Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-04-28
2002-03-05
Mertz, Prema (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S183000, C435S194000, C435S325000, C530S350000
Reexamination Certificate
active
06352833
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to methods useful for delaying or ameliorating human diseases associated with endothelial dysfunction.
BACKGROUND OF THE INVENTION
The vascular endothelium plays a critical regulatory role due to its physical location between circulating blood and vascular smooth muscle cells. In response to mechanical and hormonal signals from the blood, endothelial cells release substances which modulate platelet adhesion and aggregation and affect contraction and proliferation of vascular smooth muscle.
Under physiological conditions, the endothelium maintains the vasculature in a dilated state, inhibits vascular smooth muscle proliferation, and prevents the adhesion of circulating platelets and monocytes. Thus, the endothelium modulates the tone of the underlying vascular smooth muscle.
Activation or injury to the endothelium leads to the development of endothelial dysfunction which contributes to adhesion and aggregation of platelets, vasoconstriction, and proliferation of vascular smooth muscle cells. Endothelial dysfunction is present in a variety of conditions associated with aging, diabetes, gastrointestinal disease, hyperglycemia, hypercholesterolemia, peripheral vascular disease, stroke, hypertension, thrombosis, angina, atherosclerosis and other cardiovascular diseases.
In view of the wide range of disorders that are associated with endothelial dysfunction, it would be desirable to identify compounds that are capable of enhancing the effects of endothelial-derived substances on their target cells, including vascular smooth muscle cells and platelets.
SUMMARY OF THE INVENTION
Here we present evidence that G protein-coupled receptors are regulated by cyclic GMP-dependent protein kinase (G kinase), the principle mediator of nitric oxide-cyclic GMP (NO-cGMP) signaling which modulates inhibition of platelet aggregation and relaxation of vascular smooth muscle. The invention provides methods for identifying compounds which mimic the effects of NO-cGMP on platelets and vascular smooth muscle cells to enhance platelet inhibition and vascular smooth muscle cell relaxation.
In the first aspect, the invention features a method of assaying a test compound, the method comprising: providing a system comprising a G protein-coupled receptor-bearing cell, cyclic GMP or an analog thereof, and cyclic GMP-dependent protein kinase; contacting the test compound with the system; and determining whether the test compound affects either the activation of the cell or the phosphorylation of the G-protein-coupled receptor or fragment thereof, or the phosphorylation of proteins associated with the G-protein-coupled receptor. In a preferred embodiment of this aspect, the cell is selected from the group consisting of platelets, human erythroleukemia (HEL) cells, and vascular smooth muscle cells. Alternatively, the cell may be genetically engineered to express the G protein-coupled receptor or fragment thereof. In other embodiments of this aspect, determining whether the test compound affects either the activation of the cell or the phosphorylation of the G-protein-coupled receptor or fragment thereof, or the phosphorylation of proteins associated with the G-protein-coupled receptor may be done in vitro or in vivo
A second aspect of the invention provides a method of assaying a test compound, the method comprising: providing a system comprising a G protein-coupled receptor-bearing cell preparation, cyclic GMP or an analog thereof, and cyclic GMP-dependent protein kinase; contacting the test compound with the system; and determining whether the test compound affects either the activation of the cell or the phosphorylation of the G-protein-coupled receptor or fragment thereof, or the activation or phosphorylation of proteins associated with the G-protein-coupled receptor. In a preferred embodiment of this aspect, the cell preparation is made from cells selected from the group consisting of platelets, HEL cells, and vascular smooth muscle cells.
In other preferred embodiments of the invention, the method further comprises a G protein-coupled receptor agonist and may include measuring GTPase activity. In another preferred embodiment of the invention, the G-protein-coupled receptor may be selected from the group consisting of the thromboxane receptors, thrombin receptors, serotonin receptors, purinergic receptors, adrenergic receptors, bradykinin receptors, angiotensin receptors, cAMP receptors, dopamine receptors, muscarinic receptors, opioid receptors, opsin receptors, and isoforms thereof.
By “test compound” is meant any chemical compound, be it naturally-occurring or artificially-derived. Test compounds may include, for example, peptides, polypeptides, synthesized organic molecules, naturally occurring organic molecules, and nucleic acid molecules.
By “contacting” is meant to submit an animal, cell, lysate, or extract derived from a cell, or molecule derived from a cell to a test compound.
By “cell preparation” is meant an extract or lysate of a cell or a component thereof.
By “affects” is meant changes, either by increase or decrease.
By “determining” is meant analyzing the effect of a test compound on the test system. The readout of the analysis may be altered protein stability, levels, or biological activity. The means for analyzing may include, for example, antibody labeling, immunoprecipitation, in vivo and in vitro phosphorylation assays, protease or glucosidase digestion, GTPase assays, measures of inositol triphosphate production, intracellular calcium mobilization, and other methods known to those skilled in the art.
By “G protein-coupled receptor” is meant a receptor with seven transmembrane domains which uses a heterotrimeric G protein as an intermediary in signal transduction. G protein-coupled receptors belong to a large superfamily of receptors that share a structural motif of seven transmembrane helices. G protein-coupled receptors include adrenergic, angiotensin, cAMP, dopamine, muscarinic, opioid, opsins, purinergic, serotonin, thrombin, thromboxane, and isoforms thereof, or any other G protein-coupled receptors that are known to couple with G proteins.
By “activation” is meant the response of a cell to a substance, whether endogenously derived or exogenously administered. The response may include changes in shape (for example, contraction), aggregation, intracellular calcium mobilization, inositol triphosphate production, expression of cell surface activation markers, increase in cell proliferation markers, increases in gene expression, and other features known to those skilled in the art.
By “proteins associated with the G protein-coupled receptor” is meant any protein or peptide that forms a complex with the G protein-coupled receptor by means of covalent, non-covalent, reversible, or other form of protein-protein interaction known to those skilled in the art.
The invention provides a means of identifying test compounds that affect vascular smooth muscle relaxation, inhibition of platelet activation, or other markers of endothelial dysfunction. This is particularly useful because endothelial dysfunction has been correlated with a variety of conditions associated with aging, diabetes, gastrointestinal disease, hyperglycemia, hypercholesterolemia, peripheral vascular disease, stroke, hypertension, thrombosis, angina, atherosclerosis and other cardiovascular diseases. Thus, compounds that affect endothelial dysfunction may be used in therapy or diagnosis of such diseases.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
REFERENCES:
patent: 5482954 (1996-01-01), Kohn et al.
Pfeifer et al. (1995) Cyclic GMP-dependent Protein Kinase Blocks Pertussis Toxin sensitive Hormone Receptor Signaling Pathways in Chinese Hamster Ovary Cells. J. Biol. Chem. 270(16)9052-9059.*
Wang et al. (1998) Mechanism of platelet inhibition by nitric oxide: In vivo phosphorylation of thromboxane receptor by cyclic GMP-dependent protein kinase. Proc. Natl. Acad. Sci. USA 95:4888-4893.*
Diviani et al. (1
Clark & Elbing LLP
Mertz Prema
Murphy Joseph F.
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