COMPOSITIONS AND METHODS FOR IDENTIFYING MODULATORS OF TRPV2

Cross Reference to Related Applications

This application claims priority to Application No. 60/731,686 filed on October 31, 2005 and Application No. 60/782,656 filed on March 15, 2006, the entire contents of which are incorporated by reference herein.

Field of The Invention

The present invention relates to the regulation of thermal receptor ion channel proteins. In particular, the present invention relates to compositions and methods for screening, identifying and characterizing compounds that increase or decrease the biological activity of a TRPV2.

Background

In mammals, the sensation of pain triggered by thermal, mechanical or chemical stimuli is a useful warning and protective system. 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 thermal receptors on a sub-population of sensory neurons called nociceptors (pain-sensing neurons). Upon activation, the thermal 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 thermal receptors represent highly promising targets for developing drugs for the treatment of various painful conditions.

Several temperature-activated receptors have been identified with wide ranging temperature sensitivities from noxious heat to noxious cold. These temperature-activated receptors belong to the transient receptor potential (TRP) family of non-selective cation channels, which in C. elegans and D. melanogaster are involved in mechano- and osmoregulation. Several of these temperature-activated receptors, including TRPVl and TRPV2, are implicated in noxious heat sensation (Caterina et al., 1997, Nature, 389: 816; and Caterina et al., 1999, Nature 398: 436). TRPVl, the most extensively characterized member of the thermo-TRP family, is activated by moderate heat (-43 ⁰ C), capsaicin, protons and certain endocannabinoids, such as anandamide and 2-AG. It is well accepted that TRPVl contributes to acute thermal nociception and hyperalgesia after injury (Clapham, Nature. 2003, 426(6966): 517-24).

TRPV2, also termed VRL-I, has been proposed as a sensor of noxious temperatures (>52°C), which presumably mediates "first" pain, i.e. the rapid, acute, and sharp pain evoked by noxious stimuli (Caterina et al., 1999, supra; Story et al., Cell, 2003, 112:819-829, and references therein). TRPV2 is structurally most closely related to TRPVl (-50% sequence identity at the protein level). TRPV2 is expressed in medium- to large- diameter neurons of sensory ganglia, as well as at lower levels in brain, spinal cord, spleen and lung. Furthermore, TRPV2 is upregulated in sympathetic postganglionic neurons following injury, suggesting a potential role for TRPV2 in sympathetically mediated pain (Gaudet et al., Brain Res. 2004, 1017(l-2):155-62). Thus, modulation of TRPV2 may potentially have many therapeutic applications.

Despite great interest in TRPV2 modulation, a system for screening, identifying and characterizing TRPV2 modulators has yet to be developed. This is in part due to the lack of known, and in particular, selective TRPV2 agonists, as well as the technical difficulty of assaying these channels in a high temperature environment. In general, TRPV2 does not respond to known TRPVl agonists (Benham et al., 2003, Cell Calcium 33:479-487). However, a recent study reported that 2-aminoethoxydiphenyl borate (2- APB), a non-selective TRP modulator, was able to activate TRPVl, TRPV2, and TRPV3

(Hu et al (2004), J. Biol. Chem., 279: 35741-8), although TRPV2 activation by 2-APB was not observed by others (Chung et al. (2004), J Neurosci. 24: 5177-82).

In an effort to overcome the above-mentioned challenges, the present invention provides novel compositions and methods for screening, identifying and characterizing TRPV2 agonists.

Summary

It has now been discovered that certain cannabinoids specifically activate TRPV2 channel activity.

In one general aspect, the present invention provides a method for identifying a compound that decreases the biological activity of TRPV2, comprising the steps of: a) contacting a TRPV2 polypeptide with a cannabinoid that is capable of activating TRPV2 activity under a condition in which the TRPV2 is activated by the cannabinoid; b) contacting the TRPV2 polypeptide with a test compound; c) measuring the biological activity of the TRPV2 in the presence of both the cannabinoid and the test compound; d) repeating step a); e) measuring the biological activity of the TRPV2 in the presence of the cannabinoid but not the test compound; and f) comparing the TRPV2 activity measured from step c) with that from step e); thereby identifying the compound that decreases the biological activity of TRPV2 when the TRPV2 activity measured from step c) is less than that from step e).

In another general aspect, the present invention provides a method for identifying a compound that increases the biological activity of TRPV2, comprising the steps of: a) obtaining atomic coordinates defining a three-dimensional structure of a complex comprising a TRPV2 interacting with a cannabinoid that is capable of activating the TRPV2; b) elucidating a structural relationship between the TRPV2 and the interacting cannabinoid; c) designing a structural analog of the cannabinoid based on the structural relationship; d) synthesizing the structural analog; and e) determining the extent to which the structural analog alters the biological activity of the TRPV2, thereby identifying the compound that increases the biological activity of TRPV2.

Another general aspect of the present invention is a method for increasing the biological activity of a TRPV2, comprising the step of contacting the TRPV2 with a cannabinoid that is capable of activating the TRPV2 activity.

The present invention further provides a method for stimulating noxious thermo- sensation in a subject, comprising administering to the subject a pharmaceutical composition comprising an effective amount of a cannabinoid that is capable of activating the TRPV2 activity, thereby stimulating the noxious thermo-sensation in the subject.

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.

Description of The Figures

Figure 1 shows the subclasses of cannabinoids present in Cannabis (Thakur et al., Life Sci. 2005 Oct 17, Epub ahead of print).

Figure 2 shows non-enzymatic formation of δ9-THC from its precursor (Thakur et al., supra).

Figure 3 shows the structures of two representative endocannabinoid (Thakur et al., supra).

Figure 4 shows concentration-dependent activation of rat TRPV2 by δ9-THC in a FLIPR assay.

Figure 5 illustrates activation of both rat and human TRPV2 by δ9-THC and subsequent block of the δ9-THC-activated currents by ruthenium red from whole-cell patch clamp studies.

Figure 6 shows δ ⁹ -THC activated deletion mutants of TRPV2 recombinantly expressed from HEK293 cells: (A) the N-terminal deletion mutants; and (B) the C-terminal deletion mutants.

Figure 7 illustrates the activation of the human and rat TRPV2 chimera recombinantly expressed from HEK293 cells: (A) the chimera was expressed individually from the cells; and (B) the complementary effect of RRH and HRR when they were co-expressed from the cells.

Detailed Description

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.

The following are abbreviations that are at times used in this specification:

2- AG = 2-arachidonylglycerol

AEA = anandamide = N-arachidonoylethanolamine bp = base pair cDNA = complementary DNA

Ca ²⁺ = calcium

δ ⁹ -THC = Delta-9-tetrahydrocannabinol

DRG = dorsal root ganglion

FLIPR = fluorescence imaging plate reader kb = kilobase; 1000 base pairs

PAGE = polyacrylamide gel electrophoresis

PCR = polymerase chain reaction

SDS = sodium dodecyl sulfate

TG = trigeminal ganglion

TRPV2 = transient receptor potential cation channel, subfamily V, member 2

As used herein, the term "biological activity of a TRPV2" refers to an activity exerted by the TRPV2 protein as determined in vivo or in vitro, according to standard techniques. Such an activity can be a direct activity such as the ability of a TRP V2 to bind to a ligand, such as a cannabinoid or an analog thereof. The activity can be the conductivity of an ion channel formed by the TRP V2. The activity can also be functional changes of cell physiology, such as calcium mobilization or nociceptive response of the cell. The biological activity of a TRPV2 can be an indirect activity, such as a signal transduction activity mediated by TRPV2 via its interaction with one or more than one additional protein or other molecule(s).

"Binding affinity" refers to the ability of two or more molecular entities to bind or interact with each other. The binding can be from the formation of one or more chemical bonds that results in continual and stable proximity of the two interacting entities. The binding can also be based solely on physical affinities, which can be equally effective in co-localizing the two interacting entities. Examples of physical affinities and chemical bonds include but are not limited to, forces caused by electrical charge differences, hydrophobicity, hydrogen bonds, van der Waals force, ionic force, covalent linkages, and combinations thereof. The state of proximity between the interacting entities can be transient or permanent, reversible or irreversible. In any event, it is in contrast to and distinguishable from contact caused by natural random movement of two entities.

"Cannabinoid" includes any of various compounds that activate a cannabinoid receptor or a structural analog of the compounds.

In one embodiment, "cannabinoid" includes herbal cannabinoids, a class of compounds that were originally extracted from the plant Cannabis sativa L or a metabolite thereof. Cannabis sativa L. is one of the oldest known medicinal plants and has been extensively studied with respect to its phytochemistry. The plant biosynthesizes a total of 483 identified chemical entities belonging to different chemical classes

(EISohly, 2002, In: F. Grotenhermen and E. Russo, Editors, Cannabis and Cannabinoids, Haworth Press, Binghamton (2002), pp. 27-36.), of which the cannabinoids are the most distinctive class of compounds, known to exist only in this plant. There are 66 known plant-derived cannabinoids (Thakur et al., Life Sci. 2005 Oct 17, Epub ahead of print). The most prevalent of which are the tetrahydrocannabinols (THCs), the cannabidiols (CBDs), and the cannabinols (CBNs). The next most abundant cannabinoids are the cannabigerols (CBGs), the cannabichromenes (CBCs), and cannabinodiols (CBNDs).

Figure 1 shows the representative structures of subclasses of cannabinoids present in Cannabis sativa. Most cannabinoids contain 21 carbon atoms, but there are some variations in the length of the C-3 side chain attached to the aromatic ring. In the most common homologues, the n-pentyl side chain is replaced with an π-propyl (De Zeeuw et al., Science. 1972, 175:778-779); and Vree et al., Journal of Pharmacy and Pharmacology. 1972, 24:7-12). These analogues are named using the suffix "varin" and are designated as THCV, CBDV, or CBNV, as examples. Cannabinoids with one (Vree et al., 1972, supra) and four (Smith, 1997, Journal of Forensic Sciences 42 (1997), pp. 610-618) carbons also exist but are minor components. Classical cannabinoids (CCs) are ABC tricyclic terpenoid compounds bearing a benzopyran moiety and are insoluble in water but soluble in lipids, alcohols, and other non-polar organic solvents (Thakur et al., 2005, supra). These phenolic derivatives are more water-soluble as their phenolate salts formed under strong alkaline conditions.

One particular example of "cannabinoid" is Delta-9-tetrahydrocannabinol (Delta- 9-THC, δ ⁹ -THC), the key psychoactive ingredient of cannabis (marijuana) (Gaoni and Mechoulam 1964, Journal of the American Chemical Society 86 (1964), pp. 1646-1647). As illustrated in Figure 2, δ ⁹ -THC is formed by the decarboxylation of its non- psychoactive precursor δ ⁹ -THCA by the action of light or heat during storage or smoking or under alkaline conditions. δ ⁹ -THCA is biosynthesized by a well-established pathway involving the action of several specific enzymes.

It was discovered that δ ⁹ -THC interacts with the two known cannabinoid (CB) receptors, CBl (Devane et al., 1988, Molecular Pharmacology 34 (1988), pp. 605-613;

Gerard et al, 1990, Nucleic Acids Research 18 (1990), p. 7142; Gerard et al., 1991, Biochemical Journal 279 (1991), pp. 129-134; and Matsuda et al., 1990, Nature 346 (1990), pp. 561-564.) and CB2 (Munro et al., 1993, Nature 365 (1993), pp. 61-65). Both cannabinoid receptors belong to the super family of G-protein coupled receptors, and produce a broad spectrum of physiological effects (Grotenhermen, 2002, In: R. Grotenhermen and E. Russo, Editors, Cannabis and Cannabinoids, Haworth Press, Binghamton (2002), pp. 123-142) including antiemetic, appetite enhancing, analgesic, and lowering of intraocular pressure. The discovery of specific cannabinoid receptors inside animal ultimately led to the search and identification of endocannabinoid.

Thus, the term "cannabinoid" also includes endocannabinoid. The term "endocannabinoid" refers to a ligand to a cannabinoid receptor, wherein said ligand is endogenously produced by in the bodies of an animal. Exemplary endocannabinoids include, but are not limited to, N-arachidonoylethanolamine (AEA, anandamide) and 2- arachidonylglycerol (2-AG), the structures of which are shown in Figure 3. Anandamide was shown to bind to the CBl receptor with modest affinity (K _\ = 61 nM), have low affinity for the CB2 receptor (K ₁ = 1930 nM) (Lin et al., 1998, Journal of Medicinal Chemistry 41 (1998), pp. 5353-5361), and behave as a partial agonist in the biochemical and pharmacological tests used to characterize cannabinoid activity. It was reported that anandamide can also bind to and activate TRPVl (Di Marzo et al., Prostaglandins Leukot Essent Fatty Acids 2002; 66: 377-91). 2-AG binds weakly to both CB 1 (K ₁ = 472 nM) and CB2 (Ki = 1400 nM) receptors (Mechoulam et al., 1995, Biochemical Pharmacology 50 (1995), pp. 83-90). 2-AG was isolated from intestinal and brain tissues and is present in the brain at concentrations approximately 170-fold higher than AEA (3) (Stella et al., 1997, Nature 388 (1997), pp. 773-778).

In yet another embodiment, the term "cannabinoid" covers the synthetic cannabinoids are produced by chemical synthesis and do not occur naturally. The synthetic cannabinoids can be synthesized based on the structure of herbal cannabinoids or endocannabinoid. Synthetic cannabinoids are particularly useful in experiments to determine the relationship between the structure and activity of cannabinoid compounds, by making systematic, incremental modifications of cannabinoid molecule. Exemplary

synthetic cannabinoids includes dronabinol (synthetic THC), nabilone, and any other synthetic compounds that activate a cannabinoid receptor or a structural analog of the compounds.

A "cannabinoid that is capable of activating the TRPV2 activity" refers to any cannabinoid that is capable of binding to a TRPV2 channel and, in the absence of other stimulation, exhibits at least a 10% increase in the conductivity of the TRPV2 channel compared to the baseline. A person skilled in the art can experimentally determine whether a cannabinoid is capable of activating the TRPV2 activity. In some embodiments, "cannabinoid that is capable of activating the TRPV2 activity" is a cannabinoid which, upon binding to a TRPV2 channel, results in at least a 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% increase in the conductivity of the channel compared to the baseline. "Cannabinoid that is capable of activating the TRPV2 activity" includes, but is not limited to, /f -tetrahydrocannabinol, cannabinol, cannabidiol nabilone, CP55940, HU210, and 2-AG. Interestingly, the other endocannabinoid tested, anandamide, showed no or minimal activation effect on TRPV2 (Table 2, Example 4 infra).

A "cannabinoid receptor" or a "CB receptor" each refers to a protein that functions as a specific receptor for a cannabinoid. The "CB receptor" can be a CB 1 receptor or a CB2 receptor.

The CB 1 receptor has been detected primarily in brain, specifically in the basal ganglia and in the limbic system, including the hippocampus. They are also found in other tissues such as the cerebellum and in both male and female reproductive systems. CBl receptors appear to be responsible for the euphoric and anticonvulsive effects of cannabis. A CBl can (1) have greater than about 70% amino acid sequence identity to a human CBl receptor depicted in GenBank protein ID: NP_057167 (the longer isoform of human CBl receptor) or NP_149421 (the shorter isoform of human CBl receptor); or (2) bind to antibodies, e.g., polyclonal or monoclonal antibodies, raised against the human CBl receptor depicted in GenBank protein ID NP_057167 or NP_149421. In some embodiments, the CBl receptor has greater than about 75, 80, 85, 90, or 95 percent

amino acid sequence identity to the human CBl receptor depicted in GenBank protein ED NP_057167 or NPJ 49421. The CBl receptor includes orthologs of the CBl receptors in animals including human, rat, mouse, pig, dog and monkey, etc. The CBl receptor also includes structural and functional polymorphisms of the CBl receptor. "Polymorphism" refers to a set of genetic variants at a particular genetic locus among individuals in a population. The CBl receptor includes the structural and functional polymorphisms of the CBl receptor from human (GenBank protein ID NPJ357167 or NPJ49421), rat (GenBank protein ID: NP_036916), or mouse (GenBank protein ID: NP_031752), or etc.

The CB2 receptor has been detected almost exclusively in the immune system, with the greatest density in the peripheral blood cells. CB2 receptors appear to be responsible for the anti-inflammatory and possible other therapeutic. A CB2 can (1) have greater than about 70% amino acid sequence identity to a human CB2 receptor depicted in GenBank protein ID: NP_001832; or (2) bind to antibodies, e.g., polyclonal or monoclonal antibodies, raised against the human CB2 receptor depicted in GenBank protein ID NP_001832. In some embodiments, the CB2 receptor has greater than about 75, 80, 85, 90, or 95 percent amino acid sequence identity to the human CB2 receptor depicted in GenBank protein ID NPJ)Ol 832. The CB2 receptor includes orthologs of the CB2 receptors in animals including human, rat, mouse, pig, dog and monkey, etc. The CB2 receptor also includes structural and functional polymorphisms of the CB2 receptor. The CB2 receptor includes the structural and functional polymorphisms of the CB2 receptor from human (GenBank protein ID NPJlOl 832), rat (GenBank protein ID: NPJ)65418), mouse (GenBank protein ID: NPJ)34054), or etc.

A "cell" refers to at least one cell or a plurality of cells appropriate for the sensitivity of the detection method. The cell can be present in a cultivated cell culture. The cell can also be present in its natural environment, such as a biological tissue or fluid. Cells suitable for the present invention may be bacterial, but are preferably eukaryotic, and are most preferably mammalian.

A "compound that increases the conductivity of a TRPV2 channel" includes any compound that results in increased passage of ions through the TRPV2 channel. In one

embodiment, such a compound is an agonist for the TRPV2 channel that binds to the TRPV2 channel to increase its conductivity. Such a compound triggers, initiates, propagates, or otherwise enhances the channel conductivity. In another embodiment, such a compound is a positive allosteric modulator, which interacts with the TRPV2 channel at allosteric sites different from the agonist-binding site, and potentiates the response of the channel to an agonist.

A "compound that decreases the conductivity of a TRPV2 channel" includes any compound that results in decreased passage of ions through the TRPV2 channel. In one embodiment, such a compound is an antagonist for the TRPV2 channel that binds to the TRPV2 channel to counter, decrease or limit the action of an agonist in either a competitive or non-competitive fashion. In another embodiment, such a compound is a negative allosteric modulator, which interacts with the TRPV2 channel at allosteric sites different from the agonist or antagonist-binding site, and decreases the response of the channel to an agonist. In yet another embodiment, such a compound is an inverse agonist that binds to the TRPV2 channel and decreases the conductivity of the channel in the absence of any other compound, such as an agonist.

"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 of the natural nucleotides.

An "isolated" nucleic acid molecule is one that is substantially separated from at least one of the other nucleic acid molecules present in the natural source of the nucleic acid, or is substantially free of at least one of the chemical precursors or other chemicals when the nucleic acid molecule is chemically synthesized. An "isolated" nucleic acid molecule can also be, for example, a nucleic acid molecule that is substantially 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. A nucleic acid molecule is "substantially separated from" or "substantially free

of other nucleic acid molecule(s) or other chemical(s) in preparations of the nucleic acid molecule when there is less than about 30%, 20%, 10%, or 5% (by dry weight) of the other nucleic acid molecule(s) or the other chemical(s) (also referred to herein as a "contaminating nucleic acid molecule" or a "contaminating chemical").

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) 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 sequence 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, as by cleavage and electrophoretic or chromatographic separation.

The term "oligonucleotide" or "oligo" 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 16-25 nucleotides in length are useful, although longer oligonucleotides of greater than about 25 nucleotides may 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 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.

The terms "polypeptide," "protein," and "peptide" are used herein interchangeably to refer to amino acid chains in which the amino acid residues are linked by peptide bonds or modified peptide bonds. The amino acid chains can be of any length of greater than two amino acids. Unless otherwise specified, the terms "polypeptide," "protein," and

"peptide" also encompass various modified forms thereof. Such modified forms may be naturally occurring modified forms or chemically modified forms. Examples of modified forms include, but are not limited to, glycosylated forms, phosphorylated forms, myristoylated forms, palmitoylated forms, ribosylated forms, acetylated forms, ubiquitinated forms, etc. Modifications also include intra-molecular crosslinking and covalent attachment to various moieties such as lipids, flavin, biotin, polyethylene glycol or derivatives thereof, etc. In addition, modifications may also include cyclization, branching and cross-linking. Further, amino acids other than the conventional twenty amino acids encoded by the codons of genes may also be included in a polypeptide.

An "isolated protein" is one that is substantially separated from at least one of the other proteins present in the natural source of the protein, or is substantially free of at least one of the chemical precursors or other chemicals when the protein is chemically synthesized. A protein is "substantially separated from" or "substantially free of other protein(s) or other chemical(s) in preparations of the protein when there is less than about 30%, 20%, 10%, or 5% (by dry weight) of the other protein(s) or the other chemical(s) (also referred to herein as a "contaminating protein" or a "contaminating chemical").

Isolated proteins can have several different physical forms. The isolated protein 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 postranslationally modified by specific proteolytic cleavage events that result in the formation of fragments of the full-length nascent polypeptide. 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 polypeptide can be a non- naturally occurring polypeptide. For example, an "isolated polypeptide" can be a "hybrid polypeptide." An "isolated polypeptide" can also be a polypeptide derived from a naturally occurring polypeptide by additions or deletions or substitutions of amino acids. An isolated polypeptide can also be a "purified polypeptide" which is used herein to mean a specified polypeptide in a

substantially homogeneous preparation substantially free of other cellular components, other polypeptides, viral materials, or culture medium, or when the polypeptide is chemically synthesized, chemical precursors or by-products associated with the chemical synthesis. A "purified polypeptide" can be obtained from natural or recombinant host cells by standard purification techniques, or by chemical synthesis, as will be apparent to skilled artisans.

"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, 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 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 sequence can be introduced into host cells using any suitable method including, for example, electroporation, calcium phosphate precipitation, microinjection, transformation, biolistics 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 may be prokaryotic or eukaryotic, including bacteria such as E. coli, fungal cells such as yeast, mammalian cells such as cell lines of human, bovine, porcine, monkey 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 may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

"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 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 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 MoI. Biol., 48:443 (1970). Software for performing Needleman & Wunsch analyses is publicly available through the Institut Pasteur (France) Biological Software website: http://bioweb.pasteur.fr/seqanal/ interfaces/needle.html. 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, New York, (1989). An exemplary stringent hybridization condition comprises hybridization in 6x sodium chloride/sodium citrate (SSC) at about 45 ⁰ C, followed by one or more washes in 0.2x SSC and 0.1% SDS at 50 - 65 ⁰ C, depending upon the length over which the hybridizing polynucleotides share complementarity.

A 'TRPV2", "transient receptor potential cation channel, subfamily V, member 2", "VRL", "VRLl", "VRL-I", or "vanilloid receptor-like protein 1" each refers to a protein that forms an ion channel, the TRPV2 channel, that can be activated by high temperature and/or low osmolality, and transduces heat responses in sensory ganglia. The TRPV2 channel can also be activated by certain compounds. An activated TRPV2 channel gates the influx of Ca ²⁺ and other cations (e.g., Na ⁺ ) through the channel, resulting in membrane depolarization. A TRP V2 protein can (1) have greater than about 70% amino acid sequence identity to a human TRPV2 (hTRPV2) protein depicted in SEQ ID NO: 2 (GenBank protein ID: NP_057197); or (2) bind to antibodies, e.g., polyclonal or monoclonal antibodies, raised against a hTRPV2 protein depicted in SEQ ID NO: 2. In some embodiments, the TRPV2 has greater than about 75, 80, 85, 90, or 95 percent amino acid sequence identity to SEQ ID NO: 2. TRPV2 includes orthologs of the TRPV2 in animals including human, rat, mouse, pig, dog and monkey, etc. TRPV2 also includes structural and functional polymorphisms of the TRPV2. TRP V2 includes the

structural and functional polymorphisms of the TRP V2 from human, rat (GenBank protein ID: NP_058903, SEQ ID NO:4), mouse (GenBank protein ID: NP_035836, SEQ ID NO:6), or etc. For example, it was found that addition of a hemagglutinin A (HA) epitope tag to the end of the rat TRPV2 C-terminus did not alter the channel properties; and that deletion mutants of rat TRPV2-HA lacking the N-terminal 20, 32, and 65 and C- terminal 11, 23, or 32 amino acid residues of rat TRPV2 were still active in their responses to an elevated temperature of about 53 ⁰ C, lowered osmolality, δ9-THC or 2- APB. Therefore, TRPV2 also includes deletion or modifications of the wild-type TRPV2 that maintains the biological activity of the TRPV2, such as the deletion mutants of rat TRPV2 consisting of the amino acid sequence of SEQ ID NOs: 7 - 14. Furthermore, TRPV2 also includes chimeras between TRPV2 of different animals. For example, it was found that chimeras (SEQ ID NO: 16) between rat and human TRPV2, named RHR (i.e. Rat 1-392/ Human 391-646/ Rat 647-761), was also active in its response to an elevation of temperature of about 53 ⁰ C, δ9-THC and to 2-APB. In addition, TRPV2 further includes an active ion channel formed by the combination of two or more TRPV2 subunits, which by themselves are inactive or less active. For example, TRPV2 can be an active ion channel formed by the co-expression of the chimera RRH (Rat 1-392/ Rat 393- 646/ Human 647-764) and HRR (Human 1-390/ Rat 393-646/ Rat 647-761).

"TRPV2 activation temperature" is the temperature at which a TRPV2 channel, in the absence of other stimulation, exhibits at least a 10% increase in its conductivity compared to the baseline. A person skilled in the art can experimentally determine the activation temperature for a TRPV2 channel. In some embodiments, "TRPV2 activation temperature" is the temperature at which a TRPV2 channel exhibits at least a 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% increase in its conductivity compared to the baseline. "TRPV2 activation temperature" is typically greater than of about 52 ⁰ C. In some embodiments, the TRPV2 activation temperature is about 52 ⁰ C - 55 ⁰ C or 55 ⁰ C - 6O ⁰ C.

"TRPV2 non-activation temperature" is the temperature that falls outside of the range for a "TRPV2 activation temperature". An exemplary TRPV2 non-activation

temperature is room temperature (about 22 ⁰ C) or any temperature that is below about

52 ⁰ C.

"Vector" 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 may 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.

"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.

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, VoIs. 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, NY (2001).

It was discovered in the present invention that a group of cannabinoids are capable of activating the TRPV2 but not TRPVl activity. Thus, the present invention

provides new methods for regulating the biological activity of TRPV2 and new methods for identifying compounds that regulate biological activity of TRPV2.

In one embodiment, the TRPV2 used in the present invention is present in a cell. The cell can express TRPV2 endogeneously or recombinantly. One exemplary endogeneous cell for TRPV2 is a dorsal root ganglia (DRG) neuron or trigeminal neurons. Other examples of endogeneous cell for TRPV2 include, but are not limited to, intestine intrinsic neurons, vascular smooth muscle cells, and human hepatoblastoma (HepG2).

It will be apparent to skilled artisans that any recombinant expression methods may be used in the present invention for purposes of expressing the TRPV2. Generally, a nucleic acid encoding TRPV2 can be introduced into a suitable host cell. Exemplary nucleic acid molecules that can be used in the present invention include cDNA that encodes for the full length TRPV2 from human (SEQ ID:1, GenBank accession No: NM_016113), mouse (SEQ ID NO:5, GenBank accession No: NM_011706), or rat (SEQ ID NO:3 GenBank accession No: NM_017207).

Typically, the nucleic acids, preferably in the form of DNA, are incorporated into a vector to form expression vectors capable of directing the production of the interacting protein member (s) once introduced into a host cell. Many types of vectors can be used for the present invention. Methods for the construction of an expression vector for purposes of this invention should be apparent to skilled artisans apprised of the present disclosure. (See generally, Current Protocols in Molecular Biology, Vol. 2, Ed. Ausubel, et al., Greene Publish. Assoc. & Wiley Interscience, Ch. 13, 1988; Glover, DNA Cloning, Vol. II, IRL Press, Wash., D.C., Ch. 3, 1986; Bitter, et al., in Methods in Enzymology 153:516-544 (1987); The Molecular Biology of the Yeast Saccharomyces, Eds. Strathern et al., Cold Spring Harbor Press, VoIs. I and II, 1982; and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989.)

Generally, the expression vectors include an expression cassette having a promoter operably linked to a DNA encoding an interacting protein member. The

promoter can be a native promoter, i.e., the promoter found in naturally occurring cells to be responsible for the expression of the interacting protein member in the cells. Alternatively, the expression cassette can be a chimeric one, i.e., having a heterologous promoter that is not the native promoter responsible for the expression of the interacting protein member in naturally occurring cells. The expression vector may further include an origin of DNA replication for the replication of the vectors in host cells. Preferably, the expression vectors also include a replication origin for the amplification of the vectors in, e.g., E. coli, and selection marker(s) for selecting and maintaining only those host cells harboring the expression vectors.

The thus constructed expression vectors can be introduced into the host cells by any techniques known in the art, e.g., by direct DNA transformation, microinjection, electroporation, viral infection, lipofection, gene gun, and the like. The expression of the protein of interest may be transient or stable. The expression vectors can be maintained in host cells in an extrachromosomal state, i.e., as self- replicating plasmids or viruses. Alternatively, the expression vectors can be integrated into chromosomes of the host cells by conventional techniques such as selection of stable cell lines or site- specific recombination. In stable cell lines, at least the expression cassette portion of the expression vector is integrated into a chromosome of the host cells.

The vector construct can be designed to be suitable for expression in various host cells, including but not limited to bacteria, yeast cells, plant cells, insect cells, and mammalian and human cells. Methods for preparing expression vectors for expression in different host cells should be apparent to a skilled artisan. As described in the Example 1, infra, rat and human TRPV2 has been successfully expressed in HEK293.

Homologues and fragments of TRPV2 can also be easily expressed using the recombinant methods described above. For example, to express a protein fragment, the DNA fragment incorporated into the expression vector can be selected such that it only encodes the protein fragment. Likewise, a specific hybrid protein can be expressed using a recombinant DNA encoding the hybrid protein. Similarly, a homologue protein may be expressed from a DNA sequence encoding the homologue protein. A homologue-

encoding DNA sequence may be obtained by manipulating the native protein-encoding sequence using recombinant DNA techniques. For this purpose, random or site-directed mutagenesis can be conducted using techniques generally known in the art. To make protein derivatives, for example, the amino acid sequence of a native interacting protein member may be changed in predetermined manners by site-directed DNA mutagenesis to create or remove consensus sequences.

In other embodiments, the TRPV2 is provided in a cell membrane. The membrane preparation can be isolated from a native host cell, for example, a DRG or TG cell, which expresses TRPV2 on its cell surface. The membrane preparation can also be isolated from a recombinant host cell, for example, a CHO, HEK293, or COS cell, which expresses a TRPV2 recombinantly on its cell surface. The membrane preparation can be further prepared from the biological membranes, such as the tissue membrane, plasma membrane, cell membrane, or internal organelle membrane expressing the TRPV2 channels. Methods are known to those skilled in the art for isolation and preparation of biological membrane preparations. For example, such a method can include the steps of mechanical or enzymatic disruption of the tissue or cells, centrifugation to separate the membranes from other components, and resuspending the membrane fragments or vesicles in suitable buffer solution. Alternatively, the membrane-containing preparation can also be derived from artificial membranes. Purified TRPV2 protein can be reconstituted into lipid bilayers to form artificial membrane vesicles (see Chen et al., 1996, /. Gen. Physiol. 108:237-250). Such type of membrane vesicle can be very specific to the channel of interest, avoiding the problem of contamination from other channels. For example, such artificial membranes can include an electrode to which is tethered a lipid membrane containing ion channels and forming ion reservoirs. Methods are known to those skilled in the art to prepare artificial membrane vesicles.

In some preferred embodiments, membranes can be broken under controlled conditions, yielding portions of cell membranes and/or membrane vesicles. Cell membrane portions and/or vesicles can, in some embodiments, provide an easier format for the inventive assays and methods, since cell lysis and/or shear is not as much of a concern during the assay. Cell membranes can be derived from tissues and/or cultured

cells. Such methods of breaking cell membranes and stabilizing them are known in the art. Methods of treating tissues to obtain cell membranes are known in the art.

Preferably, human TRPV2 is used in the assays of the invention. Optionally, TRPV2 orthologs from other species such as rat or mouse, preferably a mammalian species, are used in assays of the invention.

The compound identification methods can be performed using conventional laboratory formats or in assays adapted for high throughput. The term "high throughput" refers to an assay design that allows easy screening of multiple samples simultaneously and/or in rapid succession, and can include the capacity for robotic manipulation. Another desired feature of high throughput assays is an assay design that is optimized to reduce reagent usage, or minimize the number of manipulations in order to achieve the analysis desired. Examples of assay formats include 96-well or 384-well plates, levitating droplets, and "lab on a chip" microchannel chips used for liquid handling experiments. It is well known by those in the art that as miniaturization of plastic molds and liquid handling devices are advanced, or as improved assay devices are designed, greater numbers of samples can be processed using the design of the present invention.

Any test compounds may be screened in the screening assays of the present invention to select modulators of the protein complex of the invention. By the term "selecting" or "select" compounds it is intended to encompass both (a) choosing compounds from a group previously unknown to be modulators of a protein complex or interacting protein members thereof; and (b) testing compounds that are known to be capable of binding, or modulating the functions and activities of, a protein complex or interacting protein members thereof. Both types of compounds are generally referred to herein as "test compounds" or "candidate compound". The 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, and derivatives, mimetics and analogs thereof. Preferably, they are small organic compounds, i.e., those having a molecular weight of no greater than 10,000 daltons, more

preferably less than 5,000 daltons. Preferably, the test compounds are provided in library formats known in the art, e.g., in chemically synthesized libraries (See generally, Gordan et al. /. Med. Chem., 37:1385-1401 (1994)), recombinantly expressed libraries (e.g., phage display libraries), and in vitro translation-based libraries (e.g., ribosome display libraries).

Candidate compounds comprise functional chemical groups necessary for structural interactions with polypeptides, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups and more preferably at least three of the functional chemical groups. The candidate compounds can comprise cyclic carbon or heterocyclic structure and/or aromatic or polyaromatic structures substituted with one or more 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.

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 libraries; spatially addressable parallel solid phase or solution phase libraries: synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection (Lam (1997) Anticancer Drug Des. 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 channel conductivity in which the structure of the compound is changed at one or more positions 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.

A variety of other reagents also can be included in the mixture. These include reagents such as salts, buffers, neutral proteins (e.g., albumin), detergents, etc. that can be used to facilitate optimal protein-protein and/or protein-nucleic acid binding. Such a reagent can also reduce non-specific or background interactions of the reaction components. Other reagents that improve the efficiency of the assay such as nuclease inhibitors, antimicrobial agents, and the like can also be used.

Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: Zuckermann et al. (1994). J Med. Chem. 37:2678. Libraries of compounds can be presented in solution (e.g., Houghten (1992) Biotechniques 13:412- 421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555- 556), bacteria (U.S. Patent No. 5,223,409), spores (Patent NO. 5,571,698), plasmids (Cull et al. (1992) Proc. Natl. Acad. ScL USA 89:1865-1869) or phage (see e.g., Scott and Smith (1990) Science 249:3 86-390).

The selected compounds can be tested for their ability to decrease the channel conductivity of the TRPV2, or for their ability to decrease the binding activity of the TRPV2 to a cannabinoid that is capable of activating the TRPV2. During the test, the test compound can be added to the TRPV2 prior to, after, or simultaneously with cannabinoid that is capable of activating the TRPV2. In addition, the compounds can be tested in an animal model for pain, or inflammation, etc.

Generally, a control assay is performed in which the above screening assay is conducted in the absence of the test compound. The result of this assay is then compared with that obtained in the presence of the test compound.

The test compounds may be screened in an in vitro assay to identify compounds capable of binding to a TRPV2. For this purpose, a test compound is contacted with TRPV2 under conditions and for a time sufficient to allow specific interaction between the test compound and the TRPV2 to occur, thereby resulting in the binding of the compound to the TRPV2, and the formation of a complex. Subsequently, the binding event is detected.

In one particular embodiment, the TRPV2 is immobilized on a solid support (such as a protein microchip) or on a cell surface or a membrane. For example, the protein complex can be immobilized directly onto a microchip substrate such as glass slides or onto multi-well plates using non-neutralizing antibodies, i.e., antibodies capable of binding to the complex but do not substantially affect its biological activities. A cannabinoid labeled with a detectable marker is contacted with the immobilized TRPV2. Test compounds can be contacted with the immobilized TRPV2 protein to allow binding to occur under standard binding assay conditions. To identify compound binding to aTRPV2, one can measure the detectable marker associated with TRPV2 or disassociated from the TRPV2. A test compound that binds competitively with the labeled cannabinoid to the TRPV2 will result in less binding of TRPV2 to the cannabinoid, thus less labeling associated with TRPV2.

In one embodiment, the test compound can be further evaluated for its ability to increase or decrease the ion conductivity of a TRPV2 channel. Known to those skilled in the art are methods for measuring a TRPV2 channel conductivity, for example, via the cellular depolarization/hyperpolarization or an increase in intracellular calcium ion levels. The level of intracellular calcium can be assessed using a calcium ion-sensitive fluorescent indicator, such as a calcium ion-sensitive fluorescent dye. Suitable calcium ion-sensitive fluorescent dyes include, for example, quin-2 (see, e.g., Tsien et al., J Cell BioL, 94:325, 1982), fura-2 (see, e.g., Grynkiewicz et al., J BioL Chem., 260:3440, 1985), fluo-3 (see, e.g., Kao et al., J BioL - 43 Chem., 264:8179, 1989) and rhod-2 (see, e.g., Tsien et al., J Biol. Chem., Abstract 89a, 1987). Suitable calcium ion-sensitive fluorescent dyes are commercially available from, for example, Invitrogen (Molecular Probes Products, Eugene, OR). Cellular fluorescence can also be monitored using a fluorometer or a flow cytometer having a fluorescence lamp and detector. FLIPR assay has been used routinely in measuring the ion conductivity.

The TRPV2 cation channels function to transport not only divalent cations, for example, Ca ²⁺ , but also monovalent cations, for example, Na ⁺ or K ⁺ . Therefore, assays for determining changes in the transport of monovalent cation can also be performed to measure the conductivity of a TRPV2 channel. Na ⁺ - and K^-sensitive dyes are known in the art and commercially available from, for example, Invitrogen (Molecular Probes Products, Eugene, OR).

The conductivity of a TRPV2 channel can also be measured by electrophysiologic techniques such as patch clamp. Patch clamp techniques are routinely used for studying electrical activities in cells, cell membranes, and isolated tissues. It involves forming an electrically tight, high-resistance seal between a micropipette and the plasma membrane. The current flowing through individual ion channels within the plasma membrane can then be measured. Different variants on the techniques allow different surfaces of the plasma membrane to be exposed to the bathing medium. The four most common variants include cell-attached, inside-out, outside-out and whole-cell patch clamp.

A patch-clamp method is commonly used with a voltage clamp that controls the voltage across the membrane and measures current flow. For example, in the case of whole-cell patch clamp, during the voltage clamp process, a microelectrode is inserted into a cell and current injected through the electrode so as to hold the cell membrane potential at some predefined level. A patch-clamp method can also be used in the current-clamp configuration, in which the current is controlled and the membrane potential is measured.

In another embodiment, the test compound can be further evaluated by administering it to a live animal. This can be useful to establish efficacy, toxicity and other pharmacological parameters important for establishing dosing regimens. For example, the compound can be administered to a dog to examine various pharmacological aspects of the compound in the dog. The dog testing can be particularly advantageous for identifying and establishing dosing regimens in humans, because dogs, particularly large breeds, are closer in weight to humans as compared to rats or mice and therefore provide a more suitable animal model for estimating human dosing.

The compound can also be administered to animals to assess the ability of the compound to alter nociceptive processes. Various animal models of pain exist. For example, the rat spinal nerve ligation (SNL) model of nerve injuryis a model of neuropathic pain (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 the 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 e t 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 models and inflammatory models (Brennan, TJ. et al. Pain 64:493, 1996; D'Amour, RE. 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. JNeurosci 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).

The discovery that certain cannabinoids activate TRPV2 also provides new methods for identifying additional compounds that increase the biological activity of TRPV2. Such methods can be based on rational drug design. Structural analogs or mimetics of the cannabinoid can be produced based on rational drug design with the aim of improving drug potency, efficacy and stability, and reducing side effects. Methods known in the art for rational drug design can be used in the present invention. See, e.g., Hodgson et al., Bio/Technology, 9:19- 21 (1991); U.S. Pat. Nos. 5,800,998 and 5,891,628, all of which are incorporated herein by reference.

Molecular modeling programs can be used to determine whether a small molecule can fit into a functionally relevant portion, for example, an active site, of the TRPV2 polypeptide. Basic information on molecular modeling is provided in, for example, M. Schlecht, Molecular Modeling on the PC, 1998, John Wiley & Sons; Gans et al., Fundamental Principals of Molecular Modeling, 1996, Plenum Pub. Corp.; N. C. Cohen (editor), Guidebook on Molecular Modeling in Drug Design, 1996, Academic Press; and W. B. Smith, Introduction to Theoretical Organic Chemistry and Molecular Modeling, 1996. U.S. Patents that provide detailed information on molecular modeling include U.S. Pat. Nos. 6,093,573; 6,080,576; 5,612,894; and 5,583,973.

Programs that can be useful for molecular modeling studies include, for example, GRID (Goodford, P. J., "A Computational Procedure for Determining Energetically Favorable Binding Sites on Biologically Important Macromolecules" J. Med. Chem., 28,

pp. 849-857, 1985), available from Oxford University, Oxford, UK; MCSS (Miranker, A. and M. Karplus, "Functionality Maps of Binding Sites: A Multiple Copy Simultaneous Search Method." Proteins: Structure, Function and Genetics, 11, pp. 29-34, 1991), available from Molecular Simulations, Burlington, Mass.; AUTODOCK (Goodsell, D. S. and A. J. Olsen, "Automated Docking of Substrates to Proteins by Simulated Annealing" Proteins: Structure. Function, and Genetics, 8, pp. 195-202, 1990); available from Scripps Research Institute, La Jolla, Calif.; and DOCK (Kuntz, I. D. et al., "A Geometric Approach to Macromolecule-Ligand Interactions" J. MoI. Biol., 161, pp. 269-288, 1982), available from University of California, San Francisco, CA.

In this respect, structural information on the TRPV2-cannabinoid complex is obtained. Preferably, atomic coordinates defining a three-dimensional structure of the complex can be obtained. For example, the interacting TRPV2-cannabinoid complex can be studied using various biophysical techniques including, e. g., X-ray crystallography, NMR, computer modeling, mass spectrometry, and the like. Likewise, structural information can also be obtained from protein complexes formed by interacting proteins and a compound that initiates or stabilizes the interaction of the proteins. Methods for obtaining such atomic coordinates by X-ray crystallography, NMR, and the like are known in the art and the application thereof to the target protein or protein complex of the present invention should be apparent to skilled persons in the art of structural biology. See Smyth and Martin, MoI. Pathol, 53:8-14 (2000); Oakley and Wilce, CHn. Exp. Pharmacol. Physiol, 27(3): 145-151 (2000); Ferentz and Wagner, Q. Rev. Biophys., 33:29- 65 (2000); Hicks, Curr. Med. Chem., 8(6) :627-650 (2001); and Roberts, Curr. Opin. Biotechnol, 10:42-47 (1999).

The domains, residues or moieties of a cannabinoid critical to TRPV2- cannabinoid interaction constitute the active region of the cannabinoid known as its "pharmacophore." Once the pharmacophore has been elucidated, a structural model can be established by a modeling process that may incorporate data from NMR analysis, X- ray diffraction data, alanine scanning, spectroscopic techniques and the like. Various techniques including computational analysis (e.g., molecular modeling and simulated annealing), similarity mapping and the like can all be used in this modeling process. See

e.g., Perry et al., in OSAR: Quantitative Structure-Activity Relationships in Drug Design, pp. 189-193, Alan R. Liss, Inc., 1989; Rotivinen et al., Acta Pharmaceutical Fennica, 97:159-166 (1988); Lewis et al., Proc. R. Soc. Lond., 236:125-140 (1989); McKinaly et al., Annu. Rev. Pharmacol. ToxicioL, 29:111-122 (1989). Commercially available molecular modeling systems from Polygen Corporation, Waltham, Mass., include the CHARMm program, which performs energy minimization and molecular dynamics functions, and QUANTA program, which performs construction, graphic modeling and analysis of molecular structure. Such programs allow interactive construction, modification, and visualization of molecules. Other computer modeling programs are also available from BioDesign, Inc. (Pasadena, Calif.), Hypercube, Inc. (Cambridge, Ontario), and Allelix, Inc. (Mississauga, Ontario, Canada).

A template can be formed based on the established model. Various compounds can then be designed by linking various chemical groups or moieties to the template. Various moieties of the template can also be replaced. In addition, in the case of a peptide lead compound, the peptide or mimetics thereof can be cyclized, e.g., by linking the N- terminus and C-terminus together, to increase its stability. These rationally designed compounds are further tested. In this manner, pharmacologically acceptable and stable compounds with improved potency/efficacy and reduced side effects can be developed. The compounds identified in accordance with the present invention can be incorporated into a pharmaceutical formulation suitable for administration to an individual.

In addition, the structural models or atomic coordinates defining a three- dimensional structure of the target protein or protein complex can also be used in virtual screen to select compounds capable of activating TRPV2. Various methods of computer- based virtual screen using atomic coordinates are generally known in the art. For example, U.S. Pat. No. 5,798,247 (which is incorporated herein by reference) discloses a method of identifying a compound (specifically, an interleukin converting enzyme inhibitor) by determining binding interactions between an organic compound and binding sites of a binding cavity within the target protein. The binding sites are defined by atomic coordinates.

The compounds designed or selected based on rational drug design or virtual screen can be tested for their ability to modulate (interfere with or strengthen) the interaction between the interacting partners within the protein complexes of the present invention. In addition, the compounds can further be tested in TRPV2 channel conductivity assays or animal models as described supra.

Following the selection of desirable compounds according to the methods disclosed above, the methods of the present invention further provide for the manufacture of the selected compounds. The compounds can be manufactured for further experimental studies, or for therapeutic use. The compounds identified in the screening methods of the present invention can be made into therapeutically or prophylactically effective drags for preventing or ameliorating diseases, disorders or symptoms caused by or associated with TRPV2, such as pain, or inflammation, etc.

Example 1

Expression of rat and human TRPV2 in HEK293 cells

A cDNA fragment encoding the full-length rat TRPV2 was subcloned into pCI-neo (Promega, Madison, WI) mammalian expression vector. The expression construct was then transfected into HEK293 cells with FuGeneβ transfection reagent (Roche, Indianapolis, IN) according to the vendor's protocol. Stable cell lines were selected by growth in the presence of 400 μg/ml G418. Single G418 resistant clones were isolated and purified. Stable expression of rat TRPV2 in these cells was confirmed by Western blot analysis with an anti-rat TRPV2 specific antibody (Chemicon, Temecula, CA), Ca ²⁺ imaging assay (FLIPR) and whole cell patch clamp analyses.

A cDNA fragment encoding the full-length human TRPV2 was subcloned into pCI-neo or pcDNA3 mammalian expression vectors. The expression constructs were then transfected into HEK293 cells with FuGeneό transfection reagent (Roche, Indianapolis, IN) according to 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 for 48-72 hr and either evaluated for transient expression and/ or activity, or dosed with 400 μg/ml G418 to select for stably-transfected TRPV2- expressing cell clones. Cells were maintained at 37° C and in 5% CO ₂ .

Example 2

TRPV2 is activated by δ ⁹ -tetrahydrocannabinol (δ ⁹ -THC)

To search for pharmacological activators of TRPV2, δ ⁹ -THC, a major psychoactive constituent of marijuana derived from Cannabis, was tested. The rat TRPV2-expressing HEK293 cells were seeded in a 384-well plate at a concentration of 5 x 10 ³ cells/well and incubated overnight at 37 ⁰ C. The following day, the cells were loaded with buffer and calcium dye 3 (Molecular Devices, Sunnyvale, CA) in a final volume of 50 μl and incubated for 30 minutes at 37°C/5% CO ₂ followed by 30 additional minutes at room temperature. The fluorescence intensity was measured by a fluorescent plate reader (FLIPR) before , during and after the addition of test compounds.

As shown in Figure 4A, addition of 100 μM ^ ⁹ ~THC solid linebut not buffer (dotted line), caused a robust elevation of intracellular Ca ²⁺ in rat TRPV2-expressing HEK293 cells. In contrast, no significant intracellular Ca ²⁺ elevation was observed in untransfected HEK293 cells at the same concentration of zl ⁹ -THC (dashed line), suggesting that the elevation of intracellular Ca ²⁺ was mediated by rat TRPV2. Activation of rat TRPV2 by zf-THC was dose-dependent with an EC5 ₀ value of 15.7 uM and Hill slope of 1.04 (Figure 4B).

To further confirm the zl ⁹ -THC effect on TRPV2, whole-cell patch clamp studies were performed. The extracellular solution contained (in mM): NaCl, 132; CaCl, 1.8; KCl, 5.4; MgCl ₂ , 0.8; HEPES, 10; glucose, 10; pH=7.4. The intracellular solution used to fill recording pipettes contained (in mM): CsCl, 145; EGTA, 5; HEPES, 10; glucose, 5; pH=7.4. Recordings were performed using the conventional whole-cell patch clamp technique, 2-3 days after transient transfection of human TRPV2 into HEK293 cell or 1-2 days after plating HEK293 cells stably expressing rat TRPV2 onto glass coverslips.

Currents were amplified by a patch clamp amplifier and filtered at 2 kHz (Axopatch 200B), sampled at 10 kHz using Digidata 1322A and acquired and analyzed with pClamp 9.0 (all instruments from Molecular Devices, CA). A 600 ms voltage ramp was given once every five seconds from -100 mV to +60 mV. The holding potential between voltage ramps was -100 mV. Extracellular solutions were applied to the cell at 0.5 ml/min via a gravity-fed perfusion system. AU experiments were performed at 22° C.

As shown in Figure 5B, upon application of 100 μM zf-THC, there was a significant increase of the whole-cell current amplitude (gray solid line) in HEK293 cells expressing rTRPV2 compared to control (black dotted line) at both hyperpolarized and depolarized membrane potentials. However, this effect was more pronounced at depolarized potentials. The same concentration of zd ⁹ -THC elicited no current above control level in untransfected HEK293 cells (data not shown). The J ⁹ -THC-activated current had a reversal potential near 0 mV, indicating the relatively unselective (at least for the cations used in these experiments) nature of the channel. Similar effects induced by/r'-THC were also observed in human TRPV2 (Figure 5A). Furthermore, the zf-THC- activated currents were significantly inhibited by 10 μM ruthenium red (RR), a nonselective TRP channel inhibitor (black line) in both rat and human TRP V2. Taken together, these results indicate that -_1 ⁹ -THC activates currents mediated by TRPV2 in these cells.

Example 3

TRPV2 Is Activated by Other Cannabinoids

To further explore activation of TRPV2 by cannabinoids, several different classes of cannabinoids were tested in a FLIPR calcium mobilization assay using 100 μM compound concentrations (except for anandamide which was at 120 μM) as evaluated using rat TRPV2-expressing HEK293 cells. All data was normalized to that observed for 100 μM δ9-THC. The tested compounds included: the non-psychoactive constituents of marijuana (cannabidiol and cannabinol); synthetic analogs of THC (nabilone, CP 55,940, HU210, HU211, HU-308, HU331, l l-hydroxy-δ9-THC, and 0-1821); several

endocannabinoids (anandamide, 2-arachidonoyl-glycerol (2-AG) and their analog palmitoylethanolamide (PEA)); a cannabinoid transport blocker (AM404); other synthetic cannabinoid receptor agonists (WIN55.212-2, WIN55,212-3, JWH015, JWH133, 0-1918 and CAYl 0429); and the non-selective agonist, 2- APB The ability of these compounds to activate rat TRPV2 is shown in Table 1. The data suggest that TRPV2 could be activated by more than one class of cannabinoids.

Table 1. Activation of rat TRPV2 by Cannabinoids

Example 4

δ ⁹ ~υHC selectively activates TRPV2

A selected number of cannabinoids were also tested against HEK293 cells expressing human TRPVl and canine TRPM8 in the FLIRP assay. As summarized in Table 2, 4 ⁹ -THC (240 μM) and cannabinol (600 μM) showed no significant activation of TRPVl, whereas anandamide, 2-AG and AM404 activated TRPVl, consistent with previous reports. None of these cannabinoids showed agonist effect against TRPM8.

Table 2. Activation of TRP channels by cannabinoids

Example 5

Deletion Mutants of TRPV2 Were Activated by Cannabinoids

TRPV2 deletion mutants were constructed and tested for activation by δ ⁹ -THC, 2- APB and high temperature stimulation. Methods of this Example can be used to construct and test any type of TRPV2 deletion mutants, including, but not limited to, mutants having one or more amino acid residues deleted at the N-terminal of the TRPV2, at the C-terminal of the TRPV2, and/or at any other location of the TRPV2.

DNA molecules encoding the deletion mutants were amplified by PCR using the rat TRPV2 cDNA as the template. For amino-terminal deletions, a series N-terminal forward primers encoding an initiating methionine in frame with sequences matching adjacent regions of the desired start at residues G21 (mutant N20, SEQ ID NO: 11), P33 (N32, SEQ ID NO:12), A66 (N65, SEQ ID NO: 13) and V84 (N83, SEQ ID NO: 14) paired with a C-terminal reverse primer were used for PCR amplification. While for carboxyl-terminal deletions, a forward N-terminal primer paired with a series C-terminal reverse primers ending at residues R706 (C51, SEQ ID NO:7), P729 (C32, SEQ ID NO:8), P738 (C23, SEQ ID NO:9) and E750 (Cl 1, SEQ ID NO: 10) with an in-frame stop codon at the terminal end of the open reading frame were used for amplification. After PCR amplification and purification, the DNA molecules encoding the deletion mutants were subcloned into the pCI-neo mammalian expression vector and the constructs were confirmed by DNA sequencing. The DNA molecules that encoded for the various TRPV2 deletion mutants comprised the nucleotide sequences of: SEQ ID NO: 18 (N20), SEQ ID NO: 19 (N32), SEQ ID NO:20 (N65), SEQ ID NO:21 (N83), SEQ ID NO:22 (C51), SEQ ID NO:23 (C32), SEQ ID NO:24 (C23), and SEQ ID NO:25 (Cl 1).

The deletion mutant constructs were then transfected into HEK293 cells using Fugene 6 reagent (Roche) as per the manufacturer's instructions. At 24 hours post- transfection, the cells were harvested and replated in fresh DMEM medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin and. The cells were distributed onto poly-D-Lysine coated 96-or 384- well plates at a density of approximately 40,000 and 10,000 cells per well, respectively. At approximately 48 hours post-transfection, the medium was removed from the assay plate and replaced with

Calcium 3 Dye Buffer (Molecular Devices) using the protocol available from the manufacturer. Calcium mobilization was triggered using δ ⁹ -THC or 2-APB or elevated temperature buffer and measured using either FLIPR or FLEX STATION instruments. It was found that deletion mutants of rat TRPV2 lacking the N-terminal 20, 33, 66, or lacking the C-terminal 11, 23, or 32 amino acid residues were still active in their responses to δ ⁹ -THC (Figure 6), 2-APB and an elevated temperature of about 53 ⁰ C (data not shown).

Example 6

Activation of TRPV2 Chimera

The domain-swapping chimeras between rat and human TRPV2s were also made and tested for activation by δ ⁹ -THC, 2-APB and high temperature stimulation. Methods of this Example can be used to construct and test any type of TRPV2 chimeras, including, but not limited to, the domain-swapping chimeras between TRPV2S from different animals, and the chimeras between TRPV2 and other TRPV channels such as TRPVl and TRPV3.

Based on the predicted topology and primary sequence features of TRPV2, TRPV2 are divided into 3 major domains: 1) the amino-terminal intracellular domain; 2) the transmembrane domain; and 3) the carboxyl-terminal intracellular domain. For a chimera, each domain can be of rat (R) or human (H) origin. Three rat and human TRPV2 chimera were constructed and tested herein. RRH (SEQ ID NO: 15) was a chimera comprising rat 1-392 aa, rat 393-646 aa, and human 647-764 aa. RHR (SEQ ID NO: 16) was a chimera comprising rat 1-392, human 391-646, and rat 647-761. HRR (SEQ ID NO: 17) was a chimera comprising human 1-390, rat 393-646, and rat 647-761.

DNA molecules encoding the three rat and human TRPV2 chimeras were obtained by fusion PCR. First, DNA molecules encoding the desired TRPV2 domains were amplified by PCR using the rat or human TRPV2 cDNA as the

template with synthetic primer DNA containing in-frame sequence that overlapped with the other species domain to be linked. After PCR amplification and purification, DNA molecules encoding the desired TRPV2 domains from human and rat were combined and used as templates for fusion PCR with primer DNA matching the 5' and 3' end sequences of the coding sequence for the full length TRPV2 chimera. The DNA molecules that encoded the various TRPV2 chimeras comprised the nucleotide sequences of: SEQ ID NO: 26(RRH), SEQ ID NO:27 (RHR), and SEQ ID NO:28 (HRR).

The DNA molecules encoding the TRPV2 chimera were then subcloned into the mammalian expression vector, pCI-neo and the resulting constructs confirmed by DNA sequencing. The chimeras were transiently expressed in HEK293 cells and their responses to a variety of stimulators were also tested as described in Example 5.

Chimera RHR was fully responsive to the addition of δ ⁹ -THC (Fig. 7A), ). Although the HEK cells expressing chimera RRH or HRR separately were poorly,or not active, respectively, the cells co-expressing both chimeras (RRH+HHR) were fully responsive to δ ⁹ -THC (Figure 7B. Similar responses by the above-listed chimeras to 2- APB stimulation were observed (data not shown). The gain of function study by coexpression of two inactive mutants suggests that a functional TRPV2 channel is a complex with multiple subunits and some of the critical functional domains act in trans rather than in cis.