Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
2000-03-23
2002-06-18
Eyler, Yvonne (Department: 1646)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C435S069100, C536S063000
Reexamination Certificate
active
06406908
ABSTRACT:
The present invention is directed to an isolated nucleic acid encoding human vanilloid receptor, said receptor, its preparation, cells expressing said receptor and an assay for testing compounds for their potential to decrease pain in humans.
More particularly the present invention provides in a first aspect, an isolated nucleic acid encoding a human vanilloid receptor.
Even more particularly the present invention provides an isolated nucleic acid capable of directing expression of a human vanilloid receptor, in particular a cDNA capable of directing expression of said receptor, more particularly a cDNA comprising the nucleotide sequence as depicted in SEQ ID NO:1, most particularly a cDNA capable of directing expression of the predicted protein depicted in SEQ ID NO:2.
Said isolated nucleic acid may have a nucleotide sequence having sequence identity within a range of from more than 85.5%, preferably more than 97% to 100% over the open reading frame with the nucleotide sequence as described in SEQ ID NO:1. Preferably said isolated nucleic acid comprises a nucleic acid identical over the open reading frame to the sequence as described in SEQ ID NO:1.
The nucleic acid may be prepared for example by constructing a cDNA library from mRNA derived from human neuronal cells expressing vanilloid receptor. Such cells may be the nociceptive neurones, the cell bodies thereof residing within the dorsal root ganglia. The cDNA may then be expressed in a cell line not normally expressing endogenous vanilloid receptor and by iteratively subdividing and reassaying positive clones an individual clone may be obtained comprising the desired nucleic acid.
In a further embodiment the present invention is directed to a recombinant human vanilloid receptor. An example for said human vanilloid receptor may be a protein encoded by a nucleic acid having sequence identity within a range of from more than 85.5%, preferably more than 97% to 100% over the open reading frame with the nucleotide sequence as described in SEQ ID NO:1, e.g. a protein comprising an amino acid sequence having sequence identity within a range of from more than 85.7% to 100% with the amino acid sequence as described in SEQ ID NO:2, as calculated using the ALIGN program [Myers and Miller, CABIOS (1989)]. The amino acid differences may occur at a site selected from the N-terminus, the C-terminus and the putative pore region of the channel, i.e. amino acid 597 to amino acid 696 of SEQ ID NO:2. Preferably the human vanilloid receptor comprises an amino acid sequence identical to the sequence as described in SEQ ID NO:2.
The human vanilloid receptor when expressed in mammalian cells is activated by capsaicin, temperatures greater than 42° C. and by pH less than 5.5. The activation by all these effectors can be blocked substantially or completely by the action of the capsaicin antagonist capsazepine.
The vanilloid receptor may be prepared by stably transfecting a cell line with an appropriate expression cassette comprising a nucleic acid encoding the receptor, and culturing cells of said cell line under conditions which allow expression of said receptor.
In a further embodiment the present invention is directed to a cell belonging to a cell line expressing recombinant human vanilloid receptor.
In a further embodiment the present invention is directed to a cell belonging to a cell line expressing recombinant mammalian vanilloid receptor and aequorin. Examples for useful cell lines include any cell line growing well in culture, e.g. human embryonic kidney derived cells, like HEK293 cells and Chinese hamster ovary cells, like CHO-DUKX-B11 cells [Kaufman et al., Mol. Cell biol. 5:1750-1759 (1985)], which have been transformed/transfected with an appropriate expression cassette comprising a nucleic acid encoding human or mammalian vanilloid receptor and optionally a nucleic acid encoding aequorin. The expression cassette may be derived from a vector selected from for example pIRESneo, pBKCMV and pXMT3. A very useful cell line is the CHO-DUKX-B11 cell line.
Examples for a mammalian vanilloid receptor are the rat receptor and the human receptor.
Aequorin is a protein from the jellyfish. In the presence of Ca
++
ions the complex of aequorin and coelenterazine gives off light.
In a further embodiment the present invention is directed to an assay to measure vanilloid receptor activation comprising measuring changes in aequorin luminescence of cells expressing a mammalian vanilloid receptor and aequorin.
In a further embodiment the present invention is directed to a screening assay for vanilloid receptor channel blockers comprising incubation of a cell expressing a mammalian vanilloid receptor and aequorin with the potential vanilloid receptor channel blocker, adding an activator/agonist, e.g. capsaicin, of the vanilloid receptor channel and measuring changes in aequorin luminescence.
In a further embodiment the present invention is directed to a screening assay for vanilloid receptor channel agonists comprising incubation of a cell expressing a mammalian vanilloid receptor and aequorin, adding the potential vanilloid receptor channel agonist and measuring changes in aequorin luminescence.
In a further embodiment the present invention is directed to an assay to measure vanilloid receptor activation comprising measuring changes in the fluorescence of laser activated, calcium sensitive dyes, e.g. fura 2 AM and fluo3.
In a further embodiment the present invention is directed to a screening assay for vanilloid receptor channel blockers comprising incubation of a cell expressing a mammalian vanilloid receptor and optionally aequorin with the potential vanilloid receptor channel blocker, adding an activator/agonist, e.g. capsaicin, of the vanilloid receptor and measuring changes in the fluorescence of laser activated, calcium sensitive dyes, e.g. fura 2 AM and fluo 3.
In a further embodiment the present invention is directed to a screening assay for vanilloid receptor channel agonists comprising incubation of a cell expressing a mammalian vanilloid receptor and optionally aequorin with the potential vanilloid receptor channel agonist and measuring changes in the fluorescence of laser activated, calcium sensitive dyes, e.g. fura 2 AM and fluo3.
The above assay formats allow automation and are suitable for screening.
In a further embodiment the present invention is directed to a novel vanilloid receptor channel blocker identified by a screening assay for vanilloid receptor channel blockers comprising incubation of a cell expressing a mammalian vanilloid receptor and aequorin with the potential vanilloid receptor channel blocker, adding an activator/agonist, e.g. capsaicin, of the vanilloid receptor and measuring changes in aequorin luminescence.
In a further embodiment the present invention is directed to a novel vanilloid receptor channel agonist identified by a screening assay for vanilloid receptor channel agonists comprising incubation of a cell expressing a mammalian vanilloid receptor and aequorin with the potential vanilloid receptor channel agonist and measuring changes in aequorin luminescence.
In a further embodiment the present invention is directed to a novel vanilloid receptor channel blocker identified by a screening assay for vanilloid receptor channel blockers comprising incubation of a cell expressing a mammalian vanilloid receptor and optionally aequorin with the potential vanilloid receptor channel blocker, adding an activator/agonist, e.g. capsaicin, of the vanilloid receptor and measuring changes in the fluorescence of laser activated, calcium sensitive dyes, e.g. fura 2 AM and fluo3.
In a further embodiment the present invention is directed to a novel vanilloid receptor channel agonist identified by a screening assay for vanilloid receptor channel agonists comprising incubation of a cell expressing a mammalian vanilloid receptor and optionally aequorin with the potential vanilloid receptor channel agonist and measuring changes in the fluorescence of laser activated, calcium sensitive dyes, e.g. fura 2 A
James Iain Fraser
McIntyre Peter
Andres Janet L.
Eyler Yvonne
Novartis AG
Pfeiffer Hesna J.
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