Title:  Inhibitors of glycogen synthase kinase-3 and methods for identifying and using the same

United States Patent:  6,441,053

Issued:  August 27, 2002

Inventors:  Klein; Peter S. (Wynnewood, PA); Melton; Douglas (Lexington, MA)

Assignee:  The Trustees of the University of Pennsylvania (Philadelphia, PA); President and Fellows of Harvard University (Cambridge, MA)

Appl. No.:  327660

Filed:  June 8, 1999

A method of identifying inhibitors of glycogen synthase kinase-3 is provided. The method comprises providing a mixture comprising GSK-3, a phosphate source, and a GSK-3 substrate, incubating the mixture in the presence or absence of a test compound, and assessing the activity of GSK-3 in the mixture. A reduction of GSK-3 activity following incubation of the mixture in the presence of the test compound is an indication that the test compound is an inhibitor of GSK-3.

SUMMARY OF THE INVENTION

The invention relates to a method of identifying a GSK-3 inhibitor comprising providing a mixture comprising GSK-3, a source of phosphate, a GSK-3 substrate and a GSK-3 assay buffer, incubating the mixture in the presence or absence of a test compound, and measuring the level of phosphorylation of the GSK-3 substrate, wherein a lower level of phosphorylation of the GSK-3 substrate in the presence of the test compound compared with the level of phosphorylation of the GSK-3 substrate in the absence of the test compound is an indication that the test compound is a GSK-3 inhibitor.

The method of identifying a GSK-3 inhibitor may be performed either in vitro wherein the assay mixture is cell-free, in vitro wherein live cells are included in the assay, or in vivo in an animal.

The GSK-3 may be provided in the assay mixture as a protein or as a nucleic acid, either DNA or RNA, from which GSK-3 is expressed.

In one aspect of the invention, the mixture is contained within a eukaryotic cell.

In one embodiment of the invention, at least one of the GSK-3, the GSK-3 substrate and the test compound is injected into the eukaryotic cell prior to the incubation. In another embodiment, at least two of the GSK-3, the GSK-3 substrate and the test compound are injected into the eukaryotic cell prior to the incubation. In yet another embodiment, the GSK-3, the GSK-3 substrate and the test compound are injected into the eukaryotic cell prior to the incubation.

In another embodiment of the invention, the eukaryotic cell is suspended in a solution comprising the test compound.

The eukaryotic cell is selected from the group consisting of a Xenopus laevis oocyte, a Xenopus laevis embryo cell, a mammalian cell, a Drosophila melanogaster S2 cell, a Dictyostelium discoideum cell and a yeast cell. Preferably, the eukaryotic cell is selected from the group consisting of a Xenopus laevis oocyte and a Xenopus laevis embryo cell. More preferably, the eukaryotic cell is a Xenopus laevis oocyte which even more preferably, is aXenopus laevis embryo cell, yet more preferably, aXenopus laevis embryo ventral vegetal blastomere cell.

In one aspect of the invention, the phosphate source comprises a nucleotide triphosphate selected from the group consisting of ATP and GTP and preferably comprises a detectable label which is transferred to the substrate during the incubation. More preferably, the phosphate source comprises [.gamma.³² P]-ATP.

The GSK-3 which is contained within the mixture may be endogenous in the eukaryotic cell.

Preferably, the GSK-3 is selected from the group consisting of human GSK-3.alpha., human GSK-3.beta., Xenopus laevis GSK-3.alpha., Xenopus laevis GSK-3.beta., bacterially-expressed Xenopus laevis GSK-3.beta., bacterially-expressed rat GSK-3.beta., the expression product of the Drosophila melanogaster zw3/sgg gene, and the expression product of the Dictyostelium discoideum gskA gene. More preferably, the GSK-3 is bacterially-expressed rat GSK-3.beta..

The GSK-3 substrate which is contained within the mixture may also be endogenous in the eukaryotic cell.

Preferably, the GSK-3 substrate is selected from the group consisting of glycogen synthase, phosphatase inhibitor I-2, cAMP-dependent protein kinase type II subunit, phosphatase- 1 G-subunit, ATP-citrate lyase, acetyl coenzyme A carboxylase, myelin basic protein, a microtubule-associated protein, a neurofilament protein, an N-CAM cell adhesion molecule, nerve growth factor receptor, c-Jun, JunD, c-Myb, c-Myc, L-myc, adenomatous polyposis coli tumor suppressor protein, .tau. protein, .beta.-catenin, peptide GS-2, and peptide derivatives of any of these which comprise a GSK-3 phosphorylation site. More preferably, the GSK-3 substrate comprises .tau. protein.

The test compound used in the method of the invention is selected from the group consisting of bis-indolyl maleimides, staurosporine and derivatives thereof, and protein kinase C inhibitors.

The invention also includes a GSK-3 inhibitor which is identified by a method comprising providing a mixture comprising GSK-3, a source of phosphate, a GSK-3 substrate and a GSK-3 assay buffer, incubating the mixture in the presence or absence of a test compound, and measuring the level of phosphorylation of the GSK-3 substrate, wherein a lower level of phosphorylation of the GSK-3 substrate in the presence of the test compound compared with the level of phosphorylation of the GSK-3 substrate in the absence of the test compound is an indication that the test compound is a GSK-3 inhibitor.

Also included in the invention is a method of treating a GSK-3 -related disorder in an animal comprising administering to the animal a GSK-3 inhibitor suspended in a pharmaceutically acceptable carrier. Preferably, the animal is a mammal, and more preferably, the mammal is a human.

The GSK-3 related disorder which is treated according to the method of the invention is preferably selected from the group consisting of bipolar disorder including mania, Alzheimer's disease, diabetes, and leukopenia.

Lithium is recognized as a potent stimulator of hematopoiesis, both in vivo and in vitro (Doukas et al., 1986, Exp. Hematol. 14:215-221). Treatment of cyclic hematopoiesis in the grey collie dog with lithium carbonate eliminated the recurrent neutropenia and normalized the other blood cell counts (Hammond et al., 1980, Blood 55:26-28). Furthermore, lithium has been observed to stimulate in vitro Dexter culture hemopoiesis, leading to increases in granulocyte, megakaryocytes, and pluripotent stem cell numbers. In one study in a murine Dexter culture system, exposure of Dexter cultures to 1 mM LiCl prior to culture resulted in greater hemopoiesis than was observed in Dexter cultures which were not exposed to LiCl (Quesenberry et al., 1984, Blood 63:121-127). These findings suggest that human cyclic hematopoiesis, including leukopenia, may be successfully treated with lithium.

The GSK-3 inhibitor which is used to treat a GSK-3 related disorder is preferably Ro31-8220 or structurally-related compounds.

The invention also relates to a method of reducing motility of mammalian spermatozoa comprising administering to the spermatozoa a GSK-3 inhibitor suspended in a pharmaceutically-acceptable carrier. Lithium has been demonstrated to inhibit the motility of swimming spermatozoa.

Claim 1 of 1 Claim

What is claimed is:

1. A method of treating a GSK-3 related disorder other than Alzheimer's disease in an animal comprising administering to said animal a GSK-3 inhibitor suspended in a pharmaceutically acceptable carrier, wherein said GSK-3 inhibitor is not lithium and is identified by:

(a) providing a mixture comprising GSK-3, a source of phosphate, a GSK-3 substrate, and a GSK-3 assay buffer;

(b) incubating said mixture in the presence or absence of a test compound;

(c) measuring the level of phosphorylation of said GSK-3 substrate; and

(d) determining whether the level of phosphorylation of said GSK-3 substrate is lower in the presence of said test compound than in the absence of said test compound, and further wherein the GSK-3 related disorder is selected from the group consisting of bipolar disorder, mania, and leukopenia.

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