Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-02-08
2002-09-24
Mertz, Prema (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C536S023100, C435S071200, C435S325000, C435S252300, C435S254110, C435S471000, C435S320100
Reexamination Certificate
active
06455278
ABSTRACT:
BACKGROUND OF THE INVENTION
Noxious chemical, thermal and mechanical stimuli excite peripheral nerve endings of small diameter sensory neurons (nociceptors) in sensory ganglia (e.g., dorsal root, nodose and trigeminal ganglia) and initiate signals that are perceived as pain. These neurons are crucial for the detection of harmful or potentially harmful stimuli (heat) and tissue damage (H
+
(local tissue acidosis), and/or stretch) which arise from changes in the extracellular space during inflammatory or ischaemic conditions (Wall and Melzack, 1994). The vanilloid capsaicin (8-methyl-N-vanillyl-6-nonenamide), the main pungent ingredient in “hot” capsicum peppers, is a very selective activator of thinly or unmyelinated nociceptive afferents (Szolcsanyi, 1993; Szolcsanyi, 1996). Electrophysiological studies have shown that vanilloids excite small sensory neurons by activating a plasma membrane channel that is non-selectively permeable to cations (Bevan and Szolcsanyi, 1990; Oh et al., 1996; Wood et al., 1988). The ultra potent tricyclic diterpene resiniferatoxin from Euphorbia plants (RTX; (Szolcsanyi et al., 1991)) binds with nanomolar affinity at the capsaicin binding site and has revealed a very localized distribution of capsaicin receptors to rat somatic and visceral primary sensory neurons (Szallasi, 1995).
The vanilloid receptor VR1 (Caterina et al., 1997) is thought to be a heat-sensing receptor whose threshold is decreased in the presence of protons or capsaicin (Tominaga et al., 1998). Capsaicin and protons interact at specific membrane recognition sites (vanilloid receptors) expressed almost exclusively by primary sensory neurons involved in nociception and neurogenic inflammation (Bevan and Szolcsanyi, 1990). The vanilloid (“capsaicin”) receptor VR1 is activated by capsaicin and RTX, and activation of VR1 is blocked by the antagonists capsazepine (CPZ; (Bevan et al., 1992)) and ruthenium red (RR; (Caterina et al., 1997; Wood et al., 1988)).
Hydropathicity analysis of the amino acid sequence of VR1 reveals 6 potential membrane spanning regions (S1-S6) and a putative pore-loop region between S5 and S6. A large intracellular domain contains 3 ankyrin repeat domains. (Caterina et al., 1997). This channel has significant structural similarities with the putative “store-operated” TRP calcium channel family. VR1 is a ligand-gated non-selective cation channel that shows pronounced outward rectification (Caterina et al., 1997). Importantly, VR1 is highly permeable to Ca2+, an ion known to be very important in regulating cell function ((Blackstone and Sheng, 1999; Gupta and Pushkala, 1999; van Haasteren et al., 1999)).
Searching genomic databases has revealed VRL-1, a subunit structurally related to VR1. Rat and human VRL-1 (AF129113 and AF129112, respectively) are ~49% identical and 66% similar to rat VR1 (AF029310)) (Caterina et al., 1999). Human VRL protein (AF103906) cloned by Wood and collegues (unpublished) is 99% identical to VRL-1 (AF129112). Recently, a patent application by Partiseti and Renard was published that described hVRCC (human vanilloid receptor like cation channel) which is nearly identical to AF129112 (the only difference is the deletion of Q418). We will refer to these sequences as VR2. Overall, the predicted structure of VR1 and VR2 is characteristic of a family of ion channels defined by the transient receptor potential (TRP) channels originally cloned from Drosophila melanogaster, a Ca-permeable channel that plays a role in phototransduction (Lu and Wong, 1987; Minke and Selinger, 1996). This receptor appears to also be involved in the sensation of pain-producing heat (Caterina et al., 1999). Expression of VR2 in oocytes and HEK cells usually conferred a sensitivity of the cells to noxious temperatures (>53 degC.), that was not sensitive to CPZ but was nearly completely blocked at 10 &mgr;M ruthenium red. Activation of VR2 induces a non-selective cation current with high permeability to Ca2+. Interestingly, the threshold for heat sensitivity decreased with repeated application of noxious stimuli, but not subthreshold temperatures (Caterina et al., 1999).
The rat SIC (stretch-inhibitable channel; Genbank AB015231), encoded by 529 amino acids, is thought to form an ion channel inhibited by stretch (Suzuki et al., 1999). The first 379 amino acids homologous to rat VR1. SIC lacks the large N-terminal cytoplasmic domain of the VR family but contains a sequence homologous to the A exon prior to the putative TM1. The last 163 amino acids, beginning in the middle of putative TM6 of rat SIC are similar to the corresponding amino acid sequence of the human VR3 A+B− of the present invention.
The present invention describes the cloning and function of a novel vanilloid receptor family member, VR3. This gene appears to be alternatively spliced to create at least 3 isoforms.
SUMMARY OF THE INVENTION
DNA molecules encoding 3 isoforms of the human vanilloid receptor 3 (hVR3) have been cloned and characterized. The biological and structural properties of these proteins are disclosed, as is the amino acid and nucleotide sequence. The recombinant protein is useful to identify modulators of the receptor VR3. Modulators identified in the assay disclosed herein are useful as therapeutic agents, which are candidates for the treatment of inflammatory conditions and for use as analgesics for intractable pain associated with postherpetic neuralgia, diabetic neuropathy, postmastectomy pain, complex regional pain syndromes, arthritis (e.g., rheumatoid and osteoarthritis), as well as ulcers, neurodegenerative diseases, asthma, chronic obstructive pulmonary disease, irritable bowel syndrome, and psoriasis. Uses include the treatment of central nervous system diseases, diseases of the intestinal tract, abnormal proliferation and cancer especially in the digestive system, prostate and female gonads, ulcer, liver disease, kidney disease, control of viscera innervated by the dorsal root ganglia, or to diagnose or treat any disorder related to abnormal expression of these hVR3 polypeptides, among others. In another aspect, the invention relates to methods to identify agonists and antagonists using the materials provided by the invention, and treating conditions associated with hVR3 imbalance. The recombinant DNA molecules, and portions thereof, are useful for isolating homologues of the DNA molecules, identifying and isolating genomic equivalents of the DNA molecules, and identifying, detecting or isolating mutant forms of the DNA molecules.
REFERENCES:
patent: WO 99/37765 (1999-07-01), None
Mikayama et al. Proc. Natl Acad. Sci. USA vol. 90, pp. 10056-10060, 1993.*
Voet et al. Biochemistry. John Wiley & Sons Inc. pp. 126-128 and 228-234, 1990.*
Bevan, S.; Hothi, S; Hughes, I.F.; James, H.P.; Rang, Shah K.; Walpole, C.S.J.; Yeats, J.C. Capsazepine: a competitive antagonist of the sensory neurone excitant capsaicin. Br. J. Pharmacol, 107, 544-522, 1992.
Bevan, Stuart; Szolcsanyi, Janos. Sensory neuron-specific actions of cpsaicin: mechanisms and applications. TiPS—vol. 11, Aug. 1990.
Caterina, Michael J.; Rosen, Tobias A.; Tominaga, Makoto; Brake, Anthony J.; Julius, David. A capsaicin-receptor homologue with a high threhold for noxious heat. Letters to Nature, vol. 398, Apr. 1, 1999.
Gupta, P.D.; Pushkala, K. Importance of the role of calcium in programmed cell death: a review. Cytobios 99, 83-95, 1999.
Leeman, Susan E,; Krause, James E.; Lembeck, Fred. Substance P and Related Peptides: Cellular and Molecular Physiology. Annals of the New York Academy of Sciences, vol. 632, 1991.
Minke, Baruch; Selinger; Zvi. The roles of trp and calcium in regulating photoreceptor function in Drosphila. Neurobiology, 6:459-466, 1996.
Oh, Uhtaek; Hwang, Sun Wook; Kim, Donghee. Capsaicin Activates a Nonselective Cation Channel in Cultured Neonatal Rat Dorsal Ganglion Neurons. The Journal of Neurosciences, 16 (5): 1659-1667, Mar. 1, 1996.
Szallasi M.D., Arpad. Autoradiographic visualization and pharmacological characterization of vannilloid (capsaicin)n receptors in several sp
Dubin Adrienne Elizabeth
Erlander Mark G.
Glass Charles A.
Huvar Arne
Mertz Prema
Ortho-McNeil Pharmaceutical , Inc.
Wallen III John W.
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