Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-09-28
2003-02-25
Kunz, Gary (Department: 1647)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007210
Reexamination Certificate
active
06524805
ABSTRACT:
BACKGROUND
1. Technical Field
The invention relates to methods and materials involved in the activation and inhibition of opiate, cannabinoid, and estrogen receptors. Specifically, the invention relates to mu3 opiate receptors, cannabinoid receptors, and estrogen surface receptors, and the biological responses induced by such receptors.
2. Background Information
Three general classes of cell surface opioid receptors (kappa, delta, and mu) have been described based on ligand specificity. Opioid receptors exhibiting high binding specificity for morphine have been designated mu opioid receptors. Detailed analysis of mu opioid receptors from various tissue has revealed the existence of multiple mu opioid receptor subtypes. In fact, the cDNA encoding the mu1 opioid receptor subtype has been identified. Oligonucleotides complementary to some, but not all, exons of the mu1 opioid receptor can block the effects mediated by the mu1 and mu2 receptor subtypes. Thus, the mu1 and mu2 opioid receptor subtypes appear to share exon sequences, as would be expected of splice variants. Supporting the idea of alternative splicing is the finding of a single mu gene in mouse chromosomal DNA. In addition, a novel rat brain mu opioid receptor subtype, designated rMOR1B, has been identified. This receptor is identical to the rat mu1 opioid receptor at its N-terminus but differs in its length and sequence at the C-terminus. Further, affinity studies demonstrated that the substrate specificity of rMOR1B is similar to that of the rat mu1 opioid receptor, but rMOR1B is more resistant to agonist-induced desensitization and has a different expression pattern in brain. The presence of another opiate receptor, designated mu3 opiate receptor, has been demonstrated pharmacologically. This mu3 opiate receptor is opioid peptide insensitive and opiate alkaloid selective. In addition, detailed binding analysis indicates that the mu3 opiate receptor is expressed by immune tissues (e.g., human monocytes and granulocytes).
Cannabinoids induce physiological activities similar to those induced by morphine. Cannabinoids, however, induce these activities by interacting with specific cannabinoid receptors that are structurally distinct from opioid receptors. To date, two subtypes of G-protein-coupled cannabinoid receptors have been identified: CB1 and CB2. These two cannabinoid receptor subtypes are expressed by different tissues and have different pharmacological properties. For example, the CB1 receptor is expressed in brain and endothelial tissue while the CB2 receptor is expressed in the immune system. In addition, SR 141716A is a CB1 receptor antagonist while SR 144528 is a CB2 receptor antagonist.
Estrogen, like morphine, appears to have multiple receptors. In fact, two different cDNA clones have been identified: one encoding estrogen receptor-alpha (ER-alpha) and the other encoding estrogen receptor-beta (ER-beta). In addition, many variants of ER-beta have been described, including human ER-beta isotypes 1 through 5. Unlike the mu opioid receptor subtypes, however, these estrogen receptors are intracellular nuclear receptors. Presumably, each of these intracellular estrogen receptors, upon interaction with estrogen, mediate biological responses by interacting directly with DNA. Tamoxifen is a lipophilic anti-estrogen compound that can inhibit the interaction of estrogen with intracellular nuclear receptors.
SUMMARY
The present invention relates generally to mu3 opiate receptors, cannabinoid receptors, and estrogen surface receptors (ESRs). Specifically, the invention provides methods and materials for identifying mu3 opiate receptor agonists and antagonists, cannabinoid receptor agonists and antagonists, and ESR agonists and antagonists. In addition, the invention provides an isolated nucleic acid molecule that encodes a mu3 opioid receptor, a host cell containing an isolated nucleic acid molecule that encodes a mu3 opioid receptor, and an isolated mu3 opioid receptor polypeptide. Further, the invention provides methods and materials for treating cancers, inflammatory conditions, sepsis conditions, viral infections, and cardiovascular diseases.
The present invention is based on the discovery of a cell surface receptor for estrogen. Specifically, this cell surface receptor for estrogen, designated estrogen surface receptor 1 (ESR1), exhibits ligand specificity for estrogen and 17&bgr;-estradiol (E2) as well as E2 conjugated to bovine serum albumen (E2-BSA). In addition, ESR1 is tamoxifen sensitive. In other words, tamoxifen can inhibit the stimulatory effects of ESR1 agonists such as estrogen and E2. Thus, tamoxifen is an ESR1 antagonist. Further, ESR1 is expressed by human endothelial cells. Moreover, the ESR1-mediated biological responses induced by ESR1 agonists include changes in intracellular calcium concentration and nitric oxide release. The existence of a cell surface receptor for estrogen has significant medical implications. For example, plasma membrane impermeable compounds can be used to influence the biological effects of estrogen. Taken together, the discovery of ESR1 and the biological responses mediated by ESR1 provides methods and materials for modulating calcium and nitric oxide regulated mechanisms. As described herein, modulating calcium and nitric oxide regulated mechanisms can be used to treat cancers, inflammatory conditions, sepsis conditions, viral infections, and cardiovascular diseases.
The present invention is also based on the discovery of several assays for identifying mu3 opiate receptor, cannabinoid receptor, or ESR agonists and antagonists. Specifically, the assays involve monitoring at least one biological response induced by mu3 opiate receptors, cannabinoid receptors, or ESRs. Such biological responses include changes in intracellular calcium concentration and nitric oxide release. Thus, the assays can be configured to monitor intracellular calcium concentration, nitric oxide release, or both. The assays are particularly advantageous since the biological responses induced by mu3 opiate receptors, cannabinoid receptors, or ESRs can be detected within seconds of applying an agonist. Thus, many test molecules can be screened rapidly for the ability to either stimulate or inhibit mu3 opiate receptor, cannabinoid receptor, or ESR activities. In addition, the assays are specific for the particular receptor subtype. For example, stimulation or inhibition of mu3 opiate receptor activity can be easily distinguished from effects operating through mu1 or mu2 opioid receptors. Likewise, stimulation or inhibition of CB1 activity can be easily distinguished from effects operating through CB2, and stimulation or inhibition of ESR activity can be easily distinguished from effects operating through the intracellular nuclear receptors for estrogen (e.g., ER-alpha and ER-beta). Further, any results generated from an assay that, for example, monitors intracellular calcium concentration can be easily confirmed by performing an assay that monitors nitric oxide release. Having the ability to confirm a particular test molecule's ability to stimulate or inhibit mu3 opiate receptor, cannabinoid receptor, or ESR activity provides a powerful tool for reliably identifying receptor agonists and antagonists. Taken together, the assays described herein can be used to identify mu3 opiate receptor, cannabinoid receptor, or ESR agonists and antagonists rapidly, specifically, and reliably.
In addition, the present invention is based on the discovery of an isolated nucleic acid molecule that encodes a mu3 opiate receptor. Specifically, the mu3 opiate receptor is a cell surface receptor that exhibits specificity for morphine while being opioid peptide insensitive. In addition, the interaction of morphine with the mu3 opiate receptor induces changes in intracellular calcium concentration and nitric oxide release. Isolated nucleic acid molecules that encode the mu3 opioid receptor, isolated mu3 opioid receptor polypeptides, and host cells containing such isolated nucleic acid molecules are particularly useful to r
Fimiani Caterinea
Stefano George B.
Fish & Richardson P.C. P.A.
Kunz Gary
Landsman Robert S.
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