WO2001017958A2 - Neuropeptide sf receptor assays, compounds and therapeutic methods - Google Patents
Neuropeptide sf receptor assays, compounds and therapeutic methods Download PDFInfo
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- WO2001017958A2 WO2001017958A2 PCT/US2000/024447 US0024447W WO0117958A2 WO 2001017958 A2 WO2001017958 A2 WO 2001017958A2 US 0024447 W US0024447 W US 0024447W WO 0117958 A2 WO0117958 A2 WO 0117958A2
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
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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
- G01N2333/70567—Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors
Definitions
- the present invention relates to the field of assays for compounds that interact with the receptor for Neuropeptide FF and its related peptides.
- Neuropeptide FF (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2
- NPAF octadecapeptide neuropeptide AF
- NPFF neuropeptide FF
- FMRFamide molluscan peptide FMRFamide
- NPSF neuropeptides
- NPSF neuropeptides
- a summary of the known NPSF related neuropeptides and their common abbreviations are shown below: Species Peptide Sequence Abbreviation
- NPSF neuropeptides
- binding sites for NPFF were shown to exist, no binding partner for these peptides was identified.
- the lack of a known receptor or other binding partner has hampered the design of assays and impeded efforts to discover and study compounds that mimic or alter the biological effects of NPSF or related neuropeptides.
- HG31 was previously identified as weakly homologous to the NPY receptors.
- the nucleic acid and polypeptide sequences of the human homolog of HG31 was described in U.S. Provisional Application 60/111,432 filed December 08, 1998 and EP 0 884 387 A2 published December 16, 1998. Variants of the nucleic acid and polypeptide sequences are also described in those documents.
- EP 0 884 387 A2 describes the utility of the sequences of the HG31 receptor (therein referred to as the HLW AR77 receptor) as including the treatment of infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2; pain; cancers; diabeties; obesity; anorexia; bulimia; Parkinson's disease; acute heart failure; hypotension; hypertension; urinary retention; osteoporosis; angina pectoris; myocardial infarction; ulcers; asthma; allergies; benign prostatic hypertrophy; and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles dela Tourett's syndrome, among others and diagnositc assays for such conditions.
- HG31 sequences described previously were stated to be similar to the receptors for NPY. Therefore, HG31 sequences reported in the art would be expected to be useful as "minus targets" in counter-screens in connection with screening projects designed to identify compounds that specifically interact with NPY family receptors, (see Hodgson, 1992, Bio/Technology 10:973-980, at 980). However, while the homology of HG31 to other receptors was reported, no actual ligand for the HG31 receptor was identified.
- the present invention solves this problem in the art and provides assays that employ the interaction of HG31 and NPSF and its related neuropeptides to determine whether a candidate compound is a ligand, agonist or antagonist of HG31.
- the present invention provides assays for the study of the interaction of NPSF or a related neuropeptide with the HG31 receptor.
- the assays are useful to identify whether a candidate compound can bind to the HG31 receptor under conditions in which NPSF or a related neuropeptide can bind the receptor.
- the assays are also useful to determine whether a candidate compound is an agonist or antagonist of HG31 in the presence of NPSF or a related neuropeptide.
- the above assays can be performed in a variety of formats including competitive, non-competitive and comparative assays in which the interaction of NPSF or a related neuropeptide with HG31 is assessed as a positive or negative control or compared to the result obtained with the candidate compound.
- a “compound” is an organic or inorganic assembly of atoms of any size, and includes small molecules (less than about 2500 Daltons) or large macromolecules, e.g., peptides, polypeptides, whole proteins, and polynucleotides.
- a “candidate” is a compound that may be a ligand of a HG31 receptor polypeptide.
- a candidate compound may also be an agonist or antagonist of the HG31 receptor. Whether or not the candidate is an actual agonist, antagonist or ligand of a HG31 receptor polypeptide is determined in an assay.
- an "agonist” is a compound that interacts with and activates a polypeptide of the HG31 receptor.
- An activated HG31 receptor polypeptide induces a change in a biochemical pathway linked to the receptor, e.g., can stimulate the cleavage of GTP by a G protein, activate the adenylate cyclase pathway or activate the phospholipase C- ⁇ pathway.
- an "antagonist” is a compound that interacts with and inhibits or prevents the activation of a polypeptide of the HG31 receptor.
- recombinant refers to the use recombinant genetic techniques to manipulate and/or produce polynucleotides, polypeptides, expression vectors, host cells and the like, “recombinant” is also used as an adjective to describe the products of such techniques.
- HG31 receptor and NPSF receptor are used interchangeably.
- FIGS. 1A-1C depict the nucleotide (SEQ ID NO:3) and amino acid (SEQ ID NO:4) sequences of short form of HG31.
- FIGS. 2A-2D depict the nucleotide (SEQ ID NO:5) and amino acid (SEQ ID NO:6) sequences of the long form of HG31.
- FIG.3 Activation of HG31 by human NPSF and its related peptides in the aequorin bioluminescence assay.
- HEK293/aeql7/G ⁇ l5 cells stably expressing HG31 were assayed against NPAF, NPFF, and NPSF.
- RLU Random Luminescence Units. Results shown are the means ( ⁇ SEM) of triplicate determinations.
- the effective half-maximum concentrations (EC 50 ) of NPAF, NPFF, and NPFF are 44, 39, 9.0 nM, respectively.
- FIG. 4 Activation of HG31 by NPSF and its related peptides in the melanophore assay.
- Melanophore cells were transfected with HG31 or pcDNA3 (vector control) and assayed against various peptides one day post-transfection. Results shown are the means ( ⁇ SEM) of duplicate determinations.
- the effective half- maximal concentrations (EC50) are NPSF, 1.8 nM; NPFF 9.6 nM; NPAF, 3.5 nM; LPLRF, 143 nM; PrP-20 (Prolactin-releasing peptide), 164 nM.
- FIG. 5 The nucleotide sequence (SEQ ID NO:7) and amino acid sequence (SEQ LD NO: 13) of the human NPFF gene as reported by Perry et al., FEBS Lett., 409:426-430.
- FIG. 6 Radioligand binding analysis of HG31.
- the present invention provides assays that make use of the interaction of Neuropeptide SF (NPSF) and the related mammalian neuropeptides AF (NPAF), and FF (NPFF) with the HG31 receptor.
- NPSF Neuropeptide SF
- NPAF neuropeptide AF
- NPFF FF
- the assays provide methods to identify compounds that are ligands of HG31 or are agonists or antagonists of the HG31 receptor.
- the invention includes compounds identified as agonists or antagonists of HG31 and their use in treatment of a patient. In particular, the invention includes administering an agonist or antagonist of the HG31 receptor as a therapy to pain and opioid tolerance.
- Neuropeptide SF Neuropeptide SF
- AF NPAF
- FF neuropeptide SF
- NPFF neuropeptide SF
- Their involvement in pain modulation includes inducing a vigorous abstinence syndrome in morphine-tolerant rats, regulating the density of opioid receptors, and modulating self-administration of morphine.
- Agonists and antagonists of the HG31 receptor can be useful in the treatment of pain, opioid tolerance, and other pain-related disorders. Therefore, assays of this invention are useful to discover compounds that mimic the action of these neuropeptides and to discover compounds that are agonists or antagonists of the HG31 receptor.
- HG31 was cloned as an orphan G protein-protein coupled receptor as described as U.S. Provisional Application 60/111,432 filed December, 08, 1998, and EP 0 884 287 A2 (therein referred to as HLWAR77) in December 16, 1998.
- the receptor has limited homology to other neuropeptide receptors such as those for neuropeptide Y (NPY). However, no ligand for HG31/HLWAR77 was identified.
- Cikos et al. published an orphan receptor called NPGPR that is identical to HG31/HLWAR77 except an extra 102 amino acids at the N-terminus (Cikos et al., 1999, Biochem. Biophys. Res. Commun., 256:352-356).
- HG31 in the U.S. Provisional Application 60/111,432 is referred to as the Short Form of HG31 (SEQ ID NO:4) while the form of NPGPR published by Cikos et al. is referred to as the Long Form of HG31 (SEQ ID NO:6).
- Polynucleotides and polypeptides of HG31 that can be usefully employed in the assays of the present invention include those described in U.S Provisional application 60/111,432 filed December 08, 1998, EP 0 884 387 A2 published December 16, 1998, Cikos et al., and as described herein.
- polynucleotide is a nucleic acid of more than one nucleotide.
- a polynucleotide can be made up of multiple polynucleotide units that are referred to by description of the unit.
- a polynucleotide can comprise within its bounds a polynucleotide(s) having a coding sequence(s), a polynucleotide(s) that is a regulatory region(s) and/or other polynucleotide units commonly used in the art.
- Polynucleotides of particular usefulness in the present invention are those disclosed in FIG. 1 (SEQ ID NO:3) & FIG. 2 (SEQ ID NO:5).
- HG31 polynucleotide As used herein in reference to a polynucleotide or gene sequence we may use the terms HG31 polynucleotide.
- SEQ LD NO:4 When referring expressly to a gene for a receptor having the particular polypeptide sequence recited in SEQ LD NO:4, we may refer to the gene by the proper name, the NPSF-Rla gene.
- the present invention also relates to the use of recombinant vectors and recombinant hosts, both prokaryotic and eukaryotic, which contain the substantially purified nucleic acid molecules disclosed throughout this specification.
- the DNA sequences used in this invention in whole or in part, can be linked with other DNA sequences, i.e., DNA sequences to which the HG31 sequence is not naturally linked, to form "recombinant DNA molecules" containing HG31.
- the DNA sequences can be inserted into vectors in order to direct recombinant expression of HG31.
- Such vectors may be comprised of DNA or RNA; for most purposes DNA vectors are preferred.
- Typical vectors include plasmids, modified viruses, bacteriophage, cosmids, yeast artificial chromosomes, and other forms of episomal or integrated DNA that can encode HG31.
- An "expression vector” is a polynucleotide having regulatory regions operably linked to a coding region such that, when in a host cell, the regulatory regions can direct the expression of the coding sequence.
- the use of expression vectors is well known in the art. Expression vectors can be used in a variety of host cells and, therefore, the regulatory regions are preferably chosen as appropriate for the particular host cell.
- a “regulatory region” is a polynucleotide that can promote or enhance the initiation or termination of transcription or translation of a coding sequence.
- a regulatory region includes a sequence that is recognized by the RNA polymerase, ribosome, or associated transcription or translation initiation or termination factors of a host cell. Regulatory regions that direct the initiation of transcription or translation can direct constitutive or inducible expression of a coding sequence.
- Polynucleotides useful in this invention contain full length or partial length sequences of the HG31 receptor gene.
- Polynucleotides of this invention can be single or double stranded. If single stranded, the polynucleotides can be a coding, "sense,” strand or a complementary, "antisense,” strand.
- Antisense strands can be useful as modulators of the receptor by interacting with RNA encoding the receptor. Antisense strands are preferably less than full length strands having sequences unique or highly specific for RNA encoding the receptor.
- the polynucleotides can include deoxyribonucleotides, ribonucleotides or mixtures of both.
- the polynucleotides can be produced by cells, in cell-free biochemical reactions or through chemical synthesis.
- Non-natural or modified nucleotides including inosine, methyl-cytosine, deaza-guanosine, and others known to those of skill in the art, can be present.
- Natural phosphodiester internucleotide linkages can be appropriate.
- polynucleotides can have non-natural linkages between the nucleotides.
- Non-natural linkages are well known in the art and include, without limitation, methylphosphonates, phosphorothioates, phosphorodithionates, phosphoroamidites and phosphate ester linkages.
- Dephospho-linkages are also known, as bridges between nucleotides. Examples of these include siloxane, carbonate, carboxymethyl ester, acetamidate, carbamate, and thioether bridges.
- Plastic DNA having, for example, N-vinyl, methacryloxytethyl, methacrylamide or ethyleneimine internucleotide linkages, can be used.
- PNA Peptide Nucleic Acid
- PNA is also useful and resists degradation by nucleases. These linkages can be mixed in a polynucleotide.
- nucleic acids claimed herein can be present in whole cells or in cell lysates or in a partially, substantially or wholly purified form.
- a polynucleotide is considered purified when it is purified away from environmental contaminants.
- a polynucleotide isolated from cells is considered to be substantially purified when purified from cellular components by standard methods while a chemically synthesized nucleic acid sequence is considered to be substantially purified when purified from its chemical precursors.
- a procedure using conditions of high stringency is as follows: Prehybridization of filters containing DNA is carried out for 2 hr. to overnight at 65°C in buffer composed of 6X SSC, 5X Denhardt's solution, and 100 ⁇ g/ml denatured salmon sperm DNA. Filters are hybridized for 12 to 48 hrs at 65°C in prehybridization mixture containing 100 ⁇ g/ml denatured salmon sperm DNA and 5-20 X 106 cpm of 32P-labeled probe. Washing of filters is done at 37°C for 1 hr in a solution containing 2X SSC, 0.1% SDS. This is followed by a wash in 0.1X SSC, 0.1% SDS at 50°C for 45 min. before autoradiography.
- the present invention also relates to the use of HG31 receptor polypeptides, also referred to as NPSF receptor polypeptides including fragments and mutant or polymorphic forms of HG31, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy- terminal truncations such that these provide for polypeptides or fragments thereof useful for screening for modulators, agonists and/or antagonists of HG31.
- NPSF receptor polypeptides also referred to as NPSF receptor polypeptides including fragments and mutant or polymorphic forms of HG31, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy- terminal truncations such that these provide for polypeptides or fragments thereof useful for screening for modulators, agonists and/or antagonists of HG31.
- One of skill in the art can determine whether such naturally occurring forms are mutant or polymorphic forms of HG31 by sequence comparison. One can further determine whether the encoded protein, or fragments of any HG31 protein, is biologically active by routine testing of the protein of fragment in a in vitro or in vivo assay for the binding of the HG31 receptor to NPSF or a related neuropeptide.
- Intracellular concentration of Ca2+ can be determined by a variety of methods, such as using the real-time, live cell, fluorometric imaging assaying with the FLIPR (Fluorometric Imaging Plate Reader) machine (MOLECULAR DEVICES, U.S.A.), or using reporter genes under the control of a Ca2+-responsive promoter (e.g., using vectors supplied by AURORA BIOSCLENCES, San Diego, CA, U.S.A.). It is known that there is a substantial amount of redundancy in the various codons which code for specific amino acids. Therefore, this invention is also directed to those DNA sequences encode RNA comprising alternative codons which code for the eventual translation of the identical amino acid.
- FLIPR Fluorometric Imaging Plate Reader
- the present invention discloses codon redundancy which can result in differing DNA molecules expressing an identical protein.
- this invention includes modified HG31 polypeptides which have amino acid deletions, additions, or substitutions but that still retain substantially the same biological activity, i.e., binding NPSF or related neuropeptides, as the a native HG31 polypeptide.
- assays using HG31 polypeptides having changes which do not substantially alter the physical or functional properties of the expressed protein are also included within the scope of this invention.
- a “conservative amino acid substitution” refers to the replacement of one amino acid residue by another, chemically similar, amino acid residue. Examples of such conservative substitutions are: substitution of one hydrophobic residue (isoleucine, leucine, valine, or methionine) for another; substitution of one polar residue for another polar residue of the same charge (e.g., arginine for lysine; glutamic acid for aspartic acid). In particular, substitution of valine for leucine, arginine for lysine, or asparagine for glutamine is not expected to cause a change in functionality of the polypeptide.
- DNA sequences coding for a peptide can be altered so as to code for a peptide having properties that are different than those of the naturally occurring peptide.
- Methods of altering the DNA sequences include but are not limited to site directed mutagenesis.
- altered properties include but are not limited to changes in the affinity of an enzyme for a substrate or a receptor for a ligand.
- NPSF receptors For the purposes of this invention, naturally occurring, or wild-type NPSF receptors have the amino acid sequences shown in FIGS. 1 (SEQ ID NO:4) and 2 (SEQ ID NO:6).
- a "functional equivalent" NPSF receptor polypeptide possesses a biological activity that is substantially the same as the biological activity of a wild type HG31.
- a HG31 polypeptide has "substantially the same biological activity" as the wild type if that polypeptide has a Kd for NPSF or related neuropeptides that is no more than 5-fold greater than the Kd of a HG31 polypeptide shown in FIGS. 1 (SEQ ID NO:4) or 2 (SEQ ID NO:6).
- the term “functional derivative” is intended to include those "fragments,” “mutants,” “variants,” “degenerate variants,” “analogs,” “homologues” or “chemical derivatives” of a wild type HG31 protein that exhibit substantially the same biological activity.
- fragment is meant to refer to any polypeptide subset of wild-type HG31.
- mutant is meant to refer to a polypeptide that may be substantially similar to the wild-type form but possesses distinguishing biological characteristics. Such altered characteristics include but are in no way limited to altered binding of NPSF or related neuropeptides, altered affinity for NPSF or related neuropeptides and altered sensitivity to chemical compounds affecting biological activity of the HG31 or functional derivative.
- variant is meant to refer to a molecule substantially similar in structure and function to either the entire wild-type protein or to a fragment thereof.
- a "polymo ⁇ hic" HG31 is an HG31 that is naturally found as an allele in the population at large.
- a polymo ⁇ hic form of HG31 can have a different nucleotide sequence from the particular HG31 alleles shown in FIGS. 1 (SEQ LD NO:3) and 2 (SEQ ID NO:5).
- SEQ LD NO:3 SEQ LD NO:3
- SEQ ID NO:5 SEQ ID NO:5
- a polymo ⁇ hic HG31 gene can encode the same or different amino acid sequence as those depicted herein.
- some polymo ⁇ hic forms HG31 will exhibit biological characteristics that distinguish the form from wild-type receptor activity, in which case the polymo ⁇ hic form is also a mutant.
- Polymo ⁇ hic forms encompass allelic variants.
- a protein or fragment thereof is considered purified or isolated when it is obtained at a concentration at least about five-fold to ten-fold higher than that found in nature.
- a protein or fragment thereof is considered substantially pure if it is obtained at a concentration of at least about 100-fold higher than that found in nature.
- a protein or fragment thereof is considered essentially pure if it is obtained at a concentration of at least about 1000-fold higher than that found in nature.
- Expression Vectors and Host Cells An important aspect of many assays is the step of providing a host cell expressing a recombinant NPSF receptor polypeptide on the surface thereof.
- a variety of expression vectors can be used to express recombinant HG31 receptor polypeptides in host cells.
- Expression vectors are defined herein as DNA sequences that are arranged for the transcription of cloned DNA and the translation of their mRNAs in an appropriate host.
- Such vectors can be used to express eukaryotic DNA in a variety of hosts such as bacteria, bluegreen algae, plant cells, insect cells and animal cells. Specifically designed vectors allow the shuttling of DNA between hosts such as bacteria-yeast or bacteria-animal cells.
- An appropriately constructed expression vector should contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and promoters.
- a promoter is defined as a DNA sequence operably linked to a coding region so that it interacts with cellular proteins to direct RNA polymerase to bind to DNA and initiate mRNA synthesis.
- a strong promoter is one which causes mRNAs to be initiated at high frequency.
- a promoter can be inducible.
- Expression vectors can include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses.
- mammalian expression vectors which can be suitable for recombinant NPSF receptor polypeptide expression, include but are not limited to, pcDNA3.1 (Invitrogen), pLITMUS28, pLITMUS29, pLITMUS38 and pLITMUS39 (New England Biolabs), pcDNAI, pcDNAIamp (Invitrogen), pcDNA3 (Invitrogen), pMClneo (Stratagene), pXTl (Stratagene), pSG5 (Stratagene), EBO- pSV2-neo (ATCC 37593) pBPV- 1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and 1ZD35 (AT
- bacterial expression vectors can be used to express recombinant NPSF receptor polypeptide in bacterial cells.
- Commercially available bacterial expression vectors which are suitable for recombinant human HG31 expression include, but are not limited to pQE (Qiagen), pETl la (Novagen), lambda gtll (Invitrogen), and pKK223-3 (Pharmacia).
- a variety of fungal cell expression vectors can also be used to express recombinant NPSF receptor polypeptide in fungal cells.
- Commercially available fungal cell expression vectors which are suitable for recombinant HG31 expression include but are not limited to pYES2 (INVITROGEN) and Pichia expression vector (INVITROGEN).
- insect cell expression vectors can be used to express recombinant receptor in insect cells.
- Commercially available insect cell expression vectors which are suitable for recombinant expression of HG31 receptor polypeptides include but are not limited to pBlueBacIII and pBlueBacHis2 (INVITROGEN), and pAcG2T (PHARMINGEN).
- the expression vectors generally described above and containing DNA encoding the NPSF-R1 receptor polypeptide can be used for expression of NPSF receptor polypeptide in an appropriate host cell.
- Recombinant host cells can be prokaryotic or eukaryotic, including but not limited to bacteria such as E.
- an expression vector is used to transform or transfect the appropriate cells, or cells can be obtained and cultured from an appropriate transgenic animal.
- L cells L-M(TK-) (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), Saos-2 (ATCC HTB-85), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL 171) and CPAE (ATCC CCL 209).
- the expression vector can be introduced into host cells via any one of a number of techniques including but not limited to transformation, transfection, protoplast fusion, and electroporation.
- the expression vector-containing cells are individually analyzed to determine whether they produce a HG31 receptor polypeptide. Identification of HG31 expressing cells can be done by several means, including but not limited to immunological reactivity with anti-HG31 antibodies, labeled ligand binding and the presence of host cell-associated HG31 activity.
- a cloned NPSF receptor cDNA can be recombinantly expressed by molecular cloning into an expression vector (such as pcDNA3.1, pQE, pBlueBacHis2 and pLITMUS28) containing a suitable promoter and other appropriate transcription regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce recombinant HG31 receptor polypeptide.
- an expression vector such as pcDNA3.1, pQE, pBlueBacHis2 and pLITMUS28
- Techniques for such manipulations can be found described in Sambrook, et ai, 1989, and are well known and easily available to the one of ordinary skill in the art.
- NPSF receptor polypeptides from recombinant polynucleotides can also be performed using in vitro produced synthetic mRNA.
- Synthetic mRNA can be efficiently translated in various cell-free systems, including but not limited to wheat germ extracts and reticulocyte extracts, as well as efficiently translated in cell based systems, including but not limited to microinjection into frog oocytes, with microinjection into frog oocytes being preferred.
- Expression systems can be designed to produce mutant, polymo ⁇ hic or allelic variants of the HG31 polypeptides shown in FIGS. 1 (SEQ ID NO:3) and 2 (SEQ ID NO:5). Fragments of HG31 receptor polypeptides can also be produced.
- recombinant DNA molecules including but not limited to the following can be constructed: a cDNA fragment containing the full-length open reading frame for HG31 as well as various constructs containing portions of the cDNA encoding only specific domains of the protein or rearranged domains of the protein.
- All constructs can be designed to contain none, all or portions of the 5' and/or 3' untranslated region of a HG31 cDNA.
- the expression levels and activity of HG31 receptor polypeptides can be determined following the introduction, both singly and in combination, of these constructs into appropriate host cells.
- the HG31 construct is transferred to a variety of expression vectors (including recombinant viruses), including but not limited to those for mammalian cells, plant cells, insect cells, oocytes, bacteria, and yeast cells.
- Assays of the present invention can be designed in many formats generally known in the art of screening compounds for biological activity or for binding to receptors.
- NPSF receptor polypeptides employing NPSF receptor polypeptides and NPSF or a related neuropeptide
- the assays of the present invention advantageously exploit the discovery that NPSF and related nueropeptides are high affinity ligands for HG31 receptor polypeptides that activate the HG31 receptor upon binding thereto.
- the present invention includes methods of identifying compounds that specifically bind to NPSF receptor polypeptides.
- Compounds that bind the receptor can be agonists or antagonists.
- the specificity of binding of compounds having affinity for HG31 can be shown by measuring the affinity of the compounds for recombinant cells expressing a HG31 receptor polypeptide on the surface thereof or affinity for membranes from such cells.
- Expression of HG31 receptor polypeptides and screening for compounds that bind to HG31 or that inhibit the binding of labeled NPSF or a related neuropeptide to these cells, or membranes prepared from these cells provides an effective method for the rapid selection of compounds with high affinity for HG31.
- the NPSF or a related neuropeptide can be radiolabeled but can also be labeled by other means known in the art and thereafter can be used to displace bound compounds or can be used as an activator of the HG31 in an assays. If one desires to produce a fragment of the HG31 receptor or mutant, polymo ⁇ hic or allelic variants of the receptor, one can test those products in the assays described below and compare the results to those obtained using a HG31 receptor polypeptide of SEQ ID NO:4 or SEQ ID NO:6. In this manner one can easily assess the ability of the fragment, mutant, polymo ⁇ h or allelic variant to bind compounds, be activated by agonists or be inactivated or inhibited by antagonists of the native HG31 receptor.
- the present invention includes assays by which compounds that are HG31 agonists and antagonists may be identified.
- the assay methods of the present invention differ from those described in the art because the present assays inco ⁇ orate at least one step wherein the interaction of NPSF or a related neuropeptide and the HG31 receptor polypeptide is inco ⁇ orated into the assay.
- the present invention includes a method for determining whether a candidate compound is a ligand of HG31 the method of which comprises:
- step (c) of the method is practiced are conditions that are typically used in the art for the study of protein-ligand interactions: e.g., physiological pH; salt conditions such as those represented by such commonly used buffers as PBS or in tissue culture media; a temperature of about 4°C to about 55°C.
- the NPSF or a related neuropeptide and candidate compound can be applied to the cell sequentially or concurrently.
- the assay can be conducted such that the HG31 is presaturated with NPSF or a related neuropeptide and the ability of the candidate compound to displace the bound NPSF or a related neuropeptide is assessed.
- the present invention also includes a method of using the interaction of NPSF or a related neuropeptide and a HG31 receptor polypeptide as a positive control for determining whether a compound is capable of binding to a HG31 receptor polypeptide, i.e., whether the compound is a potential agonist or an antagonist of HG31, where the method comprises:
- membranes can be prepared from the test cells and those membranes can be exposed to the compound.
- Such a modification utilizing membranes rather than cells is well known in the art and is described in, e.g., Hess et al, 1992.
- the present invention provides a method of using the interaction of NPSF or a related neuropeptide and HG31 in a competitive format for determining whether a candidate compound is capable of binding to a HG31 receptor polypeptide in membranes comprising:
- the present invention provides a method of using the interaction of NPSF or a related neuropeptide and a HG31 receptor polypeptide as a positive control for determining whether a candidate compound is capable of binding to a HG31 receptor polypeptide comprising:
- test cells by transfecting cells with an expression vector that directs the expression of HG31 on the surface of the cells;
- negative control cells i.e., cells lacking HG31 on their surface
- RNA encoding HG31 can be prepared as, e.g., by in vitro transcnption using a plasmid containing HG31 under the control of a bactenophage T7 promoter, and the RNA can be micromjected into Xenopus oocytes order to cause the expression of HG31 m the oocytes. Compounds are then tested for binding to the HG31 expressed the oocytes. Alternatively, rather than detecting binding, the effect of the compounds on the electrophysiological properties of the oocytes can be determined. As in all assays of this invention, a step using the interaction of NPSF or a related neuropeptide and HG31 is inco ⁇ orated into the assay.
- the present invention includes assays by which HG31 agonists and antagonists may be identified by their ability to stimulate or antagonize a functional response mediated by HG31.
- HG31 belongs to the class of proteins known as G- prote coupled receptors (GPCRs). GPCRs transmit signals across cell membranes upon the binding of ligand. The ligand-bound GPCR interacts with a heterotnmenc G-protein, causing the G ⁇ subunit of the G-protein complex to exchange GDP for GTP, and then disassociate from the G ⁇ and G ⁇ subunits. The G ⁇ subunit can then go on to activate a vanety of second messenger systems.
- GPCRs G- prote coupled receptors
- HG31 After being bound by NPSF or a related neuropeptide, HG31 particularly couples to Gi/o which, when activated, inhibits the adenylate cyclase, resulting in the inhibition of cyclic adenosine monophosphate (cAMP) production.
- cAMP cyclic adenosine monophosphate
- the present invention provides a method of using the interaction of NPSF or a related neuropeptide and HG31 in a functional assay for determining whether a candidate compound is an agonist of HG31, where the method compnses-
- the present invention also provides a method of using the interaction of NPSF or a related neuropeptide and HG31 in a functional assay for determining whether a candidate compound is an antagonist of HG31, where the method comprises: (a) providing test cells by transfecting cells with an expression vector that directs the expression of a HG31 receptor polypeptide on the surface of the cells;
- HG31 from step (c) is less than the amount bound by membranes from step (d), then the candidate compound is an antagonist of HG31.
- the present invention also provides a method of using the interaction of NPSF or a related neuropeptide and HG31 in a functional assay for determining whether a candidate compound is an agonist of HG31 based on the finding that activation of HG31 by NPSF or a related neuropeptide leads to inhibition of adenylate cyclase, where the method comprises:
- step (h) comparing the amount of cAMP produced in cells from (b), (c), (d), and (e). If the amount of cAMP produced by cells from Step (b) is less than the amount produced by cells from Step (d), then the candidate compound is an agonist of HG31.
- the amount of cAMP produced by cells from step (c) should be less than the amount produced by cells from step (e), which serves as a positive control.
- the present invention also provides a method of using the interaction of NPSF or a related neuropeptide and HG31 in a functional assay for determining whether a candidate compound is an antagonist of HG31 based on the finding that activation of HG31 by NPSF or a related neuropeptide leads to inhibition of adenylate cyclase, where the method comprises:
- step (c) subsequently to step (b), exposing these test cells to NPSF or a related neuropeptide;
- test cells (d) exposing another portion of the test cells to NPSF or a related neuropeptide only; (e) exposing cells from (c) and (d) to an activator of adenylate cyclase;
- the GPCR Upon the addition of an agonist of the GPCR to the transfected cells, the GPCR was activated and was able, via G ⁇ l5 or G ⁇ l6, to activate the ⁇ isoform of phospholipase C, leading to an increase in inositol phosphate levels in the cells.
- the present invention provides a method of determining whether a candidate compound is an antagonist of HG31 composing:
- step (c) subsequently or concurrently to step (b), exposing the cells to NPSF or a related neuropeptide;
- Another format for utilizing promiscuous G-proteins in connection with HG31 includes a method of identifying agonists of HG31 comprising: (a) providing cells that expresses both HG31 and a promiscuous
- Levels of inositol phosphates can be measured directly using ion- exchange chromatography or indirectly by monitoring calcium mobilization.
- Intracellular calcium mobilization is typically assayed in whole cells under a microscope using fluorescent dyes or in cell suspensions via luminescence using the aequorin assay.
- the above-described assay can be easily modified to form a method to identify antagonists of HG31.
- Such a method is also part of the present invention and comprises: (a) providing cells that expresses both HG31 and a promiscuous
- step (c) subsequently or concurrently to step (b), exposing a portion of the cells from step (b) to NPSF or a related neuropeptide, (d) measuring the level of inositol phosphates in the cells; where a decrease in the level of inositol phosphates in the cells in the presence of the candidate as compared to the level of inositol phosphates in the cells in the absence of the candidate indicates that the compound is an antagonist of HG31.
- conditions under which the binding steps of above-described methods are practiced are conditions that are typically used in the art for the study of protein-ligand interactions: e.g., physiological pH; salt conditions such as those represented by such commonly used buffers as PBS or in tissue culture media; a temperature of about 4°C to about 55°C
- the cells are transfected with expression vectors that direct the expression of HG31 and the promiscuous G-protem in the cells.
- the promiscuous G-protein is selected from the group consisting of G ⁇ l5 or G ⁇ l6.
- Expression vectors containing G ⁇ l5 or G ⁇ l6 are known in the art. See, e.g., Offermanns, Buhl et al, 1993; Amatruda et al, 1993.
- the cells express a chimenc G-protem, e.g. Gqi or Gqo Assay conditions and expression vectors using chimenc G-protems are known in the art. See. e g., Coward et al., 1999, Analytical Biochem., 270, 242-248. Additional types of functional assays that can be used to identify agonists and antagonists of HG31 include transcnption-based assays.
- Transcnption- based assays involve the use of a reporter gene whose transcnption is dnven by an mducible promoter whose activity is regulated by a particular intracellular event such as, e.g., changes in intracellular calcium levels that are caused by the interaction of a receptor with a ligand.
- Transcipti on-based assays are reviewed in Rutter et al., 1998
- the transcnption-based assays of the present invention rely on the expression of reporter genes whose transcnption is activated or repressed as a result of intracellular events that are caused by the interaction of an agonist with a HG31 receptor polypeptide.
- An extremely sensitive transcnption based assay is disclosed in
- the assay disclosed in Zlokarnik and U.S. Patent No. 5,741,657 employs a plasmid encoding ⁇ - lactamase under the control of an mducible promoter. This plasmid is transfected into cells together with a plasmid encoding a receptor for which it is desired to identify agonists.
- the mducible promoter on the ⁇ -lactamase is chosen so that it responds to at least one intracellular signal that is generated when an agonist binds to the receptor
- the level of ⁇ -lactamase in the transfected cells increases.
- This increase in ⁇ -lactamase is made measurable by treating the cells with a cell-permeable dye that is a substrate for ⁇ -lactamase
- the dye contains two fluorescent moieties. In the intact dye, the two fluorescent moieties are close enough to one another that fluorescent resonance energy transfer (FRET) can take place between them. Following cleavage of the dye into two parts by ⁇ - lactamase, the two fluorescent moieties are located on different parts, and thus can drift apart. This increases the distance between the fluorescent moieties, thus abolishing the amount of FRET that can occur between them. It is this decrease in FRET that is measured in the assay.
- FRET fluorescent resonance energy transfer
- One skilled in the art can modify the assay described in Zlokarnik and U.S. Patent No. 5,741,657 to form an assay for identifying candidate compounds that are agonists of HG31 receptor polypeptides by using an inducible promoter to drive ⁇ -lactamase that is activated by an intracellular signal generated by the interaction of agonists and the HG31 receptor.
- an inducible promoter to drive ⁇ -lactamase that is activated by an intracellular signal generated by the interaction of agonists and the HG31 receptor.
- a plasmid encoding HG31 is transfected into the cells.
- the cells are exposed tocandidate compounds for a few hours to allow the transcription and translation of the reporter enzyme D-lactamase.
- the cells are then exposed to the cell-permeable dye for a certain period to time to allow the dyes to load and to be cleaved.
- Those candidates that cause a decrease in FRET are likely to be actual agonists of the receptor.
- a portion of the transfected cells are treated with NPSF or a related neuropeptide and the decrease in FRET measured is compared to any decrease in FRET measured in cells exposed to the candidate compound. Additionally, a portion of the transfected cells are treated with NPSF or a related neuropeptide and the decrease in FRET can be monitored as a positive control.
- the present invention includes a method for identifying agonists of the HG31 receptor comprising:
- the assay descnbed above can be modified to an assay for identifying antagonists of the HG31 receptor.
- modification would involve the use of ⁇ - lactamase under the control of a promoter that is repressed by at least one intracellular signal generated by interaction of an agonist with the HG31 receptor and include conducting the assay in the presence of NPSF or a related neuropeptide.
- ⁇ - lactamase When the cells are exposed to substances suspected of being antagonists of the receptor, ⁇ - lactamase will be decreased, and FRET will be increased.
- the present invention includes a method for identifying antagonists of the HG31 receptor compnsing:
- step (e) exposing the cells from step (c) and (d) to a substrate of ⁇ - lactamase that is a cell-permeable dye that contains two fluorescent moieties where the two fluorescent moieties are on different parts of the dye and cleavage of the dye by ⁇ -lactamase allows the two fluorescent moieties to dnft apart;
- the mducible promoter that is repressed by at least one intracellular signal generated by interaction of an agonist with the HG31 receptor is a promoter that is repressed by decreases in cAMP levels or changes in potassium currents.
- the inducible promoter that is activated by at least one intracellular signal generated by interaction of an agonist with the HG31 receptor is a promoter that is activated by decreases in cAMP levels.
- ⁇ -lactamase is TEM-1 ⁇ -lactamase from Escherichia coli.
- the substrate of ⁇ -lactamase is CCF2/AM (Zlokarnik et al, 1998)
- the cells express a promiscuous G-protem, e.g., G ⁇ l5 or G ⁇ l ⁇ .
- the cells express a chimenc G-protein, e.g. Gqi or Gqo.
- Assay conditions and expression vectors using chimenc G-proteins are known in the art. See. e.g., Coward et al., 1999, Analytical Biochem., 270, 242-248.
- the mducible promoter is a promoter that is activated or repressed by NF- ⁇ B or NFAT.
- the assays descnbed above could be modified to identify inverse agonists. In such assays, one would expect a decrease in ⁇ -lactamase activity.
- inverse agonists can be identified by modifying the functional assays that were descnbed previously where those functional assays monitored decreases in cAMP levels. In the case of assays for inverse agonists, increases in cAMP levels would be observed.
- transcnption-based assays that can be used to identify agonists and antagonists of the HG31 receptor rely on the use of green fluorescent proteins or luciferase as reporter genes.
- the cells are eukaryotic cells. In another embodiment, the cells are mammalian cells.
- the cells are L cells L-M(TK-) (ATCC CCL 1.3), L cells L- M (ATCC CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) or MRC-5 (ATCC CCL 171).
- the assays described above can be carried out with cells that have been transiently or stably transfected with an expression vector that directs the synthesis of a HG31 receptor polypeptide.
- Transfection is meant to include any method known in the art for introducing HG31 into the test cells.
- transfection includes calcium phosphate or calcium chloride mediated transfection, lipofection, infection with a retro viral construct containing HG31, and electroporation.
- binding of a compound to HG31 can be measured by employing a labeled compound or agonist.
- the compound or agonist can be labeled in any convenient manner known to the art, e.g., radioactively, fluorescently, enzymatically.
- the HG31 receptor polypeptide has a full length amino acid sequence as shown in FIGS. 1 (SEQ ID NO:4) or 2 (SEQ ID NO:6).
- the melanophore screening system is descnbed in WO 92/01810, published February 6, 1992. Bnefly, melanophores are transfected to express a HG31 receptor polypeptide. In an assay for antagonists, the transformed melanophores are exposed to both an activating ligand, e.g., NPSF or a related neuropeptide, and a candidate compound Inhibition of the signal generated by the activating ligand indicates that the candidate is a potential antagonist of the receptor. In an assay for an agonist, the cells are contacted with candidate compounds and it is determined whether any compound activates the receptor to generate a signal. Activation of the receptor indicates that the candidate is a potential agonist of the receptor. Cells are exposed to an activating ligand, e.g., NPSF or a related neuropeptide, and used as a control m the later embodiment.
- an activating ligand e.g., NPSF or a related neuropeptide
- Yeast expressing mammalian adenylate cyclase Yeast expressing mammalian adenylate cyclase.
- yeast that express mammalian adenylate cyclase are descnbed in WO 95/30012, published November 9, 1995. These yeast can be engineered to co-express a HG31 receptor polypeptide m the presence of an appropnate G-protein.
- the transformed yeast are exposed to both an activating ligand, e.g., NPSF or a related neuropeptide, of HG31 and a candidate compound. Inhibition of the signal generated by the activating ligand indicates that the candidate is a potential antagonist of the receptor.
- the cells are contacted with candidate compounds and it is determined whether any compound activates the receptor to generate a signal. Activation of the receptor indicates that the candidate is a potential agonist of the receptor Cells are exposed to an activating ligand, e.g., NPSF or a related neuropeptide, and used as a control in the later embodiment.
- Yeast cells engineered to produce pheromone system protein sunogates can be used to screen for the ability of the surrogate to substitute for the cognate yeast pheromone receptor as descnbed in U.S. Patent No. 5,789,184, August 4, 1998.
- the method involves expressing a HG31 receptor polypeptide in Saccharomyces cerevisiae in which the receptor is linked to pheromone pathway
- the yeast G ⁇ subunit is generally deleted and replaced with a mammalian G ⁇ protein so that the mammalian G protein-coupled receptor can be coupled to the yeast pheromone pathway.
- Clones encoding agonist ligands for the HG31 receptor can be selected for their stimulation of the pheromone pathway.
- Clones encoding antagonist ligands for the HG31 receptor can be selected for their inhibition of the pheromone pathway in the presence of an HG31 agonist.
- libraries of chemicals can be screened for their agonist or antagonist activity by testing the chemicals directly. Phospholipase second signal screening
- Another screening technique involves expressing the HG31 receptor wherein the receptor is linked to a phospholipase C or D.
- Cells including CHO, endothelial, embryonic kidney and other cells can be used.
- ligand and candidates are screened for agonist or antagonist activities by detecting the activation or inhibition or the receptor's activation of the phospholipase second signal.
- yeast cells expressing a heterologous phospholipase is found in WO 96/40939, published December 19, 1996.
- HTPS high throughput screening
- HTPS assays are useful for a variety of large screening projects including screening preassembled chemical libraries, screening the output of combinatorial chemical synthesis of compounds and screening peptide display libraries (e.g., phage display libraries).
- NPSF-related neuropeptides as natural ligands of the o ⁇ han receptor HG31
- the complete coding sequence of the HG31 short form was amplified from a cDNA library using the two primers: sense primer, 5'-
- CTCTGCCCACCTCTTCTCTTC-3' (SEQ ID NO: 10) and antisense primer, 5'- AGAGAGGGCTTTCAGTAAATGTT-3 ' (SEQ LD NO: 11 ).
- the PCR product was purified and subcloned into pCR2.1 by TA cloning (LNVITROGEN, Carlsbad, CA, USA).
- the sequence of HG31 was verified by complete sequencing and then subcloned into pcDNA3.1(-) (INVITROGEN, Carlsbad, CA, USA), resulting in plasmid HG31/pcDNA3.1(-).
- HG31 sequence was then digested out from the pcDNA3.1 vector and sub-cloned into the vector pIRESpuromcyin (CLONTECH, PALO ALTO, CA, USA), resulting the plasmid HG3 lMRESpuromycin.
- the HEK293/aeql7 cell line was licensed in from NIH (Button and Brownstein, 1993, Cell Calcium, 14:663-671).
- the complete coding sequence of mouse promiscuous G protein G ⁇ l5 was cloned into the vector pIRES/zeocin
- HEK293/aeql7 cells were transfected into HEK293/aeql7 cells using Lipofectamine (GIBCO-BRL, Gaithersburg, MD, USA) and selected with zeocin. Individual stable colonies were isolated and tested for coupling of various receptors. One clone, #3 , showed promiscuous coupling, was named HEK293/aeql7/G ⁇ l5 and used for assays thereafter.
- HEK/293/aeql7/G ⁇ l5 were grown in Dulbecco's Modified Medium (DMEM, GIBCO-BRL, Gaithersburg, MD, USA) + 10% fetal bovine serum (heat inactivated), 1 mM sodium pyruvate, 500 ⁇ g/ ml Geneticin, 200 ⁇ g/ml zeocin, 100 ⁇ g/ml streptomycin, 100 units/ml of penicillin.
- DMEM Dulbecco's Modified Medium
- GIBCO-BRL Gibithersburg, MD, USA
- 10% fetal bovine serum heat inactivated
- 1 mM sodium pyruvate 500 ⁇ g/ ml Geneticin
- 200 ⁇ g/ml zeocin 100 ⁇ g/ml streptomycin
- penicillin 100 units/ml of penicillin.
- the HG31/pIRESpuromycin plasmid DNA was transfected into HEK293/ae
- Cells were seeded at ⁇ 20% confluency two days before the assay. For the assay, cells were washed once with DMEM + 0.1 % fetal bovine serum, and then charged for one hour at 37°C /5% CO2 in DMEM containing 5 ⁇ M coelenterazine cp (MOLECULAR PROBES, Eugene, OR, USA) and 30 ⁇ M glutathione.
- the cells were then washed once with Versene (GIBCO-BRL, Gaithersburg, MD, USA), detached using Enzyme-free cell dissociation buffer (GIBCO-BRL, Gaithersburg, MD, USA), diluted into ECB (Ham's F12 nutrient mixture (GIBCO-BRL) + 0.3 mM CaCl2, 25 mM HEPES, pH7.3, 0.1% fetal bovine serum).
- ECB Ham's F12 nutrient mixture (GIBCO-BRL) + 0.3 mM CaCl2, 25 mM HEPES, pH7.3, 0.1% fetal bovine serum).
- the cell suspension was centrifuged at 500x g for 5 min. The supernatant was removed, and the pellet was then resuspended in 10 mL ECB.
- the cell density was determined by counting with a hemacytometer and adjusted to 500,000 cells/ml in ECB.
- NPAF and NPSF Human neuropeptide NPAF and NPSF were custom-synthesized (PHOENIX PHARMACEUTICALS, INC., Belmont, CA, USA).
- Bovine neuropeptide FF was purchased from commercial sources (PENINSULA
- the peptides were diluted in ECB into 2X concentrates using 5-fold serial dilutions, and aliquoted into assay plates in triplicates at 0.1 ml/well.
- the cell suspension was injected at 0.1 ml/well, read and integrated for a total of 20 seconds using a Dynex MLX luminometer (DYNEX TECHNOLOGIES, MIDDLESEX, UK). Data were analyzed using the software
- HG31 -transfected cells showed a robust, dose-dependent response to human neuropeptide NPSF and NPAF, and bovine NPFF.
- the effective half-maximal concentrations (EC50) of NPAF, NPFF, and NPSF are 44, 39, 9 nM, indicating that HG31 is a high affinity receptor for NPSF and its related peptides.
- HG31-negative cells did not show any response to the peptides (data not shown).
- Xenopus laevis melanophores and fibroblasts were grown as described previously (Daniolos, et al, 1992. Pigment Cell Res. 3, 38- 43; and Lerner 1994. Trends Neurol. Sci. 17, 142-146). Briefly, melanophores were grown in Xenopus fibroblast-conditioned growth medium.
- the fibroblast-conditioned growth medium was prepared by growing Xenopus fibroblasts in 70% L-15 medium (Sigma), pH 7.3, supplemented with 20 % heat inactivated fetal bovine serum (GIBCO-BRL, Gaithersburg, MD, USA), 100 ⁇ g/ml streptomycin, 100 units/ml penicillin and 2 mM glutamine at 27°C.
- fibroblast-conditioned growth medium The medium from growing fibroblasts was collected, passed through a 0.2 micron filter (referred to as fibroblast-conditioned growth medium) and used to culture melanophores at 27°C. Plasmid DNA was transiently transfected into melanophores by electroporation using a BTX ECM600 electroporator (Genetronics, Inc. San Diego, CA, USA
- Melanophores were incubated in the presence of fresh fibroblast- conditioned growth medium for 1 hour prior to harvesting of cells. Melanophore monolayers were detached by trypsinization (0.25% trypsin, JHR BIOSCB ⁇ NCES, Lenexa, KS, USA), followed by mactivation of the trypsin with fibroblast- conditioned growth medium.
- the cells were collected by centnfugation at 200x g for 5 minutes at 4°C. Cells were washed once in fibroblast conditioned growth medium, centnfuged (200 x g, 5 minutes, 4°C) and resuspended at 5 x 106 cells per ml in ice-cold 70% PBS pH 7.0. 400 ⁇ l ahquots of cells in PBS were added to pre-chilled 1.5 ml tubes containing 2 ⁇ g of HG31/pcDNA3.1(-) plasmid DNA, and 22 ⁇ g of pcDNA3 plasmid vector DNA for a total of 24 ⁇ g DNA in a 40 ⁇ l total volume. Samples were incubated on ice for 20 minutes with mixing every 7 minutes.
- the media On the day following transfection, the media was replaced with fresh fibroblast-conditioned growth media and incubated for an additional day at 27°C pnor to assaying for receptor expression. On the day of ligand stimulation, medium was removed by aspiration and cells were washed with 70% L-15 media containing 15 mM HEPES, pH 7.3.
- Assays were divided into two separate parts in order to examine Gs/Gq-coupling which results in pigment dispersion in melanophores, or Gi-coupling which results in pigment aggregation.
- Assays were performed as follows. Cells were incubated m 100 ⁇ l of 70% L-15 media containing 15 mM HEPES, pH 7.3, for 1 hour the dark at room temperature, and then incubated in the presence of melatonm (2 nM final concentration) for 1 hour in the dark at room temperature to induce pigment aggregation.
- Tf the final transmittance at 590 nm.
- cell monolayers plated in 96-well microtiter plates were incubated in the presence of 100 ⁇ l/well of 70% L-15 media containing 1% fibroblast-conditioned growth medium, 2 mM glutamine, 100 ⁇ g /ml streptomycin, 100 units/ml penicillin and 15 mM HEPES, pH 7.3, for 15 minutes in the dark at room temperature to preset the cells to dispersion.
- Initial absorbance readings at 590 nm were determined, followed by the addition of ligands.
- HG31 were plated onto 96 well microtiter plates. Following the above pretreatment conditions, cells were incubated for 1 hour in the presence of a collection of 83 known peptides including NPSF. Final concentrations of the peptides were in the range of 100-500 nM.
- Pigment aggregation responses (Gi-coupled responses) were detected only with NPSF, NPAF, NPFF, LPRLF and prolactin releasing peptide -20 (PrP-20) with responses ranging from 71%, 92%, 86%, 56%, 48% respectively relative to a 100 nM melatonin control response. Background aggregation responses ranged from 10-22% of the melatonin control. Comparative background responses were seen in pcDNA3 only transfected melanophores, with no values exceeding background for the above peptides.
- Pigment dispersion responses were not detected with NPSF, NPAF, NPFF, LPRLF and prolactin releasing peptide.
- HG31 -transfected cells showed strong, dose- dependent pigment aggregation in response to NPSF, NPAF, NPFF, LPLRF (chicken brain peptide), and prolactin-releasing peptide (PrP-20), indicating that the activation of HG31 by NPSF and its related peptides is coupled to the Gi/o pathway.
- the effective half-maximum response concentrations (EC 50 ) of these peptides are: NPSF, 1.8 nM; NPAF, 3.5 nM; NPFF, 9.6 nM; LPLRF, 142 nM; PrP-20, 162 nM, indicating that HG31 is a high affinity receptor for NPSF, NPAF, and NPFF. All of the peptides were tested in the range of 6x10-11 _ l ⁇ lO-6 M.
- transgenes are genetic construct including a gene.
- the transgene is typically integrated into one or more chromosomes in the cells in an animal or its ancestor by methods known in the art. Once integrated, the transgene is carried in at least one place in the chromosomes of a transgenic animal.
- a gene is a nucleotide sequence that encodes a protein.
- the gene and/or transgene can also include genetic regulatory elements and/or structural elements known in the art.
- animal is used herein to include all mammals, except humans. It also includes an individual animal in all stages of development, including embryonic and fetal stages. Preferably the animal is a rodent, and most preferably mouse or rat.
- a "transgenic animal” is an animal containing one or more cells bearing genetic information received, directly or indirectly, by deliberate genetic manipulation at a subcellular level, such as by microinjection or infection with recombinant virus. This introduced DNA molecule can be integrated within a chromosome, or it can be extra-chromosomally replicating DNA.
- transgenic animal refers to a transgenic animal in which the genetic information was introduced into a germ line cell, thereby conferring the ability to transfer the information to offspring. If offspring in fact possess some or all of the genetic information, then they, too, are transgenic animals.
- the genetic information is typically provided in the form of a transgene carried by the transgenic animal.
- the genetic information received by the non-human animal can be foreign to the species of animal to which the recipient belongs, or foreign only to the particular individual recipient. In the last case, the information can be altered or it can be expressed differently than the native gene. Alternatively, the altered or introduced gene can cause the native gene to become non-functional to produce a "knockout" animal.
- a "targeted gene” or “Knockout” (KO) transgene is a DNA sequence introduced into the germline of a non-human animal by way of human intervention, including but not limited to, the methods described herein.
- the targeted genes of the invention include nucleic acid sequences which are designed to specifically alter cognate endogenous alleles of the non-human animal.
- HG31 receptor gene should not fully encode the same receptor endogenous to the host animal, and its expression product can be altered to a minor or great degree, or absent altogether.
- the altered HG31 gene induce a null, "knockout,” phenotype in the animal.
- a more modestly modified HG31 gene can also be useful and is within the scope of the present invention.
- ES cells can be obtained from pre-implantation embryos cultured in vitro and fused with embryos (M. J. Evans et al, Nature 292:154-156 (1981); Bradley et al, Nature 309:255-258 (1984); Gossler et al. Proc. Natl. Acad. Sci. USA 83:9065- 9069 (1986); and Robertson et al, Nature 322:445-448 (1986)).
- Transgenes can be efficiently introduced into the ES cells by a variety of standard techniques such as DNA transfection, microinjection, or by retrovirus-mediated transduction.
- the resultant transformed ES cells can thereafter be combined with blastocysts from a non-human animal.
- the introduced ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal (R. Jaenisch, Science 240: 1468-1474 (1988)). Animals are screened for those resulting in germline transformants. These are crossed to produce animals homozygous for the transgene.
- Methods for evaluating the targeted recombination events as well as the resulting knockout mice are readily available and known in the art. Such methods include, but are not limited to DNA (Southern) hybridization to detect the targeted allele, polymerase chain reaction (PCR), polyacrylamide gel electrophoresis (PAGE) and Western blots to detect DNA, RNA and protein.
- the creation and study of a transgenic animal may have a therapeutic aim.
- Gene therapy is discussed infra.
- the presence of a mutant allele or variant sequence within cells of an organism, particularly when in place of a homologous endogenous sequence, may allow the organism to be used as a model in testing and/or studying the role of the HG31 gene or substances which modulate activity of the encoded polypeptide and/or promoter in vitro or are otherwise indicated to be of therapeutic potential.
- Transgenic animals expressing HG31 as a transgene are provided as follows.
- a polynucleotide having an HG31 nucleotide sequence e.g., the nucleotide sequence of a cDNA or genomic DNA encoding a full length HG31 receptor, or a polynucleotide encoding a partial sequence of the receptor, sequences flanking the coding sequence, or both, can be combined into a vector for the integration of the polynucleotide into the genome of an animal.
- the HG31 sequence can be from a human HG31 or from the animal's HG31.
- the target cell for transgene introduction is a murine embryonic stem cell (ES).
- ES cells can be obtained from pre-implantation embryos of a variety of non-human animals cultured in vitro and fused with embryos (M. J. Evans et al, (1981); Bradley et al, (1984); Gossler et al. (1986); and Robertson et al, (1986)).
- the transgene is introduced into the murine ES cells by microinjection, however, a variety of standard techniques such as DNA transfection, or retrovirus- mediated transduction can be used.
- the injected ES cells are then combined with blastocysts from a non-human animal.
- the introduced ES cells colonize the embryo and contribute to the germ line of the resulting chimeric animal (R. Jaenisch, Science 240:1468-1474 (1988)).
- the chimeric mice are screened for individuals in which germline transformation has occuned. These are crossed to produce animals homozygous for the transgene.
- the targeted recombination events as well as the resulting mice are evaluated by techniques well known in the art, including but not limited to DNA (Southern) hybridization to detect the targeted allele, polymerase chain reaction
- PCR polyacrylamide gel electrophoresis
- PAGE polyacrylamide gel electrophoresis
- transgenic animals Three basic types of transgenic animals are created depending on the construction of the transgene vector. If the vector is designed to include a nucleotide sequence that encodes a full length human HG31 receptor and to integrate at a site other than the animal's endogenous HG31 gene, the resultant transgenic animal will express both a native and human HG31 receptors. If the vector is designed without a cognate HG31 gene and to integrate at the site of the animal's endogenous HG31 gene such that after integration the endogenous gene is altered to such an extent that the animal lacks a functional HG31 receptor, then a knockout animal is produced.
- the resultant animal lacks a native HG31 receptor and expresses a human HG31 receptor.
- Animals having a human gene and lacking an endogenous gene can also be created by crossing the first type of animal with a knockout animal to obtain animals homozygous for the knockout and homozygous for the added human HG31 gene. This can be facilitated if the human gene integrates in a chromosome different from the chromosome carrying the endogenous HG31 gene.
- Transgenic animals are a source of cells and tissues for use in assays of
- HG31 modulation, activation or inhibition Cells can be removed from the animals, established as cell lines and maintained in culture as convenient.
- Transgenic HG31 knockout animals can exhibit a variety of phenotypes including lower, little or no tolerance to opiates. In addition to being a valuable resource for the study of HG31 influenced aspects of neuropharmacology, knockout animals can be used to provide negative control cells lacking functional HG31 receptor polypeptides for use in assays.
- HEK293/aeql7/G ⁇ l5 cells were seeded in T-175 tissue culture flasks at -18 x l ⁇ 6 cells/flask for a total of 13 flasks, and were transfected using LIPOFECTAMINE-2000 (GIBCO-BRL, Gaithersburg, MD, USA) following the manufacturer's protocol. Ten flasks were transfected with HG31/pcDNA3.1(-) while the remaining three flasks were transfected with vector control (pcDNA3.1(+)). Three days after transfection, cells were washed once with PBS, scraped off into PBS, and spun down at l,000g for 5 min at 4°C. All procedures were conducted on ice from here on.
- the cell pellets were resuspended in 40 ml of harvest buffer [50 mM HEPES, pH 7.4, 1 mM EDTA, 4 complete protease inhibitor pellets (BOEHRINGER MANNHEIM, Indianapolis, IN, USA )] and homogenized for 4 bursts at 4°C using a 5 mm probe on a polytron homogenizer (POWERGEN 125, FISHER SCIENTIFIC, Pittsburgh, PA, USA). The suspension was then centrifuged at 48,000g for 20 min at 4°C. The pellet was then resuspended in harvest buffer, dispensed into microcentrifuge tubes at -10 mg of protein per tube, and centrifuged at 12,000g for at 10 min. at 4°C. The pellets were then resuspended in harvest buffer + 250 mM sucrose at a concentration of 8 mg/ml and stored at -70°C.
- Tyr'-NPFF Tetyr-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide
- SEQ ID NO: 12 was custom-synthesized by PHOENIX PHARMACEUTICALS (Belmont, CA, USA) and labeled at the N-terminal Tyr residue with 125i to a specific activity of - 2,000 Ci/mmol by WOODS ASSAY (Portland, Oregon, USA).
- the binding solution (0.2 ml in a 96-well plate) contained 80 ⁇ g (total protein) of HG31- expressing cellular membrane, 0.1 nM 125 ⁇ -Ty r l-NPFF, and varying concentrations of competitor in 25 mM Tris-HCl, pH 7.5, 5 mM MgCl2- After incubation for 1 hour at room temperature, binding reactions were harvested onto a GF/B filter (UNIFILTER-96, PACKARD, Downers Grove, IL, USA) and washed with 25 mM Tris-HCl, pH 7.5 (4 cycles, one wash for 1.0 second and another wash of 2.0 second per cycle) using a harvester (HARVESTER-9600, TOMTEC, Hamden, CT, USA).
- a harvester HARVESTER-9600, TOMTEC, Hamden, CT, USA.
- the filter was then dried for 30 minutes at 50°C and counted by gamma counting (TOPCOUNT NXT, PACKARD, Downers Grove, IL, USA). Data were analyzed and plotted using the software GRAPHPAD PRISM Version 3.0 (GRAPHPAD SOFTWARE, San Diego, CA, USA).
- HG31 -expressing membranes displayed specific binding to 125i-Ty r l-NPFF which was competed off by bovine NPFF, human NPSF, and human NPAF.
- concentration causing 50% inhibition of specific binding (IC50) for bovine NPFF, human NPSF, and human NPAF are 7.5,
- HG31 is a high affinity receptor for NPSF.
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US15343699P | 1999-09-10 | 1999-09-10 | |
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WO2001017958A9 WO2001017958A9 (en) | 2002-11-28 |
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Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU2002314955B2 (en) * | 2001-06-12 | 2006-10-26 | Ipr Licensing, Inc. | Method and apparatus for frequency selective beam forming |
WO2009033812A2 (en) * | 2007-09-11 | 2009-03-19 | Mondobiotech Laboratories Ag | Use of a combination of neuropeptide-ff and alpha-msh as a therapeutic agent |
WO2009033725A1 (en) * | 2007-09-11 | 2009-03-19 | Mondobiotech Laboratories Ag | Use of human neuropeptide as a therapeutic agent |
WO2009033724A1 (en) * | 2007-09-11 | 2009-03-19 | Mondobiotech Laboratories Ag | Use of c-type natriuretic peptide, alone or incombination with neuropeptide af, as a therapeutic agent |
WO2009043462A1 (en) * | 2007-09-11 | 2009-04-09 | Mondobiotech Laboratories Ag | Use of neuropeptide af as a therapeutic agent |
CN108976287A (en) * | 2018-08-15 | 2018-12-11 | 兰州大学 | The disulfide bond of one opioids and neuropeptide FF receptor multiple target point molecule BN-9 are cyclized analog and the preparation method and application thereof |
-
2000
- 2000-09-06 WO PCT/US2000/024447 patent/WO2001017958A2/en active Application Filing
Non-Patent Citations (2)
Title |
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PERRY ET AL.: 'A human gene encoding morphine modulating peptides related to NPFF and FMRFamide' FEBS LETTERS vol. 406, no. 3, 16 June 1997, pages 426 - 430, XP002907620 * |
VILIM ET AL.: 'Gene for pain modulatory neuropeptide NPFF: Induction in spinal cord by noxious stimuli' MOLECULAR PHARMACOLOGY vol. 55, no. 5, May 1999, pages 804 - 811, XP002907621 * |
Cited By (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU2002314955B2 (en) * | 2001-06-12 | 2006-10-26 | Ipr Licensing, Inc. | Method and apparatus for frequency selective beam forming |
WO2009033812A2 (en) * | 2007-09-11 | 2009-03-19 | Mondobiotech Laboratories Ag | Use of a combination of neuropeptide-ff and alpha-msh as a therapeutic agent |
WO2009033725A1 (en) * | 2007-09-11 | 2009-03-19 | Mondobiotech Laboratories Ag | Use of human neuropeptide as a therapeutic agent |
WO2009033724A1 (en) * | 2007-09-11 | 2009-03-19 | Mondobiotech Laboratories Ag | Use of c-type natriuretic peptide, alone or incombination with neuropeptide af, as a therapeutic agent |
WO2009043462A1 (en) * | 2007-09-11 | 2009-04-09 | Mondobiotech Laboratories Ag | Use of neuropeptide af as a therapeutic agent |
WO2009040025A3 (en) * | 2007-09-11 | 2009-05-22 | Mondobiotech Lab Ag | Use of neuropeptide sf, alone or in combination with glp-2, as a therapeutic agent |
WO2009033812A3 (en) * | 2007-09-11 | 2009-05-22 | Mondobiotech Lab Ag | Use of a combination of neuropeptide-ff and alpha-msh as a therapeutic agent |
US20100204130A1 (en) * | 2007-09-11 | 2010-08-12 | Dorian Bevec | Use of human neuropeptide as a therapeutic agent |
CN108976287A (en) * | 2018-08-15 | 2018-12-11 | 兰州大学 | The disulfide bond of one opioids and neuropeptide FF receptor multiple target point molecule BN-9 are cyclized analog and the preparation method and application thereof |
CN108976287B (en) * | 2018-08-15 | 2020-07-31 | 兰州大学 | Disulfide bond cyclized analog of opioid and neuropeptide FF receptor multi-target molecule BN-9 and preparation method and application thereof |
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WO2001017958A3 (en) | 2002-05-23 |
WO2001017958A9 (en) | 2002-11-28 |
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