Tissue Protective Cytokine Receptor Complex, Assays for Identifying Tissue Protective Compounds and Uses Thereof

Abstract

The present invention is directed methods for identifying compounds that have a tissue protective activity using a heteromultimer receptor complex that mediates the tissue protective activities. The complex consists of at least one EPO-R in complex with at least one betac Receptor. These compounds used in the assays to identify tissue protective compounds include, but are not limited to, small molecules and biologics. The compounds identified using these assays can be used to treat or prevent various diseases, disorders, or conditions of the central and peripheral nervous systems as well as those of other erythropoietin-responsive or excitable cells, tissues, and organs.

Description

[0001]This application claims benefit of priority of copending U.S. patent application Ser. No. 10 / 676,694, filed Sep. 30, 2003, which claims benefit of priority under 35 U.S.C. §119(e) to U.S. provisional patent Application No. 60 / 465,891, filed Apr. 25, 2003, the entire contents of each of which is incorporated herein by reference in their entirety.1. INTRODUCTION[0002]The present invention is directed to methods that target tissue protective cytokine receptor complexes having at least one beta common (βc) receptor (also known as CD131) and at least one erythropoietin (EPO) receptor for drug screening / discovery and methods of treatment or prophylaxis. In particular, the present invention is drawn to methods for identifying and screening for compounds that modulate the interaction of a tissue protective cytokine receptor complex and a tissue protective cytokine receptor complex ligand. The methods of the invention also encompass methods for treating or preventing a disease or disor...

AI Technical Summary

Benefits of technology

[0011]Cell-based assays can be used to identify compounds that target a tissue protective cytokine receptor complex in the presence and / or absence of a ligand for a tissue protective cytokine receptor complex. In such embodiments, a cell that expresses a tissue protective cytokine receptor complex is contacted with a test compound. To determine whether the test compound modulates the activity of a tissue protective cytokine receptor complex in the cell, a readout is observed or measured. The readout can be a simple measure of the binding of the test compound to the tissue protective cytokine receptor complex. In certain embodiments, the test compound is labeled and binding of the labeled test compound to the tissue protective cytokine receptor complex is measured by detecting the label attached to the test compound. In other embodiments, the readout is a phenotypic change such as a change in cell size, shape, or apoptosis of the cell. In yet other embodiments, the readout is expression of a reporter gene or a change in an indicator dye loaded into the cell or included in the culture media. In such embodiments, the cells employed in the assay are genetically engineered to express a reporter gene product upon activation of a tissue protective cytokine receptor complex. In other embodiments, the readout is determined by enzymatic reaction of the cell. In such embodiments, cells used in the assay after being contacted with a test compound are lysed. The cell lysates are them examined for indicators of the activation of a tissue protective cytokine receptor complex. For example, a cell lysates can be exposed to an antibody that binds the complex, which antibody can also attach to a support anchoring the complex. Other antibodies can then be added to the assay to detect whether the tissue protective cytokine receptor complex has been activated. For example, anti-phosphotyrisine antibodies can bind to phosphorylated portions of the complex and / or to downstream components of the EPO / β common receptor complex. In certain embodiments, antibodies that can bind to phosphorylated portions of the β common receptor portion of the complex and / or antibodies can bind to phosphorylated portions of the EPO receptor are used. In certain embodiments, cells expressing tissue protective cytokine receptor complexes are contacted with antibodies that can bind to phosphorylated portions of the β common receptor and / or the EPO receptor. In certain embodiments, isolated tissue protective cytokine receptor complexes are contacted with antibodies that can bind to phosphorylated portions of the β common receptor and / or the EPO receptor. In certain embodiments, antibodies that can bind to phosphorylated portions of the EPO receptor are PY-JAK2 antibodies. As downstream cytokine activation targets are identified for tissue protective cytokine receptor complexes, the readout of the screening methods of the invention can be detection of phosphorylation of downstream cellular components.

[0021]In still another embodiment, the invention provides for a method for enhancing the function of excitable cells, tissues, or organs in a human comprising modulating a tissue protective cytokine receptor complex in a human.

[0030]The term “preventing a disease, disorder, or condition” means delaying the onset, hindering the progress, hindering the appearance, protection against, inhibiting or eliminating the emergence, or reducing the incidence, of such disease, disorder, or condition. Use of the term “prevention” is not meant to imply that all patients in a patient population administered a preventative therapy will never develop the disease, disorder, or condition targeted for prevention, but rather that the patient population will exhibit a reduction in the incidence of the disease, disorder, or condition. For example, many flu vaccines are not 100% effective at preventing flu in those who administered the vaccine. Prevention also encompasses protection of excitable tissues. One skilled in the art can readily identify patients and situations for whom preventative therapy would be beneficial, such as, but not limited to, patients for whom surgery is planned, patients at risk for inherited diseases, disorders, or conditions, patients at risk for diseases, disorders, or conditions precipitated by environmental factors, or portions of the population at risk for particular diseases, disorders, or conditions such as the elderly, infants, or those with weakened immune systems, or those patients with genetic or other risk factors for a disease, disorder, or condition.

Problems solved by technology

However, it has been reported that these complexes failed to result in any physiologically relevant effect (Scott et al., 2000, Blood, 96:1588-1590).

However, neither EPO nor carbamylated EPO had an effect on recovery in the βc receptor knock-out mice.

For example, many flu vaccines are not 100% effective at preventing flu in those who administered the vaccine.

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