(en)A recombinant canine TRPV2 channel which has been prepared by cDNA cloning and polymerase chain reaction techniques is disclosed. Expression systems for these channels and an assay using the expression systems are also disclosed. The recombinant TRPV2 channel can be used in assays to evaluate compounds which directly or indirectly interact with or bind to TRPV2 channel.

1.ApplicationNumber: US-4201608-A
1.PublishNumber: US-2009165151-A1
2.Date Publish: 20090625
3.Inventor: QIN NING
FLORES CHRISTOPHER M.
NEEPER MICHAEL P.

4.Inventor Harmonized: QIN NING(US)
FLORES CHRISTOPHER M(US)
NEEPER MICHAEL P(US)

5.Country: US
6.Claims:
(en)A recombinant canine TRPV2 channel which has been prepared by cDNA cloning and polymerase chain reaction techniques is disclosed. Expression systems for these channels and an assay using the expression systems are also disclosed. The recombinant TRPV2 channel can be used in assays to evaluate compounds which directly or indirectly interact with or bind to TRPV2 channel.

7.Description:
(en)CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority to Application No. 60/893,445 filed on Mar. 7, 2007, the entire contents of which are incorporated by reference herein.


FIELD OF THE INVENTION
The present invention relates to the canine TRPV2 (cTRPV2) channel, cloning of the channel, methods of screening for modulators of the TRPV2 channel and uses thereof.
BACKGROUND OF THE INVENTION
Both heat and cold evoke thermosensation, which, if of sufficient intensity, may elicit feelings of pain. Temperature detection in mammals is a critical function in the maintenance of thermal homeostasis and protection from potentially injurious temperature extremes. Considerable efforts have been put into elucidating the biochemical mechanisms involved in the detection, transduction and transmission of hot and cold sensations in neuronal tissues. Thermal stimuli activate specialized receptors located on sensory neurons, such as those deriving from the dorsal root ganglion (DRG) and the trigeminal ganglion (TG). When these stimuli are in the noxious range (i.e., very hot or cold), they activate a certain subset of thermo-sensitive receptors on a sub-population of sensory neurons called nociceptors (pain-sensing neurons). Upon activation, the thermo-sensitive receptors (e.g., ion channels) transduce the noxious stimulus into an electrical signal that is propagated along the sensory neuron to the spinal cord, where it is relayed to the brain, ultimately leading to the perception of pain. Accordingly, these thermo-sensitive receptors represent highly promising targets for developing drugs for the treatment of various painful conditions, particularly those in which thermal hypersensitivity and/or thermally evoked pain are present.
Several thermo-sensitive receptors have been implicated in sensing temperature changes. Currently, six members from the transient receptor potential (TRP) ion channel family have been proposed to serve as thermal sensors of temperatures ranging from noxious heat to noxious cold and thus are referred to as thermo-TRPs. The thermo-TRPs, which are non-selective cation channels, are also sensitive to chemical stimuli, such as capsaicin, menthol mustard oil, etc., which are known to produce a burning or cooling sensation. Importantly, all of the thermo-TRPs are expressed in nociceptive sensory neurons, either Aδ- or C-fibers, wherein thermo-, mechano- and chemo-sensory transduction take place. TRPV1, the most extensively characterized member of this family, is activated by moderate heat (˜43° C.), capsaicin, protons and certain lipidic mediators, such as anandamide (Caterina, M. J., et al. 1997 , Nature 389 (6653), 816-824; Tominaga, et al. 1998 , Neuron 21(3), 531-543) Moreover, numerous studies using a variety of experimental approaches have implicated TRPV1 in the transduction of pain signals in animal models of hyperalgesia (Caterina, M. J., et al. 2000 , Science 288(5464), 306-313; Davis, J. B., et al. 2000 Nature 405(6783), 183-187); hence, this TRP has been aggressively pursued as a target for the alleviation of certain types of pain.
The transient receptor potential V2 (TRPV2) protein (previously called and also known as vanilloid receptor-like 1 or VRL-1) is a member of the transient receptor potential (TRP) superfamily, which has been implicated in mediating diverse cellular functions, including the transduction of thermal, chemical and mechanical stimuli. TRPV2 is closely related to TRPV1 and was identified by homologous cloning from rat and human (Caterina et al., 1999 , Nature 398(6726), 436-441) as well as mouse (Kanzaki, et al. 1999 , Nat Cell Biol 1(3), 165-170). Functional studies have revealed that rat TRPV2 responds to noxious heat, with an activation threshold above 520° C. (Caterina, Rosen et al. 1999) and that mouse TRPV2 responds to changes in osmolarity or to membrane stretch (Muraki, et al. 2003 , Circ Res 93(9), 829-838). The hypothesis that TRPV2 is an endogenous sensor of noxious heat and mechanical stretch is further supported by findings that it is expressed in medium to large diameter Aδ mechano- and thermo-sensory neurons in the dorsal root ganglion (DRG) (Caterina et al., 1999 , Nature 398(6726), 436-441).
Unlike TRPV1, however, no selective activators of TRPV2 have been reported. Recently, a novel class of selective agonists, such as Δ 9 -tetrahydrocannabinol (Δ 9 -THC) as well as other related cannabinoids, that activate human, rat and mouse TRPV2 have been identified. (See U.S. application Ser. No. 11/589,340, entitled COMPOSITIONS AND METHODS FOR IDENTIFYING MODULATORS OF TRPV2 to Qin et al., filed Oct. 30, 2006).
There is a need to identify additional thermo-sensitive receptors, as they are potential targets for the treatment of pain. There is also a need to identify thermo-sensitive receptors in different species, as they can be used as model systems to investigate the effects of test compounds. Particularly, there is a need for systems that can be used to test compounds that potentially increase or decrease the activity of a thermo-sensitive receptor responding to noxious thermal stimuli, including noxious heat. Identification and testing of such compounds would enable the treatment of various disorders associated with neuropathic pain, inflammatory pain, various chronic pains or for uses in other conditions, such as the inflammatory immune response, febrile seizures and general epilepsy and/or conditions in which thermal hypersensitivity or thermally evoked pain are present as well as other human diseases related to these thermo-sensitive receptors.
SUMMARY OF THE INVENTION
The present invention is directed to recombinant DNA and protein of canine TRPV2. The protein, which may be used in a high throughput assay, is useful to identify modulators of functional TRPV2. The present invention is also directed to modulators identified in the assays disclosed herein and their use as therapeutic agents.
Other aspects, features and advantages of the invention will be apparent from the following disclosure, including the detailed description of the invention and its preferred embodiments and the appended claims.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 illustrates the nucleotide sequence and protein translation of canine TRPV2 cDNA (SEQ ID NO: 1).
FIG. 2 illustrates the amino acid sequence of canine TRPV2 (SEQ ID NO: 2).
FIG. 3 illustrates an alignment of the canine TRPV2 nucleotide sequence and the human TRPV2 nucleotide sequence (Genbank accession #: AF129112) (SEQ ID NO: 3). The sequences share 87.0 percent similarity and 84.2 percent identity.
FIG. 4 illustrates an alignment of the canine TRPV2 amino acid sequence and the human TRPV2 amino acid sequence (Genbank accession #: AF129112) (SEQ ID NO: 4). The sequences share 85.9 percent similarity and 85.9 percent identity.
FIG. 5 illustrates the percent homology between human, murine, rat and canine TRPV2 peptide sequences.
FIG. 6 illustrates the change in cellular calcium levels with addition of 100 μM Δ 9 -tetrahydrocannabinol (THC) in HEK293 cells transiently-transfected with canine TRPV2-expressing (black line) or pcDNA3.1 vector control (gray line) plasmid DNA. RFU; Relative Fluorescent Units.



DETAILED DESCRIPTION OF THE INVENTION
All publications cited herein are hereby incorporated by reference. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention pertains. As used herein, the terms “comprising”, “containing”, “having” and “including” are used in their open, non-limiting sense.
“An activity”, “a biological activity”, or “a functional activity” of a polypeptide or nucleic acid refers to an activity exerted by a polypeptide or nucleic acid molecule as determined in vivo or in vitro, according to standard techniques. Such activities can be a direct activity, such as an association with or an enzymatic activity on a second protein or an ion enzyme activity, or an indirect activity, such as a cellular signaling activity mediated by interaction of the protein with one or more than one additional protein or other molecule(s), including, but not limited to, interactions that occur in a multi-step, serial fashion.
A “biological sample” or “sample” as used herein refers to a sample containing or consisting of cell or tissue matter, such as cells, cell associated body fluids, biological fluids, culture supernatants, DNA, RNA, or protein isolated from a subject or patient. The “subject” can be bacteria, yeast, arthropods, a mammal, such as a rat, a mouse, a canine, a human, or any other organism, that has been the object of treatment, observation or experiment. Examples of biological samples include, for example, saliva, sputum, blood, blood cells (e.g., white blood cells), amniotic fluid, plasma, semen, bone marrow, tissue or fine-needle biopsy samples, urine, peritoneal fluid, pleural fluid, and cell cultures. Biological samples can also include sections of tissues, such as frozen sections taken for histological purposes.
A “cell” refers to at least one cell or a plurality of cells appropriate for the sensitivity of the detection method. Cells suitable for the present invention can be mammalian, for example, canine cells, or other prokaryote cells.
A “clone” is a population of cells derived from a single cell or common ancestor by mitosis. A “cell line” is derived from clonal expansion of primary cells, and is capable of stable growth in vitro for many generations.
A “DNA clone” is a section of DNA that has been copied, isolated or removed from a host and inserted into a vector molecule, such as a plasmid or a phage, or a chromosome, and then replicated to form many identical copies.
The term “cDNA” as used herein means a complementary DNA (cDNA) that has been reverse transcribed from a specific RNA template through the action of the enzyme reverse transcriptase.
A “gene” is a segment of DNA involved in producing a peptide, polypeptide, or protein, and the mRNA encoding such protein species, including the coding region, non-coding regions preceding (“5′UTR”) and following (“3′UTR”) the coding region. A “gene” can also include intervening non-coding sequences (“introns”) between individual coding segments (“exons”). “Promoter” means a regulatory sequence of DNA that is involved in the binding of RNA polymerase to initiate transcription of a gene. Promoters are often upstream of (or 5′ to) the transcription initiation site of the gene. A “regulatory sequence” refers to the portion of a gene that can control the expression of the gene. A “regulatory sequence” can include promoters, enhancers and other expression control elements such as polyadenylation signals, ribosome binding site (for bacterial expression), and/or an operator.
“Nucleic acid sequence” or “nucleotide sequence” refers to the arrangement of either deoxyribonucleotide or ribonucleotide residues in a polymer in either single- or double-stranded form. Nucleic acid sequences can be composed of natural nucleotides of the following bases: thymidine, adenine, cytosine, guanine, and uracil; abbreviated T, A, C, G, and U, respectively, and/or synthetic analogs. Synthetic analogs can include nucleotide and nucleoside analogs as well as non-nucleotide and non-nucleoside analogs.
The term “oligonucleotide” refers to a single-stranded DNA or RNA sequence of a relatively short length, for example, less than 100 residues long. For many methods, oligonucleotides of about 15-40 nucleotides in length are useful, although longer oligonucleotides of greater than about 40 nucleotides can sometimes be utilized. Some oligonucleotides can be used as “primers” for the synthesis of complimentary nucleic acid strands. For example, DNA primers can hybridize to a complementary nucleic acid sequence to prime the synthesis of a complementary DNA strand in reactions using DNA polymerases. Oligonucleotides are also useful for hybridization in several methods of nucleic acid detection, for example, in Northern blotting or in situ hybridization.
The term “probe” as used herein refers to a nucleic acid that can selectively hybridize under stringent hybridization conditions to a nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide capable of TRPV2 activity and having at least 90% sequence identity to SEQ ID NO: 2. Additionally, a probe can selectively hybridize under stringent hybridization conditions to a polypeptide capable of TRPV2 activity and having at least 90% sequence identity to SEQ ID NO: 2. Nucleic acid probes can be of any practical length similar to oligonucleotides in general and can be RNA or DNA, as described above. For example, probes can be from 10-20, 21-40, 41-60, or greater than 61 nucleotides in length. Such probes are useful for the detection of TRPV2 molecules in heterogeneous mixtures or for the amplification of rare copies, genomic copies and recombinant copies in vectors, as in the case of polymerase chain reactions, South-western blots and other common molecular biology techniques. Hybridization conditions are well known in the art, for example, but not limited to, those described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989).
A “polypeptide sequence” or “protein sequence” refers to the arrangement of amino acid residues in a polymer. Polypeptide sequences can be composed of the standard 20 naturally occurring amino acids, in addition to rare amino acids and synthetic amino acid analogs. Shorter polypeptides are generally referred to as peptides.
An “isolated” or “purified” nucleic acid molecule is one that is substantially separated from other nucleic acid molecules present in the natural source of the nucleic acid. An “isolated” nucleic acid molecule can be, for example, a nucleic acid molecule that is free of at least one of the nucleotide sequences that naturally flank the nucleic acid molecule at its 5′ and 3′ ends in the genomic DNA of the organism from which the nucleic acid is derived. Isolated nucleic acid molecules include, without limitation, separate nucleic acid molecules (e.g., cDNA or genomic DNA fragments produced by PCR or restriction endonuclease treatment) substantially independent of other sequences, as well as nucleic acid molecules that are incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid molecule can include a nucleic acid molecule that is part of a hybrid or fusion nucleic acid molecule. An isolated nucleic acid molecule can be a nucleic acid molecule that is: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) synthesized by, for example, chemical synthesis; (iii) recombinantly produced by cloning; or (iv) purified, for example, by cleavage and electrophoretic or chromatographic separation.
An “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The language “substantially free of cellular material” includes preparations of protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as a “contaminating protein”). When the protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium (i.e., culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation). When the protein is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals (i.e., it is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein). Accordingly such preparations of the protein have less than about 30%, 20%, 10%, and 5% (by dry weight) of chemical precursors or compounds other than the polypeptide of interest. Isolated biologically active polypeptides can have several different physical forms. An isolated polypeptide can exist as a full-length nascent or unprocessed polypeptide, or as a partially processed polypeptide or as a combination of processed polypeptides. The full-length nascent polypeptide can be posttranslationally modified by specific proteolytic cleavage events that result in the formation of fragments of the full-length nascent polypeptide. The full-length protein or fragments of the polypeptide can be chemically modified. A fragment, or physical association of fragments can have the biological activity associated with the full-length polypeptide; however, the degree of biological activity associated with individual fragments can vary. An isolated or substantially purified polypeptide, can be a polypeptide encoded by an isolated nucleic acid sequence as well as a polypeptide synthesized by, for example, chemical synthetic methods, and a polypeptide separated from biological materials, and then purified, using conventional protein analytical or preparatory procedures, to an extent that permits it to be used according to the methods described herein.
The terms “reducing expression”, “decreasing expression”, “knocking down” and “knock down” as used herein to refer to the reduction in the expression of one or more than one gene. This reduction in gene expression can be as a result of a reduction in the rates of transcription or translation or an increase in the rates of degradation or any combination of the above. For example, TRPV2 gene expression can be reduced through the use of RNA interference technology. Suitable RNA interference techniques are described and reviewed by Sandy et. al in Biotechniques. 2005 August; 39(2): 215-24, Mammalian RNAi: a Practical Guide, which is incorporated by reference.
The term “antisense” as used herein refers to a noncoding molecule that is complementary to the bases of a coding sequence of mRNA. Introducing a transgene coding for antisense mRNA is another strategy used to reduce or knock down expression of a gene. A strand of antisense mRNA can also be introduced into the cytosol by microinjection.
Analogous molecules with modified backbones have been designed, which change various characteristics of RNA, such as its instability to degradative enzymes, thermal stability and bond strength, thereby altering gene expression. Some alternative antisense structural types include phosphorothioate, PNA (peptide nucleic acid), LNA (locked nucleic acid), and 2′-O alkyl oligos.
The term “selectively hybridize” as used herein refers to the ability of a probe to bind to a particular or intended nucleic acid or polypeptide target sequence or molecule in a heterogeneous mixture of nucleic acids or polypeptides, for example, a probe can selectively hybridize to a nucleotide sequence encoding a TRPV2 or a polypeptide capable of TRPV2 activity in a heterogeneous mixture.
“Recombinant” refers to a nucleic acid, a protein encoded by a nucleic acid, a cell, or a viral particle, that has been modified using molecular biology techniques to something other than its natural state. For example, recombinant cells can contain nucleotide sequence that is not found within the native (non-recombinant) form of the cell or can express native genes that are otherwise abnormally expressed, under-expressed, or not expressed at all. Recombinant cells can also contain genes found in the native form of the cell wherein the genes are modified and re-introduced into the cell by artificial means. The term also encompasses cells that contain an endogenous nucleic acid molecule that has been modified without removing the nucleic acid from the cell; such modifications include those obtained, for example, by gene replacement and site-specific mutation.
A “recombinant host cell” is a cell that has had introduced into it a recombinant DNA sequence. Recombinant DNA sequences can be introduced into host cells using any suitable method, including, for example, electroporation, calcium phosphate precipitation, microinjection, transformation, biolistics “gene-gun” and viral infection. Recombinant DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell. For example, the recombinant DNA can be maintained on an episomal element, such as a plasmid. Alternatively, with respect to a stably transformed or transfected cell, the recombinant DNA has become integrated into the chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the stably transformed or transfected cell to establish cell lines or clones comprised of a population of daughter cells containing the exogenous DNA. Recombinant host cells can be prokaryotic or eukaryotic, including, E. coli , fungal cells, such as yeast, mammalian cells, such as cell lines of human, bovine, porcine, canine and rodent origin, and insect cells, such as Drosophila - and silkworm-derived cell lines. It is further understood that the term “recombinant host cell” refers not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications can occur in succeeding generations, due to either mutation or environmental influences, such progeny cannot, in fact, be identical to the parent cell but are still included within the scope of the term as used herein.
The term “vector” or “construct” refers to a nucleic acid molecule into which a heterologous nucleic acid can be or is inserted. Some vectors can be introduced into a host cell allowing for replication of the vector or for expression of a protein that is encoded by the vector or construct. Vectors typically have selectable markers, for example, genes that encode proteins allowing for drug resistance, origins of replication sequences, and multiple cloning sites that allow for insertion of a heterologous sequence. Vectors are typically plasmid-based and are designated by a lower case “p” followed by a combination of letters and/or numbers. Starting plasmids disclosed herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids by application of procedures known in the art. Many plasmids and other cloning and expression vectors that can be used in accordance with the present invention are well known and readily available to those of skill in the art. Moreover, those of skill readily can construct any number of other plasmids suitable for use in the invention. The properties, construction and use of such plasmids, as well as other vectors, in the present invention will be readily apparent to those of skill from the present disclosure.
The term “expression” as used herein refers to a multi-step process that includes transcription and translation of a gene and is often followed by post-translational modification, folding, assembly and trafficking/targeting of the resulting protein. The amount of protein that a cell expresses depends on the tissue, the developmental stage of the organism and the metabolic or physiologic state of the cell.
The term “expression vector” refers to a vector that comprises regulatory sequences necessary for transcription and translation of a cloned gene or genes and thus can transcribe and clone DNA. An expression vector can include one or more than one regulatory sequence, which can be selected based on the type of host cells used, operably linked to a gene. Regulatory sequences include promoters, enhancers and other expression control elements, for example, poly (A)+ sequences. Other expression vector components can include, but are not limited to, one or more than one of the following: a signal sequence, an origin of replication, one or more than one selection gene and a transcription termination sequence.
“Sequence” means the linear order in which monomers occur in a polymer, for example, the order of amino acids in a polypeptide or the order of nucleotides in a polynucleotide.
“Sequence identity or similarity”, as known in the art, is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. As used herein, “identity”, in the context of the relationship between two or more nucleic acid sequences or two or more polypeptide sequences, refers to the percentage of nucleotide or amino acid residues, respectively, that are the same when the sequences are optimally aligned and analyzed. For the purposes of comparing a queried sequence against, for example, the amino acid sequence SEQ ID NO: 2, the queried sequence is optimally aligned with SEQ ID NO: 2 and the best local alignment over the entire length of SEQ ID NO: 2 (331 amino acids) is obtained.
Analysis can be carried out manually or using sequence comparison algorithms. For sequence comparison, typically one sequence acts as a reference sequence, to which a queried sequence is compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, sub-sequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
Optimal alignment of sequences for comparison can be conducted, for example, by using the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol., 48:443 (1970). Software for performing Needleman & Wunsch analyses is publicly available through the Institut Pasteur (France) Biological Software website. The NEEDLE program uses the Needleman-Wunsch global alignment algorithm to find the optimum alignment (including gaps) of two sequences when considering their entire length. The identity is calculated along with the percentage of identical matches between the two sequences over the reported aligned region, including any gaps in the length. Similarity scores are also provided wherein the similarity is calculated as the percentage of matches between the two sequences over the reported aligned region, including any gaps in the length. Standard comparisons utilize the EBLOSUM62 matrix for protein sequences and the EDNAFULL matrix for nucleotide sequences. The gap open penalty is the score taken away when a gap is created; the default setting using the gap open penalty is 10.0. For gap extension, a penalty is added to the standard gap penalty for each base or residue in the gap; the default setting is 0.5.
Hybridization can also be used as a test to indicate that two polynucleotides are substantially identical to each other. Polynucleotides that share a high degree of identity will hybridize to each other under stringent hybridization conditions. “Stringent hybridization conditions” has the meaning known in the art, as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989). An exemplary stringent hybridization condition for an oligonucleotide probe comprises hybridization in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by one or more than one wash in 0.2×SSC and 0.1% SDS at 50-65° C., depending upon the length over which the hybridizing polynucleotides share complementarity.
A “reporter gene” refers to a nucleic acid sequence that encodes a reporter gene product. As is known in the art, reporter gene products are typically easily detectable by standard methods. Exemplary suitable reporter genes include, but are not limited to, genes encoding luciferase (lux), β-galactosidase (lacZ), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-glucuronidase, neomycin phosphotransferase, and guanine xanthine phosphoribosyl-transferase proteins.
The term “marker” as used herein refers to a specific substance from a subject that has a particular molecular feature that makes it useful for measuring the progress of disease or the effects of treatment. Biological samples comprising the marker are taken at periodic intervals from the subject to measure the progress of disease or the effects of treatment. The first measure of the marker is compared to the second measure and/or subsequent measures. In another example, the measure of the marker can be compared to a reference, a standard or a negative control.
TRPV's are vanilloid sub-family members of the 6 transmembrane domain transient receptor potential (TRP) superfamily. Structural, biochemical and pharmacological analyses of the TRPV genes and proteins has led to the discovery of 6 distinct members, including TRPV1 and TRPV2, which are thermo-sensitive to noxious heat. Activation of TRPV's mediates several receptor subtype-specific physiological processes that include but are not limited to the regulation of cellular calcium levels, excitability and transmitter release as well as sensory transduction and homeostasis.
The term “TRPV2 channel,” as used herein includes any receptor that can be naturally occurring, cloned, mutated, recombinantly expressed, chemically modified or substituted with amino acid analogs while maintaining TRPV channel activity.
The term “epitope” as used herein refers to a site on a large molecule against which an antibody may be produced and to which it will bind. Epitopes can be naturally occurring or chemically or synthetically produced.
A “compound that decreases the activity of TRPV2 channel” includes any compound that results in decreased signaling through the TRPV2 channel. In one embodiment, such a compound can be a partial agonist. In another embodiment, such a compound can be an antagonist, which can be either competitive or non-competitive. In another embodiment the compound can be both a partial agonist and an antagonist.
A “compound that increases the activity of TRPV2 receptor” includes any compound that results in increased signaling through the TRPV2 channel. Such a compound can be an agonist or a partial agonist.
A kit is any manufacture (e.g., a package or container) comprising at least one reagent, the manufacture being promoted, distributed, or sold as a unit for performing the methods of the present invention.
A putative homolog of TRPV2 from canine (SEQ ID NO: 1) has now been cloned. The TRPV2 of canine provides a means for identification of TRPV2 antagonists and agonists with potential utility for treating certain types of pain and painful conditions, including but not limited to neuropathic pain and inflammatory pain. An alignment of the canine TRPV2 nucleotide sequence and the human TRPV2 nucleotide sequence is shown in FIG. 3 . FIG. 3 shows an 86 percent sequence identity between these two sequences. An alignment of the canine TRPV2 amino acid sequence and the human TRPV2 amino acid sequence is shown in FIG. 4 . FIG. 4 shows an 84 percent sequence identity between these two sequences.
The present invention relates to novel TRPV2 nucleic acids, polypeptides and proteins encoded by these nucleic acids, recombinant TRPV2 materials, and methods involving the production, detection, and utilization of these materials.
Inhibition of the function or expression of TRPV2 proteins can be advantageous for screening and validating drug candidates both in vitro and in vivo. Since TRPV2 is implicated in noxious heat sensation, it is anticipated that inhibition of TRPV2 activity is also relevant for therapeutic applications where neuropathic, inflammatory and chronic pains occur.
In attempts to clone the TRPV2 orthologous, a PCR-based strategy was employed. Oligonucleotide primers were synthesized according to the sequences set forth in SEQ ID NO: 5 and SEQ ID NO: 6. These primers were successfully used to amplify a portion of the TRPV2 sequence from position 761 to position 2200 SEQ ID NO: 1. The full length cDNA was then cloned by both 5′ and 3′ RACE-PCR. In the present invention, the TRPV2 gene was cloned from the cDNA of canine dorsal root ganglion. The TRPV2 gene was sequenced, and the nucleotide sequence is shown as SEQ ID NO: 1 (see also, FIG. 1 ). SEQ ID NO: 1 encodes a 765 residue polypeptide (see, SEQ ID NO: 2), as shown in FIG. 2 .
In the present invention, the TRPV2 nucleic acid was also subcloned into an expression vector and transformed into a host cell for expression of the TRPV2 protein. This recombinant TRPV2 cell system was shown to express a functional TRPV2 protein that responded to Δ 9 -THC in a dose-dependent manner.
In other diagnostic assays which are capable of detecting the expression of TRPV2, the level of expression of mRNA corresponding to the TRPV2 gene can be detected utilizing commonly used molecular biological methods, for example, Northern blotting, in situ hybridization, nuclease protection assays, RT-PCR (including real-time, quantitative PCR), high density arrays and other hybridization methods. Accordingly, in another embodiment, an assay capable of detecting the expression of one or more than one TRPV2 gene in a biological sample is provided, which comprises contacting a biological sample with an oligonucleotide capable of hybridizing to a TRPV2 nucleic acid. The oligonucleotide is generally from 10-20 nucleotides in length for PCR/primer extension experiments. Longer oligonucleotides of approximately 40-50 nucleotides are more regularly utilized for in situ or blot hybridizations. Sequences even longer than 50 nucleotides can also be employed in the detection methods of this invention. RNA can be isolated from the tissue sample by methods well-known to those skilled in the art as described, for example, in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (1996). One preferred method for detecting the level of mRNA transcribed from the TRPV2 genes is by RT-PCR. Details of RT-PCR techniques are well known and also described, for example, in Pfeffer et al. Efficient one-tube RT-PCR amplification of rare transcripts using short sequence-specific reverse transcription primers. BioTechniques 18, 204-206. (1995) and Giannoni et al. An improved reverse transcription-polymerase chain reaction method to study a lipoprotein gene. Journal of Lipid Research 35 (2): 340 (1994).
Optionally, all or a portion of the TRPV2 nucleic acid sequence can be used to probe nucleic acid preparations from other species to determine the presence of similar sequences. For example, all or a portion of the TRPV2 nucleic acid can be used as a probe to identify cDNA or genomic nucleic acid sequences from other species that are similar to the TRPV2 sequence. Positive clones can be identified as those that hybridize to the TRPV2 probe.
The invention also relates to isolated nucleic acid fragments. Isolated nucleic acids comprising fragments of SEQ ID NO: 1 are useful for a variety of purposes. For example, these sequences can be used as oligonucleotide probes for the detection of TRPV2 nucleic acids or for the detection of sequences that flank TRPV2 nucleic acids. These sequences can be used as oligonucleotide primers for the amplification of TRPV2 nucleic acids. For many methods, oligonucleotides of about 16-25 nucleotides, or from about 26-35 nucleotides in length are useful, although longer oligonucleotides of greater than about 35 nucleotides can also be utilized. Some oligonucleotides can be used as “primers” for the synthesis of complimentary nucleic acid strands. For example, DNA oligonucleotide primers can hybridize to a complimentary nucleic acid sequence to prime the synthesis of a complimentary DNA strand in reactions using DNA polymerases. Oligonucleotides are also useful for hybridization in several methods of nucleic acid detection, for example, in Northern blotting or in situ hybridization. Fluorescently-labeled oligonucleotides, for example, Cy3-dCTP-labeled fluorescent cDNA probes, are particularly useful in hybridization methods using microarrays. Oligonucleotides can also be used for the preparation of chimeric nucleic acids that encode a portion or all of the TRPV2 polypeptide fused to another polypeptide sequence, for example, one or more than one motif or domain of the TRPV2 sequence recombined with one or more than one motif or domain from one or more than one heterologous sequence. In some embodiments, this can affect the activity of the TRPV2 channel. Oligonucleotides can be used in other methods; for example, RT-PCR using SEQ ID: 5 and SEQ ID: 6 or SEQ ID: 7 and SEQ ID: 8 can be used to identify TRPV2 expression. Oligonucleotides can further be used in techniques such as RNAi to reduce TRPV2 expression. In addition to nucleic acid sequences encoding TRPV2 polypeptides, the invention also includes TRPV2 polypeptides, TRPV2 polypeptide variants, fragments of TRPV2 polypeptides and TRPV2 polypeptides having additional amino acids.
TRPV2 polypeptide variants are polypeptides capable of TRPV2 activity in which substitutions have been made in the amino acid sequence from the sequence shown in SEQ ID NO: 2. These substitutions can be as a result of naturally occurring mutations during DNA synthesis, transcription or translation, the use of amino acid analogs, chemical modifications or other molecular biology techniques well known in the art.
Fragments of TRPV2 polypeptides include polypeptides capable of TRPV2 activity, which are shorter in length than the sequence shown in SEQ ID NO: 2. These fragments include any polypeptide capable of TRPV2 activity, which are less than around 95 amino acids in length. For example, a fragment of TRPV2 can be from 65 to about 735 amino acids in length and still be capable of TRPV2 activity. Fragments of TRPV2 polypeptides may be created by naturally occurring mutations during DNA synthesis, transcription or translation. Those skilled in the art will readily recognize that the use of enzymes that cleave either DNA or proteins, chemical modifications or other molecular biology techniques can be employed to create fragments of TRPV2 polypeptides.
TRPV2 polypeptides or TRPV2 polypeptide fragments can be generated using any sort of synthetic or molecular biological technique. Standard synthetic peptide techniques can be used to generate smaller TRPV2 polypeptide fragments. Techniques for the synthesis of peptide fragments are well known and are described in, for example, Barany and Merrifield, Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A., Merrifield, et al., J. Am. Chem. Soc., 85: 2149-2156 (1963), and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill. (1984).
TRPV2 polypeptides that have additional amino acids and are longer in length than the sequence shown in SEQ ID NO: 2 are also envisioned within the scope of the invention. As described herein, the TRPV2 polypeptide can also have additional amino acid residues at its amino terminus, its carboxyl terminus or both. Such additional residues are useful for a variety or purposes, including, for example, immunodetection, purification, cellular trafficking, enzymatic activity, etc. These proteins can include any polypeptide capable of TRPV2 activity, which is greater than 10 amino acids in length. For example a recombinant TRPV2 can be from 1 to about 775 amino acids in length and still be capable of TRPV2 activity. Recombinant TRPV2 polypeptides can be created by naturally occurring mutations during DNA synthesis, transcription or translation, the use of enzymes that either ligate DNA, such as T4 ligase, or fuse proteins.
Additionally fragments of TRPV2 and TRPV2 polypeptides having additional amino acids can comprise variant sequences in which substitutions have been made in the amino acid sequence from the sequence shown in SEQ ID NO: 2. These substitutions can be as a result of naturally occurring mutations during DNA synthesis, transcription or translation, the use of amino acid analogs, chemical modifications or other molecular biology techniques well known in the art.
A nucleic acid sequence can encode a TRPV2 fusion protein, which can include additional amino acid residues providing coordinates for bonding, such as ionic, covalent, hydrogen or Van der Waals or combinations thereof with organic or inorganic compounds. Useful additional amino acid sequences include, for example, poly-histidine residues useful for protein purification via Ni+-coupled residue, constant domains of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CH1, CH2, CH3), albumin, hemagluttinin (HA) or myc affinity epitope tags useful for the formation of immuno-complexes for detection or purification (antibodies against these moieties can be obtained commercially), polypeptides useful for detection such as the green fluorescent protein (GFP), enzymes, such as beta-galactosidase (β-Gal), glutathione S-transferase (GST), chloramphenicol acetyltransferase (CAT), luciferase, and alkaline phosphatase (A), signal sequences for protein trafficking and protease cleavage sequences useful for separating additional amino acid sequences from the TRPV2 sequence, if desired. Those skilled in the art will readily recognize that chemical modifications or other molecular biology techniques can be employed to create recombinant proteins or protein fusions. Fragments of TRPV2 and TRPV2 polypeptides having additional amino acids can comprise variant sequences in which substitutions have been made in the amino acid sequence as compared with the sequence shown in SEQ ID NO: 2. These substitutions can be as a result of naturally occurring mutations during DNA synthesis, transcription or translation, the use of amino acid analogs, other molecular biology techniques, or chemical modifications well known in the art.
Due to the degeneracy of the genetic code, more than one codon can be used to encode a particular amino acid, and therefore, a TRPV2 amino acid sequence (for example, SEQ ID NO: 2) can be encoded by any one of a plurality of nucleic acid sequences. Isolated nucleic acid includes sequences wherein one or more than one codon in the sequence is replaced by one or more than one codon of a different sequence but that encodes the same amino acid residue are herein referred to as “conservative codon substitutions”. Therefore, the invention encompasses nucleic acid sequences encoding SEQ ID NO: 2, which have one or more than one conservative codon substitution. One of skill in the art would be able to determine a particular nucleic acid sequence having one or more than one conservative codon substitution and encoding SEQ ID NO: 2, based on the sequence information provided herein. Conservative codon substitutions can be made in the nucleic acid sequence encoding the TRPV2 polypeptide, for example, the codons TTT and TTC (collectively referred to as TTT/C) can encode a Phe (phenylalanine) residue; other codon substitutions are as follows: TTA/G and CTT/C/A/G: Leu; ATT/C: Ile; ATG: Met; GTT/C/A/G: Val; TCT/C/A/G: Ser; CCT/C/A/G: Pro; ACT/C/A/G: Thr; GCT/C/A/G: Ala; TAT/C: Tyr; CAT/C: His; CAA/G: Gln; AAT/C: Asn; AAA/G: Lys; GAT/C: Asp; GAA/G Glu; TGT/C: Cys; GT/C/A/G: Arg; AGT/C: Ser; AGA/G; Arg; GGT/C/A/G:Gly. Conservative codon substitutions can be made at any position in the nucleic acid sequence that encodes the TRPV2 polypeptide.
The isolated nucleic acids of the invention can also include nucleic acid sequences that encode the TRPV2 polypeptide having additional amino acid residues. In some embodiments, the additional amino acids are present at the amino terminus, the carboxyl terminus, within the TRPV2 sequence or combinations of these locations. TRPV2 polypeptides having these types of additional amino acid sequences can be referred to as “TRPV2 fusion proteins”. In some cases, it may be more appropriate to refer to them otherwise as “chimeric” or “tagged” TRPV2 proteins, or the like, depending on the nature of the additional amino acid sequences. Nonetheless, one will be able to discern a TRPV2 polypeptide having additional amino acid sequences given the sequence information provided herein. The additional amino acid residues can be few in number, for example, from one to about 20 additional amino acid residues, or longer, for example, greater than about 20 additional amino acid residues. The additional amino acid residues can serve one or more than one function or purpose including, for example, serving as epitopes for protein (e.g., antibody) or small molecule binding; serving as tags for intracellular and extracellular trafficking; providing additional enzymatic or other activity; or providing a detectable signal.
Recombinant techniques can be used for the expression of TRPV2 including but not limited to fragments of TRPV2, variants of TRPV2 and fusions of TRPV2 with other proteins from host cells transformed with a TRPV2 nucleic acid. These methods include, for example, in vitro recombinant DNA techniques and in vivo genetic recombination (for example, see the techniques described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd Edition, Cold Spring Harbor Press, NY (2001); and Ausubel et al., eds., Short Protocols in Molecular Biology, 4th Edition, John Wiley & Sons, Inc., NY (1999)).
TRPV2 can be produced by introducing an expression vector encoding a TRPV2 polypeptide into a cell and culturing the cells to express the polypeptide. When a purified TRPV2 polypeptide is desired, a step can also be performed to isolate and, if desired, purify the TRPV2 polypeptide.
In another embodiment, the invention provides a recombinant nucleic acid construct that includes all or a portion of the TRPV2 coding sequence operably linked to a regulatory sequence. These recombinant nucleic acid constructs include recombinant expression vectors suitable for expression of the TRPV2 nucleic acid in a host cell. Recombinant expression vectors include one or more than one regulatory sequence that can be selected based on the type of host cells used for TRPV2 expression, operably linked to a TRPV2 nucleic acid sequence. Regulatory sequences include promoters, enhancers and other expression control elements, for example, poly (A)+ sequences. Regulatory sequences can be specific for prokaryotic cells, for example, bacterial cells, such as E. coli , or for eukaryotic cells, such as yeast cells, insect cells or mammalian cells (for example, HEK, CHO or COS cells). Regulatory sequences can be located cis or trans relative to the TRPV2 nucleic acid sequence. Regulatory sequences can include constitutive expression sequences that typically drive expression of the nucleic acid under a wide variety of growth conditions and in a wide variety of host cells, tissue-specific regulatory sequences that drive expression in particular host cells or tissues and inducible regulatory sequences that drive expression in response to a secondary factor. The choice and design of the expression vector can depend on such factors as the particular host cell utilized and the desired levels of polypeptide expression. Genes facilitating selection of transformed cells encode proteins that confer resistance to antibiotics or other toxins, for example, ampicillin, neomycin, methotrexate or tetracycline, complement auxotrophic deficiencies or supply critical nutrients not available from complex media.
Recombinant nucleic acid constructs used for expression of the TRPV2 polypeptide can also include constructs that can be transcribed and translated in vitro, for example, constructs having a T7 promoter regulatory sequence.
Vectors suitable for the expression of TRPV2 are known in the art and commercially available. Suitable vectors include, for example, pcDNA3.1, pCI-neo, pET-14b, pcDNAl Amp and pVL1392, which are available from Novagen and Invitrogen and can be used for expression in E. coli , COS cells and baculovirus infected insect cells, respectively.
In another embodiment, the invention provides a recombinant cell that includes a TRPV2 nucleic acid. Recombinant cells include those wherein a nucleic acid sequence has been introduced. Typically, recombinant cells are created by introducing a particular nucleic acid into cells using molecular biological techniques. However, recombinant cells also include cells that have been manipulated in other ways to promote the expression of a desired nucleic acid sequence. For example, regions that are proximal to a target nucleic acid sequence can be altered to promote expression of the target nucleic acid, or genes that act to regulate the expression of a target nucleic acid can be introduced into a cell. Recombinant cells, after periods of growth and division, may not be identical to the starting parent cell; however, these cells are still referred to as recombinant cells and are included within the scope of the term as used herein.
Host cells suitable for harboring and providing the machinery for TRPV2 expression include any prokaryotic or eukaryotic cells. Examples of suitable prokaryotic host cells are eubacteria, such as gram-negative or gram-positive organisms, for example, Enterobacteriaceae such as Escherichia , for example, E. coli, Enterobacter, Salmonella , for example, Salmonella typhimurium , as well as Bacilli, such as B. subtilis, Pseudomonas , and Streptomyces . Many higher eukaryotic host cells can be used, including insect cells, such as Drosophila S2 and Spodoptera Sf9 cells, mammalian cells, such as U937, THP-1, Chinese Hamster Ovary (CHO) cells, canine kidney (COS) cells, canine kidney (MDCK) cells, human cervical carcinoma (HeLa) cells, and human embryonic kidney (HEK) cells as well as plant cells.
Growth of the transformed host cells can occur under conditions that are known in the art. The conditions will generally depend upon the host cell and the type of vector used. Suitable induction conditions, such as temperature and chemicals, can be used and will depend on the type of promoter utilized. Examples of suitable media for the propagation of prokaryotes include Luria Broth (LB), also known as Miller's L Broth. Examples of suitable media for the propagation of eukaryotes include, Minimal Essential Medium (MEM), RPMI-1640, Dulbecco's Modified Eagle's Medium (DMEM) and F12 Medium.
Nucleic acids, including expression constructs, can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing a foreign nucleic acid molecule (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, virus mediated introduction, biolistics or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (supra) and other laboratory manuals.
Recombinant cells can be useful for the production of a TRPV2 polypeptide for purification purposes or for functional studies involving the TRPV2 polypeptide. For example, a recombinant TRPV2 cell can be used to test a number of compounds for their ability to alter the activity of the TRPV2 polypeptide. The recombinant TRPV2 cell can also be used to test how altering various properties of the TRPV2 polypeptide, for example, altering the amino acid sequence of the TRPV2 polypeptide, affects TRPV2 activity.
A variety of methods can be used for purification of the TRPV2 polypeptide. For example, crude purification can be performed using ammonium sulfate precipitation, centrifugation or other known techniques. A higher degree of purification can be achieved by suitable chromatographic techniques, including, for example, anion exchange, cation exchange, high performance liquid chromatography (HPLC), gel filtration, hydrophobic interaction chromatography and affinity chromatography, for example, immunoaffinity chromatography using antibodies directed against the TRPV2 protein. If needed, steps for refolding the TRPV2 proteins can be used to obtain the active conformation of the protein when the protein is denatured during intracellular synthesis, isolation or purification, such as resuspending the protein in 6M urea and dialyzing out the urea to facilitate protein refolding.
The invention also provides novel antibodies that selectively bind to the proteins of the present invention as well as fragments thereof. Such antibodies can be used to quantitatively or qualitatively detect the protein or peptide molecules of the present invention. As used herein, an antibody selectively binds a target protein or polypeptide fragment when an antibody binds the protein or polypeptide fragment and does not significantly bind to non-target proteins (i.e., the antibody does not bind non-TRPV2 related polypeptides or proteins).
As used herein, an antibody is defined in terms consistent with that recognized within the art: antibodies are multi-subunit proteins produced by any vertebrate organism in response to an antigen challenge. The antibodies of the present invention include monoclonal antibodies and polyclonal antibodies, as well as fragments of such antibodies, including, but not limited to, Fab or F(ab′)hd 2, Fc, and Fv fragments. Many methods are known for generating and/or identifying antibodies to a given target peptide. Several such methods are described by Harlow, Antibodies, Cold Spring Harbor Press, (1989). In general, to generate antibodies, an isolated peptide is used as an immunogen and is administered to a mammalian organism, such as a rat, rabbit or mouse. Either the full-length protein, an antigenic peptide fragment, or a fusion protein can be used.
Monoclonal antibodies can be produced by hybridomas, which are immortalized cell lines capable of secreting a specific monoclonal antibody. The immortalized cell lines can be created in vitro by fusing two different cell types, usually lymphocytes, one of which is a tumor cell.
Monoclonal antibodies can be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, for example, the hybridoma technique (Kohler and Milstein, Nature, 256:495-497, 1975); the human B-cell hybridoma technique (Kosbor et al., Immunology Today, 4:72, 1983); and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985). Such antibodies can be of any immunoglobulin class, including IgG, IgM, IgE, IgA, IgD or any subclass thereof. The hybridoma producing the monoclonal antibody of this invention can be cultivated in vitro or in vivo.
Antibodies can be prepared from regions or discrete fragments of the protein containing the desired amino acid sequence. Antibodies can be prepared from any region of the peptide as described herein. However, regions that include those involved in function/activity and/or protein/binding partner interaction are preferred. An antigenic fragment will typically comprise at least 10 contiguous amino acid residues. The antigenic peptide can comprise, however, at least 12, 14, 20 or more amino acid residues. Such fragments can be selected on a physical property, such as fragments corresponding to regions that are located on the surface of the protein (e.g., hydrophilic regions) or can be selected based on sequence uniqueness or based on the position of particular amino acid residue(s) or amino acid residue variants of the polypeptides provided by the present invention.
Detection of an antibody of the present invention can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material is luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 I, 131 I, 35 D or 3 H.
Immunoprecipitation of Ab2 using the antibodies of the present invention for detection of proteins interacting with TRPV2 is also envisioned within the scope of the present invention. Immunoprecipitation can also be used, for example, to discover new disease targets. The TRPV2 antibody can be linked to any immobilizing surface, such as Protein G-sepharose or Protein A-sepharose for the purpose of TRPV2 purification, to identify interacting proteins and potential drug targets. Additionally, the process can be used to determine the molecular weight and isoelectric point of immunoprecipitated proteins by one-dimensional or two-dimensional SDS-PAGE or LC MS/MS. Immunoprecipitation can be used to verify that an antigen of interest is synthesized by a specific tissue (i.e., that a labeled protein can be identified in tissues or cells cultured with labeled precursors). This procedure can be used to determine whether a protein contains carbohydrate residues by evaluating whether immunoprecipitated antigen from cells cultured with labeled monosaccharides is labeled. Characterization of the type of covalent modification, such as phosphorylation or glycosylation wherein the carbohydrates present on glycoproteins, can be performed by evaluating the incorporation of different labeled monosaccharides into immunoprecipitated proteins during cell culture and testing whether inhibitors of glycosylation alter the molecular weight of the immunoprecipitated protein. Immunoprecipitation can be used to determine precursor-product relationships by performing pulse-chase labeling followed by immunoprecipitation. Immunoprecipitation can be used to quantify synthesis rates of proteins in culture by determining the quantity of immunoprecipitated, labeled protein. Methods and reagents for immunoprecipitation are well known in the art and can be obtained from eBioscience, San Diego, Calif. or Upstate USA, Inc. Charlottesville, Va. The invention includes, antibodies useful for immunohistochemistry (IHC) and western blotting applications, for example, human TRPV2-specific antibodies against different regions within the canine TRPV2 protein.
In practicing the present invention, many conventional techniques in molecular biology, microbiology and recombinant DNA are used. These techniques are well-known and are explained in, for example, Current Protocols in Molecular Biology, Vols. I, II, and III, F. M. Ausubel, ed. (1997); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001). The compositions, kits, and methods of the invention have the following uses, among others: 1) selecting a composition or therapy for inhibiting, modulating, or activating TRPV2 in a patient; 2) assessing the efficacy of one or more than one test compound for inhibiting, modulating, or activating TRPV2 in a patient; 3) assessing the efficacy of a therapy for inhibiting, modulating, or activating TRPV2 in a patient; 4) treating a patient afflicted with certain types of pain or painful conditions, including but not limited to neuropathic pain and inflammatory pain.
These types of compounds can be identified using a system that includes a TRPV2 polypeptide or a TRPV2 nucleic acid. Compounds can be tested directly in vivo, for example, in a rat, mouse, canine, or in model systems thereof or directly in bacterial cultures or in eukaryotic cell cultures. These methods comprise assaying for the ability of various compounds to increase or decrease the expression of the TRPV2 protein or the activity of the TRPV2 protein as shown in Example 1. The compound screening and identification methods can be performed using conventional laboratory formats or in assays adapted for high throughput.
Candidate compounds encompass numerous chemical classes, including but not limited to, small organic or inorganic compounds, natural or synthetic molecules, such as antibodies, proteins or fragments thereof, antisense nucleotides, interfering RNA (iRNA) and ribozymes. Preferably, the candidate compounds are small organic compounds (i.e., those having a molecular weight of more than 100 Daltons yet less than about 1000 Daltons). Candidate compounds comprise functional chemical groups necessary for structural interactions with polypeptides and can include at least an amine, carbonyl, hydroxyl or carboxyl group, two of the functional chemical groups or three or more of the functional chemical groups. The candidate compounds can comprise cyclic carbon or heterocyclic structure and/or aromatic or polyaromatic structures substituted with one of the above identified functional groups. Candidate compounds also can be biomolecules, such as peptides, saccharides, fatty acids, sterols, isoprenoids, purines, pyrimidines, derivatives or structural analogs of the above or combinations thereof and the like. Where the compound is a nucleic acid, the compound typically is a DNA or RNA molecule, although modified nucleic acids having non-natural bonds or subunits are also contemplated. Modified nucleic acids can comprise nucleoside analogs, nucleotide analogs, non-nucleoside analogs, non-nucleotide analogs and others.
Candidate compounds are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides, synthetic organic combinatorial libraries, phage display libraries of random peptides and the like. Candidate compounds can also be obtained using any of the numerous approaches in combinatorial library methods known in the art, including biological library methods; spatially addressable, parallel solid phase or solution phase library methods, synthetic library methods requiring deconvolution, the “one-bead one-compound” library method, and synthetic library methods using affinity chromatography selection (Lam, Anti-Cancer Drug Design, 1997, 12:145). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural and synthetically produced libraries and compounds can be readily modified through conventional chemical, physical and biochemical means.
Further, known pharmacological agents can be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidation, etc. to produce structural analogs of the agents. Candidate compounds can be selected randomly or can be based on existing compounds that bind to and/or modulate the function of TRPV2 activity. Therefore, a source of candidate agents is one or more than one library of molecules based on one or more than one known compound that increases or decreases TRPV2 protein expression and/or G-protein coupled receptor activity in which the structure of the compound is changed at one or more than one position of the molecule to contain more or fewer chemical moieties or different chemical moieties. The structural changes made to the molecules in creating the libraries of analog activators/inhibitors can be directed, random, or a combination of both directed and random substitutions and/or additions. One of ordinary skill in the art in the preparation of combinatorial libraries can readily prepare such libraries based on the existing compounds.
Assays for the identification of TRPV2 modulators, such as antagonists and agonists, can be carried out manually or using an automated system. Automated systems are preferred if high throughput screenings are performed. For example, one type of automated system utilizes multi-well culture plates, for example, 96-well, 384-well or 1536-well culture plates, wherein each well can contain cells that express TRPV2 endogenously, recombinant cells having a nucleic acid encoding the TRPV2 protein or the purified TRPV2 protein.
The purified TRPV2 protein, antibodies against TRPV2 polypeptides disclosed herein and the various constructs used to express TRPV2 can be used in studies for understanding the mechanism of action of an identified TRPV2 modulator such as an agonist or antagonist as well as understanding the function of the protein.
After a compound has been identified that meets the desired criteria for modulating TRPV2 expression or activity, the compound can then be administered to a live subject, such as a living cell or other organism, or to cultured cells or tissues expressing TRPV2 to assess the efficacy. This can be useful to establish toxicity and other pharmacological parameters of the compound important for determining dosing regimens. For example, after a compound is identified using an ex vivo system containing a TRPV2 polypeptide, the compound can be administered to a culture of cells or an organism to examine various pharmacological aspects of the compound. Additionally, animal models can be used in conjunction with the nucleotides, antibodies and kits described herein to assess the toxicity of a compound. The TRPV2 systems as described herein are particularly advantageous for identifying and establishing dosing regimens in humans, because animals, particularly large specie, such as canines and primates, can be used, which are closer in weight to humans, compared with rats or mice, and therefore provide a more suitable animal model for estimating human dosing.
In another embodiment, the invention provides a method of identifying a compound useful for treating certain types of pain or painful conditions, including but not limited to neuropathic pain and inflammatory pain, comprising the steps of: (a) contacting a sample expressing TRPV2 with a test compound; and (b) determining whether the test compound increases or decreases the expression or activity of TRPV2. In some embodiments, the method further comprises the steps of: (a) administering the test compound to an animal; and (b) determining the extent to which the test compound affects the nociceptive status of the animal.
Various animal models of pain exist, for example, the spinal nerve ligation (SNL) model of nerve injury, which is a neuropathic pain model in rats developed by Kim and Chung (Pain, 50:355-363, 1992). Other suitable animal models of pain can be utilized in connection with the teachings herein. Commonly studied rodent models of neuropathic pain include the chronic constriction injury (CCI) or Bennett model; neuroma or axotomy model; and the partial sciatic transection or Seltzer model (Shir et al., Neurosci. Lett., 115:62-67, 1990). Exemplary neuropathic pain models include several traumatic nerve injury preparations (Bennett et al., Pain 33: 87-107, 1988; Decosterd et al., Pain 87: 149-58, 2000; Kim et al., Pain 50: 355-363, 1992; Shir et al., Neurosci Lett 115: 62-7, 1990), neuroinflammation models (Chacur et al., Pain 94: 231-44, 2001; Milligan et al., Brain Res 861: 105-16, 2000) diabetic neuropathy (Calcutt et al., Br J Pharmacol 122: 1478-82, 1997), virus-induced neuropathy (Fleetwood-Walker et al., J Gen Virol 80: 2433-6, 1999), vincristine neuropathy (Aley et al., Neuroscience 73: 259-65, 1996; Nozaki-Taguchi et al., Pain 93: 69-76, 2001), and paclitaxel neuropathy (Cavaletti et al., Exp Neurol 133: 64-72, 1995), as well as acute nociceptive tests models and inflammatory models, such as those in which inflammation is produced by an injection of carrageenan, complete Freund's adjuvant (CFA), zymosan or capsaicin (Brennan, T. J. et al. Pain 64:493, 1996; D'Amour, F. E. and Smith, D. L. J Pharmacol 72: 74-79, 1941; Eddy, N. B. et al. J Pharmacol Exp Ther 98:121, 1950; Haffner, F. Dtsch Med Wochenschr 55:731, 1929; Hargreaves, K. et al. Pain 32: 77-88, 1988; Hunskaar, S. et al. J Neurosci Meth 14:69, 1985; Randall, L. O. and Selitto, J. J. Arch. Int. Pharmacodyn 111: 409-419, 1957; Siegmund, E. et al. Proc Soc Exp Bio Med 95:729, 1957). In another aspect, the animal model involves a dog, or a primate, for example, a canine or a human.
Therapeutic efficacy and toxicity can be determined by standard pharmaceutical procedures in cell cultures or experimental animals by calculating, for example, the ED 50 (the dose therapeutically effective in 50% of the population or the dose that produces 50% of the maximal desired effect) and the LD 50 (the dose lethal to 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and can be expressed as the ratio, LD 50 /ED 50 . The data obtained from cell culture assays using TRPV2 polypeptide, cells expressing TRPV2 and/or animal studies, such as canine or primate studies, are used in formulating a range of dosage for human use. The dosage contained in such compositions preferably gives rise to a range of circulating concentrations that include and/or exceed the ED 50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient and the route of administration.
The exact dosage will be determined by the one administering the dose, in light of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active agent or to maintain the desired effect, for example, control of pain levels. Factors that can be taken into account include the general health, age, weight and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities and tolerance/response to therapy.
The pharmaceutical compositions containing a compound that has been identified as modulating TRPV2 expression or activity can be administered by any number of routes, including, but not limited to, oral, intravenous, intramuscular, intraarticular, intraarterial, intramedullary, intrathecal, epidural, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, inhalational, intraocular, intra-aural or rectal means.
In addition to the active ingredients, these pharmaceutical compositions can contain suitable, pharmaceutically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations that can be used pharmaceutically or that facilitate absorption or distribution of the active compounds. Further details on techniques for formulation and administration can be found in Remington's Pharmaceutical Sciences, 20 th edition, Maack Publishing Co., Easton, Pa. (2000). Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for ingestion by the patient.
Example 1
The present invention is directed to an isolation of a cDNA from canine dorsal root ganglion (DRG) tissue (SEQ ID NO: 1) encoding a full length TRPV2 (SEQ ID NO: 2), its preparation, characterization and functional expression, the establishment of a high throughput assay for testing the ability of compounds to alter the function of TRPV2 and the evaluation for the potential to decrease pain in animals and humans. In order to clone TRPV2 from canine, messenger RNA was first isolated from canine DRG, a reverse transcriptional PCR (RT-PCR) and a Rapid Amplification of cDNA End (RACE-PCR) technique were used. The cloning result revealed that the open reading frame cTRPV2 contains 2298 bp encoding a polypeptide of 765 amino acids and having a calculated molecular mass of 85.4 kDa. A Kyte-Doolitle hydrophilicity analysis of primary sequence predicts the presence of six putative hydrophobic domains clustered near the carboxyl terminus, which may form a pore of the ion channel. The nucleotide sequence of canine TRPV2 is 83%, 82% and 86% identical to the mouse, rat and human TRPV2, respectively. (See FIG. 4 ).
A. Isolation of Poly(A + ) RNA
As a first step in the cloning of cTRPV2, poly(A + ) RNA was isolated from 100 μg of total RNA from canine DRG (Custom made by Analytical Biological Service Inc. DE) using an Oligotex spin column (Qiagen Inc., CA). Briefly, 150 μl of RNase-free water, 250 μl of buffer OBB, containing 20 mM Tris (pH=7.5), 1M NaCl, 2 mM EDTA and 0.2% SDS, and 15 μl of a suspension of Oligotex beads were added to 100 μl of total RNA solution (1 μg/μl). The RNA/Oligotex bead mixture was then heated at 70° C. for 3 min, to disrupt any secondary structure of the RNA, followed by incubation at room temperature for 10 min. The poly (A + ) RNA/Oligotex particle complex was centrifuged and washed twice with 400 μl of buffer OW2, containing 10 mM Tris (pH=7.5), 150 mM NaCl and 1 mM EDTA, and then transferred to a spin column for the elution step. The poly(A + ) RNA was eluted from Oligotex bead using 200 μl of prewarmed (70° C.) buffer OEB, containing 5 mM Tris (pH=7.5). Finally, canine DRG poly(A + ) RNA was precipitated by ethanol in the presence of 20 μg of glycogen and 150 mM sodium acetate and resuspended in 10 μl of RNase-free water.
B. Synthesis of Double-Stranded cDNA
4 μl (1 μg) of canine DRG poly(A + ) RNA and 1 μl of cDNA synthesis primer, a 52-mer oligo with sequence of 5′-TTCTAGAATTCAGCGGCCGC(T) 30 N -1 N-3′, N -1 =G, A or C; and N=G, A, C or T (Clontech, Calif. SEQ ID NO: 12), were mixed, incubated at 70° C. for 2 min and then cooled on ice for 2 min. The first strand cDNA synthesis (reverse transcription) was performed at 42° C. for 1 hour using 20 units of AMV reverse transcriptase in the presence of 1 mM dNTP mixture and first strand synthesis buffer, containing 50 mM Tris (pH=8.5), 8 mM MgCl 2 , 30 mM KCl and 1 mM DTT, in 10 μl. The second strand cDNA synthesis was performed by adding an enzyme cocktail, consisting of 24 units of E. coli DNA polymerase I, 5 units of E. coli DNA ligase I unit of E. coli RNase H, 0.25 mM of dNTP mixture (0.25 mM of each dATP, dCTP, dGTP, and dTTP) and second strand buffer, containing 100 mM KCl, 10 mM ammonium sulfate, 5 mM MgCl 2 , 0.15 mM β-NAD, 20 mM Tris (pH=7.5) and 50 μM bovine serum albumin, in 80 μl. The reaction was first carried out at 16° C. for 90 min followed by addition of 20 units of T4 DNA polymerase, with continued incubation at the same temperature for 45 min. The reaction was terminated by adding 10 mM EDTA and 8 μg of glycogen. Phenol and chloroform extractions were performed, followed by ethanol precipitation. Double-stranded cDNA was then suspended in 200 μl of TE buffer and stored at −20° C.
C. PCR Amplification of Near Carboxyl Terminus of cTRPV2
A TRPV2 cDNA fragment was successfully amplified by PCR using two primers designated as forward primer dv2-6 (5′-TGATGATCGCAGACAACT CAGCCGAGAACA-3′) (SEQ ID NO: 5), corresponding to nucleotides 761-790 of SEQ ID NO 1, and reverse primer dv2-8 (5′-GGACAGCTGGGCCTGATGGC TCTTCA-3′) (SEQ ID NO: 6), corresponding to nucleotides 2175-2200 of SEQ ID NO 1. The PCR reaction was performed in a final volume of 50 μl, containing 5 μl of canine DRG double-stranded cDNA, 5 μl of 10× reaction buffer (provided with Advantage2 DNA polymerase), 200 μM dNTPs, 200 nM forward primer dv2-6, 200 nM reverse primer dv2-8 and 1 μl of 50× Advantage™2 DNA polymerase mixture (Clontech, Calif.). PCR was performed by an initial denaturing step at 940° C. for 1 min, followed by 30 cycles of: (a) denaturing at 94° C. for 30 sec, (b) annealing at 55° C. for 30 sec and (c) extension at 72° C. for 60 sec. Agarose gel electrophoresis was performed, which revealed that the PCR product was approximately 1.44 kb. After PCR, the 1.44 kb PCR fragment was purified and subcloned into pCRII TA cloning vector (Invitrogen, CA) following the vendor's protocol. Six independent clones were picked and subjected to DNA sequencing analysis. The sequence results revealed that the PCR amplified fragment was 82%, 81% and 87% identical to the comparable region, i.e., near carboxyl termini of mouse, rat and human TRPV2, respectively.
D. RACE-PCR of 5′ and 3′ Ends of cTRPV2 Sequence
To obtain the complete 5′ and 3′ cDNA sequences of the cTRPV2 gene, RACE-PCR technology was performed. First, both 5′- and 3′-RACE-Ready cDNAs were synthesized separately with SMART™ RACE DNA Amplification Kit (BD Clontech, Calif.), according to the manufacturer's instructions. To prepare cDNA for 5′ RACE, in one 0.5 ml tube, 3 μl of dog DRG poly(A + ) RNA obtained in step A above was mixed with 1 μl of 5′-CDS primer and 1 μl of SMART II A oligo. To prepare cDNA for 3′ RACE, 3 μl of canine DRG poly(A + ) RNA was mixed with 1 μl 3′-CDS primer and 1 μl RNase free water in another 0.5 ml tube and then incubated at 70° C. for 2 min, followed by cooling on ice for 2 min. Next, 2 μl of 5× First Strand buffer, 1 μl 20 mM DTT, 1 μl of 10 mM dNTP mix and 1 μl PowerScript Reverse Transcriptase were added to each tube, and synthesis was performed at 42° C. for 90 min. The reactions were stopped by adding 200 μl of TE buffer and heating the sample to 72° C. for 7 min. The reaction products were stored at −20° C. All of the oligos, 5′-CDS, 3′-CDS and SMARTII were provided by Clontech.
For RACE-PCR, two primers were synthesized based on the above 1.44 kb cDNA sequence the cTRPV2 cDNA. The forward primer for 3′ RACE-PCR was dv2-6; the reverse primer for 5′-RACE-PCR was named dv2-2 (5′-GATTTTGCCCTCCTTGGCAGCCAG-3′) (SEQ ID NO: 7), which corresponds to nucleotides 901-924 of SEQ ID NO: 1. Both 5′ and 3′-RACE PCRs were performed in a final volume of 50 μl, containing 2.5 μl of cDNA template (either 5′- or 3′-RACE-Ready cDNA, as described above), 5 μl of 10× reaction buffer, 200 μM dNTPs, 200 nM Universal Primer Mix (UPM) (Clontech), 200 nM cTRPV2 specific primer (dv2-2 for 5′-RACE PCR or dv2-2 for 3′-RACE PCR) and 1 μl of 50× Advantage™-HF2 DNA polymerase mixture (Clontech). The thermal cycler parameter for the RACE-PCR was 25 cycles of 94° C. for 30 sec and 68° C. for 3 min. After the RACE-PCR reaction, the products were diluted 50-fold and used for the next round nested PCR. For nested PCR, two oligos were synthesized. They were reverse primer dv2-9 (5′-TTGCCAGAGTTCCGGTCGATCTGAAGCA-3′) (SEQ ID NO: 8), used for the 5′-nested-PCR and forward primer dv2-10 (5′-TCCAAGACCAGCTGCGCTTCCGC GGCGT-3′) (SEQ ID NO: 9) used for the 3′ nested-PCR. Both 5′ and 3′-nested-PCRs were performed in a final volume of 50 μl, containing 5 μl of a 1:50 dilution of the previous RACE-PCR product, 5 μl of 10× reaction buffer, 200 μM dNTPs, 200 nM Universal Primer Mix (UPM) (Clontech), 200 nM cTRPV2 specific primer (dv2-9 for 5′ nested-PCR or dv2-10 for 3′ nested-PCR) and 1 μl of 50× Advantage2™ DNA polymerase mixture (Clontech). The thermal cycler parameters for the RACE-PCR were 20 cycles of 94° C. for 30 sec and 680° C. for 3 min. The nested-PCR products were purified and directly subcloned into pCRII TA cloning vector (Invitrogen). Eight to eighteen independent clones from either 5′-RACE/nested or 3′-RACE/nested PCR were picked and subjected to DNA sequencing analysis.
E. Sequence of Full-Length cTRPV2 cDNA:
The sequence of the full-length canine TRPV2 cDNA was confirmed by synthesizing PCR primers based on the sequence of the 5′ and 3′ ends obtained by RACE-PCR. Full length cTRPV2 cDNA was amplified from canine DRG double-stranded cDNAs prepared in step B above by high-fidelity DNA polymerase using two sets of primers. The first primer set was forward primer dv2-1 (5′-ATGGCCTCGC CCTCCAGCTC TCCCA-3′) (SEQ ID NO: 10) and reverse primer dv2-2 for amplification of a 924 bp cDNA fragment encoding cTRPV2 amino terminal 308 residues; the second primer set was forward primer dv2-6 and reverse primer dv2-11 (5′-TTAGTGGGACTTCA GGAGCTGGAGG GGC-3′) (SEQ ID NO: 11) for amplification of a 1.54 kb cDNA fragment encoding cTRPV2 carboxyl terminal 512 residues. The PCR was performed in a final volume of 50 μl, containing 5 μl of above canine DRG double-stranded cDNAs (prepared as above), 5 μl of 10× reaction buffer, 200 μM dNTP, 200 nM forward primer (either dv2-1 or dv2-6) and reverse primer (either dv2-2 or d2-11), and 1 μl of 50× Advantage™-HF2 DNA polymerase mixture (Clontech, Calif.). The PCR was performed by an initial denaturing step at 94° C. for 2 min, followed by 35 cycles of denaturing at 94° C. for 30 sec and annealing and extension at 70° C. for 5 min. After PCR, the 2.3 kb PCR fragment was purified and subcloned into pCRII TA cloning vector, following the same cloning protocol as in step C above. Several independent clones were picked and subjected to DNA sequencing analysis. The clones NQC881 and NQC918 were used for further subcloning and studying. The sequence results revealed that the open reading frame nucleic acid sequence of cTRPV2 cDNA SEQ ID NO 1 was 83%, 82% and 86% identical to the cDNA sequences of mouse (Accession number: BC005415), rat (Accession number: AF129113) and human (Accession number: AF129112; SEQ ID NO: 3) TRPV2, respectively. (See FIG. 4 ).
F. Sequence Analysis
5′- and 3′-RACE-PCR facilitated the determination of the 68 bp sequence of the 5′ untranslated region; 3′-RACE-PCR allowed for the determination of the 431 bp sequence of the 3′ untranslated region, including the 37-mer poly(A + ) tail. No in-frame stop codon was identified in either 5′ or 3′ untranslated region.
The predicted cTRPV2 open reading frame consists of a 2298 bp sequence that is predicted to encode a polypeptide of 765 amino acids (SEQ ID NO: 2), having a calculated molecular mass of 85.4 kDa. A Kyte-Doolitle hydrophilicity analysis (not shown) of primary sequence predicts the presence of six putative hydrophobic (or transmembrane) domains clustered at the carboxyl half of the molecule and believed to form the pore of the ion channel.
The cTRPV2 amino acid sequence was aligned with the human (GenBank Acc. No.: AF129112; SEQ ID NO: 4), rat (GenBank Acc. No.: AF129113), and mouse (GenBank Acc. No.: BC005415) sequences, which revealed 84%, 81% and 83% sequence identity, respectively ( FIG. 4 ), using the Gap program from Seqweb version 2 of Accelrys. The Gap program uses the algorithm of Needleman and Wunsch (J Mol. Biol., 48:443 (1970)) to find the alignment of two complete sequences, by maximizing the number matches and minimizing the number of gaps. A search of canine genomic sequence with TRPV2 cDNA revealed that cTRPV2 is located at chromosome 5, spanning 17435 bp with a total of 14 exons.
Example 2
Recombinant Expression of cTRPV2
A. Cloning of Canine TRPV2 into a Mammalian Expression Vector
For expression of cTRPV2 in mammalian cell lines, the full-length cDNA of cTRPV2 was subcloned into pcDNA3.1 (neo) by performing a three-way ligation. First, the clone NQC881 was digested with NotI and XhoI to yield a 0.9 kb 5′ fragment, and the clone NQC918 was digested with XhoI and EcoRI to yield a 1.45 kb 3′ fragment. The 0.9 kb 5′ and 1.45 kb 3′ cTRPV2 fragments were purified and ligated with pcDNA3.1 that was predigested with NotI and EocRI, creating the expression construct cTRPV2/pcDNA3.1.
B. Transient Transfection of Expression Construct into Mammalian Cells
The expression construct, cTRPV2/pcDNA3.1 was then transfected into human embryonic kidney cells (HEK293, ATCC CRL-1573) with FuGene6 transfection reagent (Roche, Indianapolis, Ind.), according to the vendor's protocol. TRPV2-expressing HEK293 cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin and 100 μg/ml streptomycin at 37° C. with 5% CO 2 , and were analyzed using a calcium influx assay 48-72 hr. post-transfection.
Example 3
Calcium Influx Functional Assay of cTRPV2
To demonstrate the functionality of the cTRPV2 gene, HEK293 cells were transiently transfected with plasmids encoding either cTRPV2 or vector control and then evaluated in a FLIPR assay to measure changes in intracellular calcium levels of the cells in each well upon addition of agonist. Transiently transfected cells were seeded in 384-well plates at a density of 1×10 4 cells/well and incubated overnight at 37° C. The following day, the cells were loaded with buffer and calcium dye, as recommended by the vendor (Molecular Devices, Sunnyvale, Calif.), in a final volume of 40 μl and incubated for 30 minutes at room temperature. The fluorescence intensity was measured by FLIPR before, during and after the addition of Δ 9 -THC, which was added to the cells at a final concentration of 100 μM. The results are shown in FIG. 6 .
Example 4
A Screening Assay for a Desensitizer or Inactivator of a TRPV2 Channel
Generally, upon prolonged exposure of an ion channel to an activating stimulus (e.g., an agonist) or in response to a direct desensitizing or inactivating stimulus, the channel may assume alternate conformations that are variably less activatable in response to an activating stimulus. These less activatable or inactivatable conformations may be referred to functionally as being desensitized or inactivated, and compounds that produce these states as being desensitizers or inactivators, respectively. Such conformations may be induced or stabilized by or in the presence of these so-called desensitizers or inactivators, and may arise by the preferential action of the desensitizer or inactivator upon an open or upon a closed channel. In addition, such conformations may be reversible, across variable time courses and conditions, or may be irreversible, pending de novo synthesis of nascent channels. Compounds that are identified as desensitizers or inactivators, either being reversible or irreversible, may be useful in the treatment of certain conditions, including pain conditions, in which decreased TRPV2 activity would be therapeutic.
Therefore, in another embodiment, the invention provides a method of identifying reversible and irreversible desensitizers or inactivators of cTRPV2 activity. The first method is designed to identify compounds that induce and/or stabilize the channel in a desensitized or inactivated state from a closed state and comprises the steps of: (a) providing a recombinant cell comprising a nucleic acid encoding a cTRPV2 protein, (b) contacting the recombinant cell at a temperature above the threshold for activation (typically above about 52° C.) with a test compound for varying lengths of time, (c) contacting the recombinant cell with agonist for activation in the presence of a test compound for varying lengths of time, (d) extensively washing out the test compound and (e) at varying time points, determining the extent to which the test compound diminishes TRPV2 activity in response to a subsequent exposure to a TRPV2-activating stimulus. The second method is designed to identify compounds that induce and/or stabilize the channel in a desensitized or inactivated state from an open state and comprises EITHER the steps of: (a) providing a recombinant cell comprising a nucleic acid encoding a cTRPV2 protein, (b) contacting the recombinant cell at a temperature above the threshold for activation (typically above about 52° C.) or with a TRPV2 agonist, (c) contacting the recombinant cell with a test compound for varying lengths of time, (d) extensively washing out the test compound and agonist and (e) at varying time points, determining the extent to which the test compound diminishes TRPV2 activity in response to a subsequent exposure to a TRPV2-activating stimulus OR the steps of: (a) providing a recombinant cell comprising a nucleic acid encoding a cTRPV2 protein, (b) incubating the recombinant cell at a temperature below the threshold for activation, (c) contacting the recombinant cell with a test compound for varying lengths of time, (d) extensively washing out the test compound and (e) at varying time points, determining the extent to which the test compound diminishes TRPV2 activity in response to a subsequent exposure to a TRPV2-activating stimulus.