanine to serine substitution at residue 240 of the amino acid sequence set out in SEQ ID No.2, or its equivalent.
- the present invention also provides a nucleotide encoding a polypeptide of the invention and a host cell comprising a nucleotide of the invention operably linked to a transcriptional control sequence capable of directing the expression of a polypeptide of the invention in the host cell.
- a nucleotide of the invention comprises a thymidine residue at position 719 of the sense strand of the coding region of the nucleotide sequence set out in SEQ ID No. 1.
- the present invention provides a pair of oligonucleotide primers, each comprising at least 10 contiguous nucleotides of the polynucleotide of the invention, which primers are suitable for use in a polymerase chain reaction for amplifying the ECL-2 region of a PAR-2 gene.
- the present invention further provides the use of said primers in a method of identifying an individual having a polymorphism in the ECL-2 region of one or both PAR-2 gene alleles.
- the present invention provides a method of identifying a compound that modulates the activity of a polypeptide of the invention which method comprises contacting said polypeptide with said a candidate compound and determining whether the candidate compound modulates the activity of said polypeptide.
- the present invention provides a method of identifying a compound that selectively inhibits a polypeptide of the invention which method comprises:
- the present invention further provides a compound identified by the above methods of the invention and its use in treating a patient comprising a polypeptide of the invention.
- the present invention provides a compound that modulates the activity of a polypeptide of the invention for use in a method of treating a condition characterized by inflammation.
- the compound inhibits the activity of a polypeptide of the invention.
- the present invention provides a method of identifying a patient having an increased risk of inflammatory disorders which method comprises determining the presence or absence of a polypeptide of the invention or a polynucleotide of the invention in a biological sample obtained from said patient.
- PAR-2 The sequence of wild type PAR-2 has been published for human, mouse and rat PAR-2 (5, 37, 36, respectively). These sequences are available in public databases under Genbank accession numbers P55085 and Z49994 (human), P55086 (mouse) and Q63645 (rat). Other PAR-2 sequences may be obtained from other species such as other primate species and rodent species. Such PAR-2 sequences will generally be at least 60, 70 or 80% identical at the amino acid level over at least 50 or 100 amino acids with the human sequence shown as SEQ ID NO 2. In particular, homology should typically be considered with respect to those regions of the sequence known to be essential for PAR-2 function rather than non-essential neighbouring sequences. Although homology can also be considered in terms of similarity (i.e.
- homology in terms of sequence identity. Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences.
- % homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues (for example less than 50 contiguous amino acids).
- the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension. Calculation of maximum % homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties.
- a suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A.; Devereux et al, 1984, Nucleic Acids Research 12:387). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al, 1999 ibid - Chapter 18), FASTA (Atschul et al, 1990, J. Mol.
- a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance.
- An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs.
- GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). It is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
- % homology preferably % sequence identity.
- the software typically does this as part of the sequence comparison and generates a numerical result.
- PAR-2 sequences other than the human, mouse and rat sequences referred to above can, for example, be obtained using standard cloning techniques or by searching public databases.
- the present invention provides variant PAR-2 polypeptides of the invention which differ from the wild type sequences in that the extracellular loop 2 (ECL-2) has at least one amino acid difference from the corresponding ECL-2 amino acid sequence of the wild type polypeptide.
- the amino acid change is such that the variant PAR-2 polypeptide has the following properties: (i) it has reduced sensitivity to trypsin as compared with wild type PAR-2:
- the extracellular loop 2 of human PAR-2 is located at approximately residues 212 to 245 of the sequence shown as SEQ ID No. 2, or an equivalent region in PAR-2 sequences from other species.
- a variant PAR-2 polypeptide of the invention will have at least one amino acid difference in this region from the wild type sequence.
- the comparison should be made with the corresponding wild type sequence.
- wild type is known in the art and is generally taken to mean the phenotype that is characteristic of most of the members of occurring naturally and contrasting with the phenotype of a mutant.
- the wild type sequence is taken to be the human wild type amino acid sequence shown as SEQ ID NO 2.
- the amino acid difference is located between (and including) residues 236 to 245 of the sequence shown as SEQ ID NO. 2 (all further references to particular amino acid numbering should be taken to include the corresponding/equivalent residues in other PAR-2 sequences - the term "or its equivalent" refers to the amino acid in other PAR-2 sequences that corresponds to a particular numbered residue of the human sequence set out in SEQ ID. No. 2). More preferably, the difference is a deletion, substitution or insertion, preferably a substitution, at residue 240.
- a variant PAR-2 polypeptide of the invention comprises a non-aromatic amino acid at residue 240, preferably serine.
- a variant PAR-2 polypeptide of the invention does not comprise an aromatic residue , such as phenylalanine, at residue 240, or its equivalent in other PAR-2 sequences.
- Variant polypeptides of the invention can be tested to determine their sensitivity to trypsin and trans-cinnamoyl-LIGRLO-NH 2 using, for example, the assays described in the Examples.
- trypsin sensitivity is measured by exposing cells that express the variant PAR-2 polypeptide to trypsin and measuring the calcium response (see reference 31 for details of the calcium cell-signaling assay).
- Sensitivity to the selective PAR-2 agonist trans-cinnamoyl-LIGRLO-NH 2 can also be determined using a similar assay. A comparison is then typically made with wild type PAR-2 tested under similar conditions.
- a polypeptide of the invention is at least two-fold less sensitive to trypsin than is wild type PAR-2, more preferably at least three-fold less sensitive. Further, a polypeptide of the invention is preferably at least two-fold more sensitive to trans- cinnamoyl-LIGRLO-NHi than is wild type PAR-2, more preferably at least three or four- fold more sensitive. In addition a polypeptide of the invention is preferably no more than 20 or 40-fold more sensitive to trans-cinnamoyl-LIGRLO-NH 2 than wild type PAR-2.
- Variant polypeptides of the invention are also preferably activated by TLIGRL-NH 2 . Activation can again, typically be tested using a calcium cell-signaling assay, such as the assay described in reference 31.
- Polypeptides of the invention also include fragments of the above variant full length sequences provided that they comprise at least the region of ECL-2 that comprises the amino acid difference. Typically, polypeptide fragments will comprise at least six amino acids.
- variants and derivatives of the above are variants and derivatives of the above.
- variants or derivatives in relation to the amino acid sequences of the present invention includes any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acids from or to the sequence providing that the resulting sequence comprises an ECL-2 region with at least one amino acid difference to the corresponding wild type sequence.
- polypeptides of the invention may be modified for use in the present invention. Typically, modifications are made that maintain the biological activity of the sequence.
- Amino acid substitutions may be made, for example from 1, 2 or 3 to 10, 20 or 30 substitutions. Amino acid substitutions may include the use of non-naturally occurring analogues, for example to increase blood plasma half-life of a therapeutical ly administered polypeptide.
- Polypeptides of the invention are typically made by recombinant means, for example as described below. However they may also be made by synthetic means using techniques well known to skilled persons such as solid phase synthesis. Proteins of the invention may also be produced as fusion proteins, for example to aid in extraction and purification. Examples of fusion protein partners include glutathione-S-transferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and ⁇ -galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences. Preferably the fusion protein will not hinder the function of the protein of interest sequence. Polypeptides of the invention may also be purified from cells/tissue taken from individuals that naturally express polymorphic forms of PAR-2.
- Polypeptides of the invention may be in a substantially isolated form. It will be understood that the protein may be mixed with carriers or diluents which will not interfere with the intended purpose of the protein and still be regarded as substantially isolated.
- a protein of the invention may also be in a substantially purified form, in which case it will generally comprise the protein in a preparation in which more than 90%, e.g. 95%, 98% or 99% of the protein in the preparation is a protein of the invention.
- Polynucleotides of the invention comprise nucleic acid sequences encoding the polypeptide sequences of the invention. It will be understood by a skilled person that numerous different polynucleotides can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that skilled persons may. using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides of the invention to reflect the codon usage of any particular host organism in which the polypeptides of the invention are to be expressed.
- a particularly preferred polynucleotide of the invention comprises a thymidine residue at position 719 of the sense strand of the coding region of the nucleotide sequence set out in SEQ ID No. 1.
- Polynucleotides of the invention may comprise DNA or RNA. They may be single- stranded or double-stranded. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the present invention, it is to be understood that the polynucleotides described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of the invention.
- both strands of the duplex are encompassed by the present invention.
- the polynucleotide is single-stranded, it is to be understood that the complementary sequence of that polynucleotide is also included within the scope of the present invention.
- Polynucleotides encoding wild type PAR-2 or naturally occurring variants can be obtained in a number of ways.
- Other variants of the sequences described herein may be obtained for example by probing DNA libraries made from a range of individuals, for example individuals from different populations.
- other homologues particularly homologues found in mammalian cells e.g. hamster, bovine or primate cells
- Variants and strain/species homologues may also be obtained using degenerate PCR which will use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of the present invention.
- conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants/homologues. Sequence alignments can be performed using computer software known in the art. For example the GCG Wisconsin PileUp program is widely used.
- the primers used in degenerate PCR will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.
- polynucleotides may be obtained by site directed mutagenesis of characterised sequences, such as SEQ ID. No 1. This may be useful where for example silent codon changes are required to sequences to optimise codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides.
- Polynucleotides of the invention may be used to produce a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors.
- a primer e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors.
- Such primers, probes and other fragments will be at least 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length, and are also encompassed by the term polynucleotides of the invention as used herein.
- Primers and probes may conveniently be used to genotype individuals to establish their PAR-2 status as described below.
- Polynucleotides such as a DNA polynucleotides and probes according to the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques.
- primers will be produced by synthetic means, involving a step wise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art.
- Longer polynucleotides will generally be produced using recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques. This will involve making a pair of primers (e.g. of about 15 to 30 nucleotides) flanking a region of the sequence which it is desired to clone, bringing the primers into contact with mRNA or cDNA obtained from an animal or human cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA.
- the primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector
- Polynucleotides of the invention can be incorporated into a recombinant replicable vector.
- the vector may be used to replicate the nucleic acid in a compatible host cell.
- the invention provides a method of making polynucleotides of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector.
- the vector may be recovered from the host cell.
- Suitable host cells include bacteria such as E. coli, yeast, mammalian cell lines and other eukaryotic cell lines, for example insect Sf9 cells.
- a polynucleotide of the invention in a vector is operably linked to a control sequence that is capable of providing for the expression of the coding sequence by the host cell.
- the vector is an expression vector.
- operably linked means that the components described are in a relationship permitting them to function in their intended manner.
- a regulatory sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences.
- control sequences may be modified, for example by the addition of further transcriptional regulatory elements to make the level of transcription directed by the control sequences more responsive to transcriptional modulators.
- Vectors of the invention may be transformed or transfected into a suitable host cell as described below to provide for expression of a protein of the invention. This process may comprise culturing a host cell transformed with an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the protein, and optionally recovering the expressed protein.
- the vectors may be for example, plasmid or virus vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter.
- the vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian vector. Vectors may be used, for example, to transfect or transform a host cell.
- Control sequences operably linked to sequences encoding the protein of the invention include promoters/enhancers and other expression regulation signals. These control sequences may be selected to be compatible with the host cell for which the expression vector is designed to be used in.
- promoter is well-known in the art and encompasses nucleic acid regions ranging in size and complexity from minimal promoters to promoters including upstream elements and enhancers.
- the promoter is typically selected from promoters which are functional in mammalian, cells, although prokaryotic promoters and promoters functional in other eukaryotic cells may be used.
- the promoter is typically derived from promoter sequences of viral or eukaryotic genes. For example, it may be a promoter derived from the genome of a cell in which expression is to occur. With respect to eukaryotic promoters, they may be promoters that function in a ubiquitous manner (such as promoters of a-actin, b-actin, tubulin) or, alternatively, a tissue-specific manner (such as promoters of the genes for pyruvate kinase).
- Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR) promoter, the rous sarcoma virus (RSV) LTR promoter or the human cytomegalovirus (CMV) IE promoter.
- MMLV LTR Moloney murine leukaemia virus long terminal repeat
- RSV rous sarcoma virus
- CMV human cytomegalovirus
- the promoters may also be advantageous for the promoters to be inducible so that the levels of expression of the polynucleotide of the invention can be regulated during the life-time of the cell. Inducible means that the levels of expression obtained using the promoter can be regulated.
- any of these promoters may be modified by the addition of further regulatory sequences, for example enhancer sequences.
- Chimeric promoters may also be used comprising sequence elements from two or more different promoters described above.
- Vectors and polynucleotides of the invention may be introduced into host cells for the purpose of replicating the vectors/polynucleotides and/or expressing the proteins of the invention encoded by the polynucleotides of the invention. It is preferred to use eukaryotic cells, for example yeast, insect or mammalian cells, in particular mammalian cells to express a polypeptide of the invention.
- eukaryotic cells for example yeast, insect or mammalian cells, in particular mammalian cells to express a polypeptide of the invention.
- Vectors/polynucleotides of the invention may introduced into suitable host cells using a variety of techniques known in the art, such as transfection, transformation and electroporation. Where vectors/polynucleotides of the invention are to be administered to animals, several techniques are known in the art, for example infection with recombinant viral vectors such as retroviruses, herpes simplex viruses and adenoviruses, direct injection of nucleic acids and biolistic transformation.
- retroviruses such as retroviruses, herpes simplex viruses and adenoviruses
- Host cells comprising polynucleotides of the invention may be used to express proteins of the invention.
- Host cells may be cultured under suitable conditions which allow expression of the proteins of the invention.
- Expression of the proteins of the invention may be constitutive such that they are continually produced, or inducible, requiring a stimulus to initiate expression.
- protein production can be initiated when required by, for example, addition of an inducer substance to the culture medium, for example dexamethasone or IPTG.
- Proteins of the invention can be extracted from host cells by a variety of techniques known in the art, including enzymatic, chemical and/or osmotic lysis and physical disruption.
- the invention also provides monoclonal or polyclonal antibodies to polypeptides of the invention or fragments thereof.
- the present invention further provides a process for the production of monoclonal or polyclonal antibodies to polypeptides of the invention.
- these antibodies are specific for the polypeptides of the invention i.e. have little or no cross-reactivity with wild type PAR-2.
- polyclonal antibodies are desired, a selected mammal (e.g., mouse, rabbit, goat, horse, etc.) is immunised with an immunogenic polypeptide comprising a variant ECL-2 sequence epitope(s). Serum from the immunised animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to a PAR-2 epitope contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffmity chromatography. Techniques for producing and processing polyclonal antisera are known in the art. In order that such antibodies may be made, the invention also provides polypeptides of the invention or fragments thereof haptenised to another polypeptide for use as immunogens in animals or humans.
- Monoclonal antibodies directed against ECL-2 epitopes in the polypeptides of the invention can also be readily produced by one skilled in the art.
- the general methodology for making monoclonal antibodies by hybridomas is well known.
- Immortal antibody- producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein- Barr virus.
- Panels of monoclonal antibodies produced against orbit epitopes can be screened for various properties; i.e.. for isotype and epitope affinity.
- An alternative technique involves screening phage display libraries where, for example the phage express scFv fragments on the surface of their coat with a large variety of complementarity determining regions (CDRs). This technique is well known in the art.
- Antibodies both monoclonal and polyclonal, which are directed against ECL-2 epitopes are particularly useful in diagnosis.
- Monoclonal antibodies in particular, may be used to raise anti-idiotype antibodies.
- Anti-idiotype antibodies are immunoglobulins which carry an "internal image" of the antigen of the agent against which protection is desired. Techniques for raising anti-idiotype antibodies are known in the art. These anti-idiotype antibodies may also be useful in therapy.
- the term "antibody”, unless specified to the contrary, includes fragments of whole antibodies which retain their binding activity for a target antigen. Such fragments include Fv. F(ab') and F(ab') 2 fragments, as well as single chain antibodies (scFv). Furthermore, the antibodies and fragments thereof may be humanised antibodies, for example as described in EP-A-239400.
- Antibodies may be used in method of detecting polypeptides of the invention present in biological samples by a method which comprises:
- Suitable samples include extracts tissues such as brain, breast, ovary, lung, colon, pancreas, testes, liver, muscle and bone tissues or from neoplastic growths derived from such tissues.
- Antibodies of the invention may be bound to a solid support and/or packaged into kits in a suitable container along with suitable reagents, controls, instructions and the like.
- F240S polymo ⁇ h of PAR-2 is present at an allelic frequency of 8.4% in a group of 125 individuals, with 15.2% of individuals carrying at least one variant allele. Furthermore, we have shown that the F240S polymo ⁇ h displays differential responses to PAR-activating agonists compared to wild type PAR-2.
- the altered properties of the polymo ⁇ h may be highly significant in terms of drug regimes based on using antagonists that target PAR-2 since antagonists designed to target the wild type PAR-2 protein may be ineffective or indeed deleterious when administered to individuals possessing an S allele.
- FS heterozygotes and/or SS homozygotes may have increased susceptibility to diseases, such as inflammatory diseases. Consequently. determination of an individual's PAR-2 status may be used to identify individuals at risk from diseases associated with variant PAR-2.
- the present invention provides a method of determining the PAR-2 genotype of an individual which method comprises determining all or part of the sequence of the ECL-2 region within the individual's PAR-2 genes.
- the sequence of the nucleotide codon that specifies amino acid 240, or its equivalent is determined, more preferably the sequence of the polymo ⁇ hic site at nucleotide 719 is determined.
- the sequence may be determined for genomic DNA or mRNA. preferably genomic DNA. A variety of techniques for doing this are known in the art such as RFLP analysis or direct genomic sequencing - both of which are described in the Examples. Determination of the sequence in the present context does not necessarily mean that the exact nucleotide sequence be established since, for example, the presence or absence or a restriction fragment of a particular size can be sufficient (see the Examples).
- Nucleic acids from the individual of interest are typically obtained from a biological sample taken from the individual, such as a blood sample or other tissue sample.
- genotyping may be performed using PCR primers that flank the ECL-2 region.
- the present invention provides a pair of oligonucleotide primers, each comprising at least 10 contiguous nucleotides of the PAR-2 nucleotide sequence and which flank the ECL-2 region. These primer pairs are provided for use in establishing the PAR-2 genotype of an individual. PCR primers may be packaged into kits together with other components such as buffers, instructions etc.
- Genotyping may also be carried out using a protein-based approach, for example using antibodies to PAR-2 polypeptides of the invention (see section F).
- the F240S PAR-2 polymo ⁇ h has different properties to the wild type PAR-2 such that agonists/antagonists that may be used to affect the activity of the wild type PAR-2 receptor may not be effective against the polymo ⁇ h. Accordingly, it would be desirable to identify agonists/antagonists that are effective in those patients possessing at least one S allele or other polymo ⁇ h having altered properties compared with wild type.
- the present invention provides screening methods for identifying compounds that modulate the activity of a polypeptide of the invention, for example stimulate or inhibit a polypeptide of the invention.
- candidate compounds can be tested for binding to a polypeptide of the invention. Binding assays are well known in the art.
- candidate compounds are tested in competition assays with a known agonist or antagonist.
- the agonist or antagonist may be labeled with a detectable label.
- varying concentrations of the candidate compound are supplied along with a constant concentration of labeled agonist or antagonist and the inhibition of a binding of labeled agonist/antagonist to the receptor can be evaluated using known techniques.
- More sophisticated functional assays comprise contacting a candidate substance with a polypeptide of the invention and determining whether the polypeptide is stimulated or inhibited.
- one suitable assay is the calcium signaling assay described in the Examples and reference 31.
- the effect of candidate compounds on agonist-induced responses can be measured in the cells recombinantly expressing a variant PAR-2 receptor of the inventions.
- Assay systems for the effect of activation of receptor on these cells include calcium mobilization and voltage clamp which are described herein in further detail. These assays permit an assessment of the effect of the candidate compound on the receptor activity rather than simply the ability to bind to the receptor. Agonist-induced increases Ca release by cells expressing variant PAR-2 receptors can be assessed using known techniques. One such protocol is described in US Patent No. 5.874.400, inco ⁇ orated herein by reference. Using these types of assays, the ability of a candidate compound to activate the receptor can be tested directly. In an alternative to measuring calcium mobilization, the voltage clamp assay can be used as a measure for receptor activation.
- Agonist-induced inward chloride currents are measured in voltage-clamped cells expressing variant PAR-2 receptors essentially as previously described (Julius et al, Science (1988) 241 :558-563) except that the single electrode voltage-clamp technique is employed.
- Antagonists can be tested in a similar manner except that a known agonist is used alone in the control samples and coadministered to cells along with the candidate compound in the test samples.
- Suitable known agonists include trypsin. tryptase, TFLLR-NH2. trans-cinnamoyl- LIGRLO-NH? together with a number of agonists described in US Patent No. 5.874.400. Suitable known antagonists for use in the assay methods of the invention are also described in US Patent No. 5,874.400.
- modulators of the activity of polypeptides of the invention that are specific for those peptides and have little or no affect on wild type PAR-2.
- modulators of the activity of polypeptides of the invention that also modulate wild type PAR-2.
- assays of the invention may further include the step of determining the effect of an agonist or antagonist of a polypeptide of the invention on wild type PAR-2. typically using similar assay techniques.
- Candidate compounds of the invention may also be tested using an animal model to determine whether, for example, they can affect inflammatory responses caused by the administration of PAR-2 agonists (see reference 21).
- Suitable candidate substances include peptides. especially of from about 5 to 30 or 10 to 25 amino acids in size, based on the sequence of the PAR-2. in particular the tethered ligand sequences present in PAR-2 or variants of such peptides in which one or more residues have been substituted.
- Other peptide candidates include the peptide agonists and antagonists described in US Patent No. 5.874.400 or variants of such peptides in which one or more residues have been substituted.
- Peptides from panels of peptides comprising random sequences or sequences which have been varied consistently to provide a maximally diverse panel of peptides may be used.
- Suitable candidate substances also include antibody products (for example, monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies and CDR-grafted antibodies) which are specific for a polypeptide of the invention.
- antibody products for example, monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies and CDR-grafted antibodies
- combinatorial libraries, peptide and peptide mimetics. defined chemical entities, oligonucleotides, and natural product libraries may be screened for activity as modulators of PAR-2 activity.
- the candidate substances may be used in an initial screen in batches of, for example 10 substances per reaction, and the substances of those batches which show- inhibition tested individually.
- Candidate substances which show activity in in vitro screens such as the competition binding assays described assays can then be tested in whole cell systems, also described above.
- Antagonists/agonists of polypeptides of the present invention may be used to treat more effectively patients suffering from various disorders, which patients possess one or more PAR-2 polymo ⁇ hs according to the present invention.
- antagonists of polypeptides of the present invention may be used to treat inflammatory diseases or conditions characterized by inflammation for example, asthma, chronic obstructive pulmonary disease, arthritis, inflammatory bowel disease, psoriasis and eczema. They may also be used to treat disorders such as multiple sclerosis and to raise blood pressure.
- agonists of the polypeptides of the invention may, for example, be used as antihypertensives .
- compositions of the invention may preferably be combined with various components to produce compositions of the invention.
- the compositions are combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition (which may be for human or animal use).
- Suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline.
- composition of the invention may be administered by direct injection.
- the composition may be formulated for parenteral, intramuscular, intravenous, subcutaneous, oral or transdermal administration. More recently, alternative means for systemic administration of peptides have been devised which include transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other detergents.
- penetrants such as bile salts or fusidic acids or other detergents.
- oral administration may also be possible. Administration of these compounds may also be topical and/or localized, in the form of salves, pastes, gels and the like.
- each compound may be administered at a dose of from 0.01 to 30 mg/kg body weight, preferably from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight.
- the codon of the amino acid 240 modified by the mutation is represented is underlined.
- FIG. 1 Location of the polymo ⁇ hism and alignment of the ECL-2 amino acid residues from mouse, rat and human PAR-2. Note the conservation at the position of the Phe240 and neighboring amino acid residues. Amino acid numbers are from the human receptor.
- FIG. 3 Calcium signaling in the human wild-type and PAR 2 F240S receptors in response to trypsin and PAR2-APs.
- Wild type responses are designated by: — ⁇ — , and PAR 2 F240S, - - ⁇ - -.
- Cells were lifted with non-enzymatic cell dissociation fluid, loaded with Fluo-3 (22 ⁇ M) prior to incubation for 35min at RT.
- Cells were challenged with different concentrations PAR agonists and responses were monitored by fluorescence spectrophotometry (excitation 480nm, emission 530nm). Responses were normalized to the peak height obtained with 2 ⁇ M calcium ionophore (% A23187).
- Each data point represents the mean ⁇ SEM of three to four separate experiments each performed in duplicate.
- FIG. 5 Calcium signaling in human and rat wild-type and PAR 2 F24OS Receptors in response to TLIGRL-NH2 and tc-YGPKF-NH .
- Cells were lifted with non-enzymatic cell dissociation fluid, loaded with Fluo-3 (22 ⁇ M) prior to incubation for 35min at RT.
- Cells were challenged with the agonists shown and responses were monitored by fluorescence spectrophotometry (excitation 480nm, emission 530nm). Responses were normalized to the peak height obtained with SFLLR-NH 2 (50 ⁇ M).
- RNA Human colon tissue resected at surgery from patients with colonic cancer was used as a source of RNA. Briefly, freshly collected colon tissue was homogenized before addition of Trizol reagent (Gibco, Paisley, Scotland, UK) and chloroform. The RNA was precipitated at -20°C in isopropanol overnight. The RNA pellet was recovered by centrifugation at 4°C, washed in 80 % ethanol, air dried and suspended in DEPC-treated water and quantified spectrophotometrically at 260 nm. One microgram of total cellular RNA was reverse transcribed (RT) by AMV reverse transcriptase at 42°C for 1 hr using poly d(T) ⁇ 5 as a primer.
- RT reverse transcribed
- the cDNA encoding human PAR-2 was subcloned into pcDNA3.1(+), amplified using p Bluescript (Stratagene, Cambridge, UK) and purified using a Qiagen maxiprep kit (Qiagen Ltd, Hertfordshire, UK). The product was sequenced to ensure correct orientation of the reading frame.
- Genomic DNA was extracted from the blood of 125 normal Caucasian individuals by standard techniques (33).
- Specific PAR-2 primers corresponding to nucleotides 699-718 in extracellular loop II (T2D-sense 5'-GCT CTT GGT GGG AGA CAG GT-3') and nucleotides 926-949 in extracellular loop III (T4U-antisense 5'-GGC TCT TAA TCA GAA AAT AAT GCA-3') were used to generate a PCR product of 250 bp from the genomic DNA samples.
- a SAU 96 I site was designed into the T2D-sense primer and was accomplished by replacing nucleotide 716 (T) for G (see primer T2D above, G(716) is underlined).
- a control Sau96I restriction site was present 100 bp from the 3' end of the PCR product.
- the PCR reaction was performed in a Gene Amp 2400 PCR System with 300 ng DNA, 150 ng of each primer, 200 ⁇ M dNTP's, 2.5 mM MgCl, and 1 U Taq polymerase in a 50 ⁇ l reaction volume starting at 94°C for 5 min, 35 cycles of denaturing at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s, with a final extension at 72°C for 5 min.
- the resulting PCR product of 250 bp was digested overnight at 37°C using 6U/reaction of Sau 96 I.
- the products were run on an acrylamide gel, stained in ethidium bromide for 5 min and observed by illumination under ultra violet light.
- the presence of a 150 bp and 100 bp band signified the absence of the polymo ⁇ hic alleles.
- the presence of a 130 bp and 100 bp band in the absence of the 150 bp band demonstrated the presence of both polymo ⁇ hic alleles.
- oligonucleotides corresponding to nucleotides 389-409 in extracellular loop I S ID-sense 5'-TGA AGA TTG CCT ATC ACA TAC-3'
- nucleotides 926-949 in extracellular loop III T4U-antisense 5'-GGC TCT TAA TCA GAA AAT AAT GCA-3'
- the PCR reaction was performed as described in the RFPL analysis except the annealing temperature was set to 54°C.
- Kirsten sarcoma transformed rat kidney epithelial cells (KNRK, American Tissue Type Culture Collection, Bethesda, MD, U.S.A) in fresh medium (DMEM, 5 % FCS (vol/vol), 100 ⁇ M sodium pyruvate, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, and 250 ng/ml amphotericin B) were seeded into 60 mm petri dishes and incubated overnight in 95 % air. 5 % CO 2 at 37°C.
- transfection was performed using the Lipofectamine method according to the manufactuerer ' s protocol (Gibco/BRL) with 8 ⁇ g of each construct per petri dish.
- Transfected cells were subcloned in geneticin (0.6 mg/ml) containing medium and clones were initially selected by their ability to produce a calcium signal in response to trypsin and the PAR-2 selective peptide tc-NHi.
- geneticin 0.6 mg/ml
- clones were initially selected by their ability to produce a calcium signal in response to trypsin and the PAR-2 selective peptide tc-NHi.
- To obtain permanent cell lines for each receptor cells expressing high levels of PAR-2 were isolated by fluorescence-activated cell sorting using the B5 anti-PAR-2 polyclonal rabbit antibody (as described elsewhere (34)).
- the calcium cell-signaling assay was performed as described previously (31). Briefly, cells were seeded in 80 cm 2 flasks (Gibco/BRL) and incubated until 90 % confluence was achieved. Following two washes with PBS (without calcium and magnesium) cells were incubated with 5 ml of non-enzymatic cell dissociation fluid, and centrifuged at 200 g for 10 min. The cell pellet was resuspended in 1 ml of DMEM, 10 % FCS and 0.25 mM sulphinpyrazone.
- the reading frame for PAR-2 was cloned from cDNA derived from human colon tissue using specific primers synthesized on the basis of the 5' and 3' extremities of the published sequence (4).
- a comparison of the obtained sequence with the published sequence revealed a transition from a Thymine to a Cytosine at nucleotide position 719.
- PAR?F240S is less sensitive to the PAR-2 agonists Trypsin, SLIGKV-NH 2 and SLIGRL- NH?, but more sensitive to tc-NH ⁇ .
- the concentration effect curves for trypsin and several PAR-2 selective activating peptides are shown in Figure 3.
- trypsin was 3 orders of magnitude more potent than the PAR-2AP, SLIGKV-NH 2 , tc-NH 2 and SLIGRL-NH 2 , and the relative rank order of potencies were trypsin»»tc-NH 2 >SLIGRL-NH 2 >SLIGKV-NH 2 .
- This order of agonist potencies is in keeping with a previous report (31). Trypsin stimulated a detectable calcium elevation at 1 nM reaching a near maximum response at 100 nM.
- the effective response to the PAR-2AP tc-NH 2 was between 1 - 100 ⁇ M, SLIGKV-NH 2 from 5 - 200 ⁇ M and SLIGRL-NH 2 was effective from 2-100 ⁇ M ( Figure 3a, b, c, and d respectively).
- the potencies of all the PAR-2 agonists were significantly different ( Figure 3). Trypsin was nearly 4 fold less potent ( Figure 3 and Table 2), activating the PAR 2 F240S receptor between 5 and 500 nM, resulting in a rightward shift in the concentration-effect curve (Figure 3a).
- the parent tethered ligand peptide, SLIGKV- NH 2 was also found to be less potent in this system by over 2 fold, activating the PAR 2 F240S receptor between 10 and 500 ⁇ M ( Figure 3b and Table 2). However, in both systems the maximal response to SLIGKV-NH 2 appeared be lower than the maximal response obtained with the other PAR-2 agonists (-50 % versus -60% relative to calcium ionophore).
- the -C-NH2 peptide was 4 fold more potent (Figure 3c and Table 2), activating the PAR 2 F240S receptor between 0.2 and 20 ⁇ M compared to 1 and 50 ⁇ M in the wild- type receptor.
- the potency of SLIGRL- NH 2 was reduced by a similar degree to that of SLIGKV-NH2 ( ⁇ 2.5 fold) activating PAR 2 F240S between 5 and 200 ⁇ M ( Figure 3 and Table 2).
- the relative order of potencies for these agonists in the PAR 2 F24OS system which differ quantitatively from those for the wild-type receptor were: trypsin»tc- NH 2 »SLIGRL-NH 2 >SLIGKV-NH 2 .
- PAR 2 F240S is More Sensitive to the Selective PAR-1 Agonist TFLLR-NH? but Displays no Difference in Sensitivity to SFLLR-NH 2 and Cit-NH 2 .
- PAR-2F Receptor Systems The activities of each agonist in the PAR-2S receptor system was expressed relative to the activity of each agonist in the PAR-2F receptor, according to the equation: REC, /EC w iid-type-
- the concentration of each agonist causing a response in the PAR 2 F240S receptor (ECP AR2F240S ) was divided by the concentration of that agonist required to cause the same calcium signal (relative to A23187) in the wild- type receptor (EC w ii d - tyPe ).
- Values are averages obtained from four points along the parallel region of the dose response curves shown in Figures 3 and 4. Values greater than 1.0 designate a sensitivity that is lower in the PAR 2 F240S receptor than in the wild-type receptor.
- Rat-PAR?F240S Displays Identical Changes in Agonist Sensitivity as the Human PAR 2 F240S.
- F240 residue is important for PAR-2 agonist specificity.
- a permanent cell line in KNRK cells was established.
- the relative potencies for the PAR-activating agonists on the rat-PAR 2 F240S receptor are displayed in Table II.
- trypsin was -2 fold less potent in the rat-PAR-2 receptor system provoking a response between 1 - 20 nM,(data not shown).
- SLIGRL-NH 2 was found to be -3 fold less potent in this system.
- TC-NH2 was 3 fold more potent in the rat-PAR 2 F240S receptor as was TFLLR-NH2.
- TLIGRL-NH: and tc-YPGKF-NH2 can selectively activate PAR : F240S hut not the wild- type PAR-2 receptor.
- TLIGRL-NH2 (50 ⁇ M) was virtually without effect, however TLIGRL-NH2 (50 ⁇ M) provoked a response similar to that of SFLLR-NH 2 (50 ⁇ M) in the PAR 2 F240S receptor.
- TLIGRL- NH: (50 ⁇ M) stimulated a small response, but this response was half that observed in the rat-PAR 2 F240S cell line.
- the PAR-4 peptide GYPGKF-NH 2 (400 ⁇ M) was without effect in rat and human wild-type and PAR F240S systems (data not shown).
- the tc-YGPKF- NH 2 peptide was found to have no effect in either of the rat or human wild-type cell lines ( Figure 5d).
- the main finding of this study is the discovery of a human PAR-2 genetic polymo ⁇ hism that displays differential activation in response to trypsin and other PAR agonists.
- the PAR 2 F240S receptor displayed reduced sensitivity to trypsin.
- SKLIGV-NH 2 and SLIGRL- NH 2 whilst an increase in sensitivity to tc-NH 2 and su ⁇ risingly the PAR-1 selective agonist TFLLR-NH 2 .
- the peptide TLIGRL-NH 2 can be used selective agonist of PAR 2 F240S.
- constructing the same mutation in rat- PAR-2 echoed these findings suggesting that F240 may participate directly in regulating agonist specificity.
- the changes in trypsin and other agonist potencies will have important implications for the development of PAR-2 antagonists and raise the possibility that this polymo ⁇ hism may be implicated in disease.
- SKLIGV-NH2 and SLIGRL-NH 2 were also less potent (about 2.5 and 2.8 fold respectively).
- the loss of potency by trypsin might occur due to the exposed tethered ligand docking less efficiently onto the body of the receptor as a result of a conformational change induced by the phenylalanine to serine mutation.
- the tethered ligand may dock efficiently onto the receptor but fail to induce a comparable conformational change, and hence activation, as that induced in the wild-type receptor.
- tc-NH 2 was 4 fold more potent at activating the PAR 2 F240S receptor.
- the main structural difference between the tc-NH 2 compound and the other PAR-2AP (including the natural tethered ligand) is the attached large aromatic tr ⁇ r ⁇ -cinnamoyl group.
- the loss of the benzene group from the F240 to S240 mutation may allow the tr ⁇ '-cinnamoyl group to dock more efficiently thereby inducing greater receptor activation.
- binding assays for PAR prove problematic, it is difficult to assess whether the differential agonist potencies observed are due to a change in ligand affinity or efficacy.
- TLIGRL-NH2 a peptide that is selective for our PAR 2 F240S receptor
- PAR2F24OS can tolerate conservative changes at position one of the activating petide. thereby providing convincing evidence this receptor may interact with peptide agonists in a different manner to that of the wild-type receptor.
- PAR-2 plays a key role in inflammation (19.21.22.38). PAR-2 is expressed at high levels in the gastrointestinal tract and has been demonstrated to regulate ion transport, gut motility and prostaglandin release (39-42). Interestingly, the bacterial protease Gingipain-R derived from Porphyromonas gingivahs activates PAR-2 (43), and this activation may provide one of the first inflammatory signals of pathogen invasion. Based on our current results, the presence of the PAR-2S polymo ⁇ hism may be predictive of. or contribute to, certain inflammatory and intestinal disorders.
- PAR 2 F240S receptor may have a deleterious effect in the lung, as PAR-2 has been reported to have a protective role in the lung by stimulating the release of the powerful bronchodilator prostaglandin E 2 (44).
- PAR 2 F24OS may be involved in diseases of the airways such as asthma.
- PAR-2 antagonists have called into question the role of this receptor in disease and the possibility of PAR-2 antagonists as novel anti-inflammatory agents (45.46). To date only one true antagonist has been developed for PAR-1 (47) and as yet none are available for PAR-2.
- Protease-activated receptor 3 is a second thrombin receptor in humans. Nature 386:502-506.
- Basolateral proteinase- activated receptor induces chloride secretion in M-l mouse renal cortical collecting duct cells. J Physiol (Lond) 521 Pt 1 :3-17.
- PAR-2 proteinase activated receptor-2
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FR2841559A1 (en) * | 2002-06-28 | 2004-01-02 | Pfizer | MODIFIED RECEPTORS, PREPARATION AND USES |
EP1511765A1 (en) * | 2002-06-10 | 2005-03-09 | University Of Edinburgh | Par-2-activating peptide derivative and pharmaceutical composition using the same |
GB2450747A (en) * | 2007-07-06 | 2009-01-07 | Univ Sheffield | Treatment of sensorineural hearing loss |
WO2012090207A3 (en) * | 2010-12-30 | 2012-10-04 | Hadasit Medical Research Services & Development Limited | Par1 and par2 c-tail peptides and peptide mimetics |
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US5874400A (en) * | 1993-07-26 | 1999-02-23 | Cor Therapeutics | Recombinant C140 receptor, its agonists and antagonists, and nucleic acids encoding the receptor |
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Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
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EP1511765A1 (en) * | 2002-06-10 | 2005-03-09 | University Of Edinburgh | Par-2-activating peptide derivative and pharmaceutical composition using the same |
EP1511765A4 (en) * | 2002-06-10 | 2005-10-12 | Univ Edinburgh | Par-2-activating peptide derivative and pharmaceutical composition using the same |
US7541339B2 (en) | 2002-06-10 | 2009-06-02 | The University Of Edinburgh | Par-2-activating peptide derivative and pharmaceutical composition using the same |
FR2841559A1 (en) * | 2002-06-28 | 2004-01-02 | Pfizer | MODIFIED RECEPTORS, PREPARATION AND USES |
WO2004003202A1 (en) * | 2002-06-28 | 2004-01-08 | Pfizer Inc | Modified par receptors, their preparation and their uses for selecting compounds which modulate par activity |
GB2450747A (en) * | 2007-07-06 | 2009-01-07 | Univ Sheffield | Treatment of sensorineural hearing loss |
WO2012090207A3 (en) * | 2010-12-30 | 2012-10-04 | Hadasit Medical Research Services & Development Limited | Par1 and par2 c-tail peptides and peptide mimetics |
US9745347B2 (en) | 2010-12-30 | 2017-08-29 | Hadasit Medical Research Services And Development Ltd. | PAR1 and PAR2 c-tail peptides and peptide mimetics |
US10611798B2 (en) | 2010-12-30 | 2020-04-07 | Hadasit Medical Research Services And Development Ltd. | PAR1 and PAR2 c-tail peptides and peptide mimetics |
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