Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2001-02-02
2003-10-07
Kemmerer, Elizabeth (Department: 1647)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007800, C435S174000, C514S002600
Reexamination Certificate
active
06630312
ABSTRACT:
BACKGROUND
Insulin resistance refers to a decreased capacity of circulating insulin to regulate nutrient metabolism. Individuals with insulin resistance are predisposed to developing Type 2 diabetes, and insulin resistance is an integral feature of its pathophysiology. Greater than normal levels of insulin are secreted to overcome target tissue resistance, which leads to the eventual failure of pancreatic &bgr; cells in predisposed individuals.
Insulin resistance also occurs in hypertension, cardiovascular disease and dyslipidemia, suggesting an etiologic relationship that is referred to as the metabolic syndrome or syndrome X. The prevalence of insulin resistance is remarkably high, particularly in ageing adult populations (National Diabetes Data Group,
Diabetes in America
(National Institutes of Diabetes and Digestive Diseases, National Institutes of Health, USA, 1994), and rising—most rapidly in the young (Mokdad et al. (2000)
Diabetes Care
23:1278-1283). Nevertheless, only rare genetic causes have been identified. Environmental factors, including sedentary lifestyle, obesity, and increased age induce insulin resistance, whereas exercise and weight loss reverse it.
SUMMARY
The present invention is based, in part, on the discovery that aspirin reverses insulin resistance in liver and fat cells, e.g., by targeting IKK-&bgr;. It has been discovered that insulin sensitivity is improved in vivo by modulating, e.g., reducing, IKK&bgr; activity, e.g., by decreased protein expression. Thus, IKK-&bgr; is a target for identifying compounds for the treatment of disorders associated with insulin resistance.
Accordingly, in one aspect, the invention features a method of identifying, evaluating or making a compound or agent, e.g., a candidate compound or agent, for treatment of a disorder characterized by insulin resistance. The method includes evaluating the ability of a compound or agent to interact with, e.g., bind, IKK-&bgr;, to thereby identify a compound or agent for the treatment of a disorder characterized by insulin resistance.
In a preferred embodiment, the disorder is diabetes, e.g., Type I or Type II diabetes; hyperglycemia; hyperinsulinemia; dyslipidemia; obesity; polycystic ovarian disease; hypertension, cardiovascular disease, or syndrome X.
In a preferred embodiment, the compound is: a polypeptide, e.g., a randomly generated polypeptide which binds IKK-&bgr;; an antibody, e.g., an intrabody or a randomly generated antibody which binds IKK-&bgr; or modulates IKK-&bgr; activity; a small molecule, e.g., a small molecule which binds IKK-&bgr; or modulates IKK-&bgr; activity.
In a preferred embodiment, the method further includes contacting the identified compound with IKK-&bgr;, e.g., purified IKK-&bgr;, to thereby evaluate the interaction, e.g., binding, between the compound and IKK-&bgr;.
In a preferred embodiment, the method further includes contacting the identified compound with a cell, e.g., a fat cell or a liver cell, to thereby evaluate the effect of the compound on an IKK-&bgr; activity of the cell. The compound can be evaluated based on one or more of: the ability of the compound to modulate, e.g., reduce or reverse, insulin resistance in a cell; the ability of the compound to modulate glucose and/or lipid homeostasis; the ability of the compound to modulate phosphorylation, e.g., of a component of the insulin signaling cascade (e.g., the ability to increase tyrosine phosphorylation of a component of the insulin signaling cascade and/or decrease Ser/Thr phosphorylation of a component of the insulin signaling cascade).
In a preferred embodiment, the method further includes administering the identified compound to a subject to evaluate the effect of the compound on insulin resistance. In a preferred embodiment, the subject is a mouse (e.g., a NOD mouse, an ob/ob mouse, a db/db mouse) or a rat (e.g., a Zucker fatty rat, a streptozotocin rat).
In another aspect, the invention features a method of identifying a compound or agent for treatment of a disorder characterized by insulin resistance. The method includes contacting IKK-&bgr;, or a cell expressing IKK-&bgr; with a test compound; and determining whether the test compound interacts with, e.g., binds to IKK-&bgr;, and/or modulates the activity of IKK-&bgr;, to thereby identify a compound.
Methods for identifying a compound or an agent can be performed, for example, using a cell free assay. For example, the IKK-&bgr; can be immobilized to a suitable substrate, e.g., glutathoine sepharose beads or glutathoine derivatized microtitre plates, using a fusion protein which allows for IKK-&bgr; to bind to the substrate, e.g., a glutathoine-S-transferase/IKK-&bgr; fusion protein.
In a preferred embodiment, the ability of a test compound to bind IKK-&bgr; can be determined by detecting the formation of a complex between IKK-&bgr; and the compound. The presence of the compound in complex indicates the ability to bind IKK-&bgr;.
In a preferred embodiment, IKK-&bgr; is further contacted with aspirin.
In another preferred embodiment, a compound is identified using a cell based assay. These methods include identifying a compound based on its ability to modulate, e.g., inhibit, an IKK-&bgr; activity of the cell. For example, the ability of a compound to modulate one or more of, e.g., glucose and/or lipid homeostasis, insulin resistance in a cell, e.g., a fat cell or a liver cell, and/or phosphorylation of a component of the insulin signaling cascade can be determined.
In a preferred embodiment, the method further includes contacting the identified compound with IKK-&bgr;, e.g., purified IKK-&bgr;, to thereby evaluate binding between the compound and IKK-&bgr;.
In a preferred embodiment, the method further includes contacting the identified compound with a cell, e.g., a fat cell or a liver cell, to thereby evaluate the effect of the compound on an IKK-&bgr; activity of the cell. For example, the ability of a compound to modulate one or more of, e.g., glucose and/or lipid homeostasis, insulin resistance in a cell, e.g., a fat cell or a liver cell, and/or phosphorylation of a component of the insulin signaling cascade can be evaluated.
In a preferred embodiment, the method further includes administering the identified compound to a subject to evaluate the effect of the compound on insulin resistance. In a preferred embodiment, the subject is a mouse (e.g., a NOD mouse, an ob/ob mouse, a db/db mouse) or a rat (e.g., a Zucker fatty rat, a streptozotocin rat).
In a preferred embodiment, the compound is: a polypeptide, e.g., a randomly generated polypeptide which interacts with, e.g., binds, IKK-&bgr;; an antibody, e.g., an intrabody or a randomly generated antibody which interacts with IKK-&bgr;; a small molecule, e.g., a small molecule which interacts with IKK-&bgr;.
In a preferred embodiment, the compound is a compound other than aspirin.
In a preferred embodiment, the disorder is diabetes, e.g., Type I or Type II diabetes; hyperglycemia; hyperinsulinemia; dyslipidemia; obesity; polycystic ovarian disease; hypertension; cardiovascular disease; or syndrome X.
In another aspect, the invention features a method of identifying a compound or agent for treatment of diabetes, e.g., Type I or Type II diabetes. The method includes contacting IKK-&bgr;, or a cell expressing IKK-&bgr;, with a test compound; and determining whether the test compound binds to IKK-&bgr;, to thereby identify a compound for treatment of diabetes.
Methods for identifying a compound or an agent can be performed, for example, using a cell free assay. For example, the IKK-&bgr; can be immobilized to a suitable substrate, e.g., glutathoine sepharose beads or glutathoine derivatized microtitre plates, using a fusion protein which allows for IKK-&bgr; to bind to the substrate, e.g., a glutathoine-S-transferase/IKK-&bgr; fusion protein.
In a preferred embodiment, the ability of a test compound to bind IKK-&bgr; can be determined by detecting the formation of a complex between IKK-&bgr; and the compound. The presence of the compound in complex indicates the ability to bind
DeBerry Regina M.
Fish & Richardson PC
Joslin Diabetes Center, Inc.
Kemmerer Elizabeth
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