Identification and use of selective inhibitors of glycogen synthase kinase 3

The invention provides for use of selective inhibitors of GSK3 for treatment of diseases that are mediated by GSK3 activity, including non-insulin dependent diabetes mellitus (NIDDM) and Alzheimer's disease. Also described are methods of identifying inhibitors of GSK3 activity. The selective GSK3 inhibitor can be a peptide, peptoid, small organic molecule, or polynucleotide.

FIELD OF THE INVENTION 
This invention provides materials and methods relating to identification of 
selective inhibitors of glycogen synthase kinase 3 (GSK3), and also 
concerns methods of treating a condition mediated by GSK3 activity by 
administrating a selective inhibitor of GSK3. The biological conditions 
treatable include non-insulin dependent diabetes mellitus and Alzheimer's 
disease. 
BACKGROUND OF THE INVENTION 
Glycogen synthase kinase 3 (GSK3) is a proline-directed serine/threonine 
kinase originally identified as an activity that phosphorylates glycogen 
synthase as described in Woodgett, Trends Biochem Sci, 16: 177-181 (1991). 
GSK3 consists of two isoforms, .alpha. and .beta., and is constitutively 
active in resting cells, inhibiting glycogen synthase by direct 
phosphorylation. Upon insulin activation, GSK3 is inactivated, thereby 
allowing the activation of glycogen synthase and possibly other 
insulin-dependent events. Subsequently, it has been shown that GSK3 is 
inactivated by other growth factors or hormones that, like insulin, signal 
through receptor tyrosine kinases. Examples of such signaling molecules 
include IGF-1 and EGF as described in Saito et al, Biochem J, 303: 27-31 
(1994), Welsh et al, Biochem J, 294: 625-629 (1993), and Cross et al, 
Biochem J, 303: 21-26 (1994). GSK3 has been shown to phosphorylate 
.beta.-catenin as described in Peifer et al, Develop Biol 166:543-56 
(1994). Other activities of GSK3 in a biological context include GSK3's 
ability to phosphorylate tau protein vitro as described in Mandelkow and 
Mandelkow, Trends in Biochem Sci 18: 480-83 (1993), Mulot et al, Febs Lett 
349: 359-64 (1994), and Lovestone et al, Curr Biol 4: 1077-86 (1995), and 
in tissue culture cells as described in Latimer et al, Febs Lett 365: 42-6 
(1995). Selective inhibition of GSK3 may be useful to treat or inhibit 
disorders mediated by GSK3 activity. 
SUMMARY OF THE INVENTION 
The invention is a method for treating a biological condition mediated by 
GSK3 activity by administering an effective amount of a pharmaceutical 
composition comprising a selective GSK3 inhibitor to a subject having a 
condition mediated by GSK3 activity or susceptible to such a condition. 
The biological condition can be, for example, non-insulin dependent 
diabetes mellitus (NIDDM) or Alzheimer's disease. The selective inhibitor 
of GSK3 activity can be a small organic molecule, a peptide, a peptoid, or 
a polynucleotide. The method can include administration of a second 
therapeutic agent, for example, lithium ion. The invention is also in 
vitro method of identifying an inhibitor of GSK3 kinase activity. A 
candidate inhibitor can be a peptide, a peptoid, a small organic molecule, 
or polynucleotide. The invention thus provides pharmaceutical composition 
comprising an inhibitor identified by this in vitro method. 
The invention is also a transgenic fruit fly having a transgene encoding a 
GSK3 inhibitory polypeptide, for example p110*, under the regulatory 
control of an eye specific promoter, where the transgenic fruit fly 
exhibits an enhanced rough eye mutant morphology The invention provides a 
method of screening for an inhibitor of GSK3 activity by administering to 
this transgenic fruit fly a candidate inhibitor of GSK3, and identifying a 
functional inhibitor by its ability to enhance a rough eye mutant 
morphology. 
The invention provides a pharmaceutical composition for treating a 
condition mediated by GSK3 activity having a pharmaceutically acceptable 
carrier and an effective amount of selective inhibitor of GSK3 activity. 
The invention also provides a method of treating a subject having NIDDM or 
Alzheimer's disease by administering this pharmaceutical composition 
subject, a method of promoting activation of an insulin signaling pathway 
by contacting a cell characterized by insulin resistance with an effective 
amount of a selective inhibitor of GSK3, and a method of reducing tau 
hyperphosphorylation and polymerization in a population of cells 
exhibiting tau polymerization by contacting said cells with an effective 
amount of a selective inhibitor of GSK3. The selective inhibitor of GSK3 
used in these methods can be a small molecule. 
DETAILED DESCRIPTION 
All patents, patent publications, and scientific articles cited herein are 
incorporated by reference. 
This invention provides materials and methods for identifying inhibitors of 
the proline directed serine/threonine kinase GSK3. The invention also 
provides for use of selective inhibitors of GSK3 to treat biological 
conditions mediated by GSK3 activity, for example, Alzheimer's disease 
(AD) and non-insulin dependent diabetes mellitus (NIDDM). 
Screening Assays 
The screening assays described herein can be used to screen for selective 
inhibitors of GSK3. A selective inhibitor of GSK3 is an inhibitor that 
inhibits GSK3 at a much lower concentration of inhibitor than that 
required for any inhibitory effect on any other kinases. Other kinases are 
tested in comparison to GSK3 related assays that are used to identify the 
GSK3 inhibitor being tested for selectivity. Preferably, the GSK3 
inhibitor selectively inhibits GSK3 and does not appreciably inhibit any 
other kinase. 
Currently there are no known high throughput screening assays for 
inhibitors of GSK3. To address this problem, the inventors have developed 
methods for identifying these inhibitors. The methods include methods to 
assay for GSK3 kinase activity in an in vitro assay, in a cell-based 
assay, in a binding assay, and an in vivo Drosophila assay. Additionally, 
the inventors use assays specific for GSK3 in NIDDM, including assays 
using stable tissue culture cells, assays using differentiated cell lines, 
and assaying using human muscle primary myocytes. 
A. In Vitro Kinase Assay 
General aspects of the kinase activity assays are conducted as described in 
U.S. Pat. No. 4,568,649 EP 0154,734, and JP 84/52452, incorporated by 
reference in full. These references describe kinase activity assays 
conducted for kinases other than GSK3. Many of the components of the in 
vitro kinase assay can also be used in the other assays for identifying 
selective inhibitors of GSK3. 
The in vitro assay provides a high throughput method for screening for 
selective inhibitors that act on the polypeptide GSK3 and is suitable for 
an initial screen of candidate inhibitors. First the assay can be 
conducted for GSK3 inhibition as compared to a control reaction in the 
absence of a candidate inhibitor. To provide information as to whether the 
GSK3 inhibition identified is selective for GSK3, any positive inhibitors 
so identified can then also be tested for their potential to inhibit other 
kinases, including kinases that are close in structure and mode of action 
to GSK3, and those kinases that are distant from GSK3 in structure and 
mode of action. Any kinases can be used for this comparative purposes, for 
example the known kinases CDC2, p70S6 kinase, Akt kinase, cAbl, cSRC, and 
PI 3-kinase. Selective inhibition of GSK3 is identified where the IC50 of 
an inhibitor for GSK3 is substantially lower than the IC50 of the same 
inhibitor for another kinase tested in a related assay. The IC50 is that 
concentration of inhibitor that inhibits 50% of the enzyme activity (for 
example GSK3 activity or the activity another kinase). That is, the 
concentration of inhibitor that reduces the enzyme activity to 50% of 
normal. Preferably the IC50 of the inhibitor of GSK3 is at least 10 fold 
lower than the IC50 for any other kinases tested for that inhibitor to be 
identified as a selective inhibitor of GSK3. For example, the IC50 of a 
candidate inhibitor can be 1 nM for GSK3 inhibition and 10 nM or greater 
for effecting inhibition of any of the other kinases tested. 
The term "glycogen synthase kinase 3" or "GSK3" as used herein refers to 
GSK3.alpha. or GSK3.beta.. GSK3 is a protein originally identified by its 
phosphorylation of glycogen synthase as described in Woodgett et al, 
Trends Biochem Sci, 16: 177-181 (1991). Synonyms of GSK3 are tau protein 
kinase I (TPK I), FA kinase and kinase FA. Mammalian forms of GSK3 have 
been cloned as described in Woodgett, EMBO J. 9(8): 2431-2438 (1990), and 
He et al, Nature 374: 617-22 (1995) and Stambolic and Woodgett, Biochem. 
J. 303: 701-704 (1994). Inhibitors of GSK3 can be inhibitors of any of the 
known forms of GSK3, including either GSK3.alpha. or GSK3.beta. or both. 
GSK3 polypeptide as used herein includes the native protein and also can 
further include truncations, variants, alleles, analogs and derivatives of 
a native GSK3 protein. Such polypeptides possess one or more of the 
bioactivities of the GSK3 protein, including kinase activities such as 
polymerizing tau protein, or phosphorylating glycogen synthase, for 
example. Thus, GSK3 polypeptides from which inhibitors are screened can 
have sequence identity of at least 40%, preferably 50%, preferably 60%, 
preferably 70%, more preferably 80%, and most preferably 90% to the amino 
acid sequence of the native protein, wherever derived, from human or 
nonhuman sources. The polynucleotides encoding a GSK3 polypeptide can have 
60%, preferably 70%, more preferably 80%, more preferably 90% and most 
preferably 95% sequence identity to a native polynucleotide sequence of 
GSK3. Also included, therefore, are alleles and variants of the native 
polynucleotide sequence so that the polynucleotide encodes an amino acid 
sequence with substitutions, deletions, or insertions, as compared to the 
native sequence. 
The term "peptide substrate" refers to a peptide or a polypeptide or a 
synthetic peptide derivative that can be phosphorylated by GSK3 activity 
in the presence of an appropriate amount of ATP or a phosphate donor. 
Detection of the phosphorylated substrate is generally accomplished by the 
addition of a labeled phosphate that can be detected by some means common 
in the art of labeling, such as radiolabeled phosphate. The peptide 
substrate may be a peptide that resides in a molecule as a part of a 
larger polypeptide, or may be an isolated peptide designed for 
phosphorylation by GSK3. 
Additionally, a synthetic peptide substrate designed for a purpose 
appropriate to the assay can be used, for example the CREB peptide. CREB 
peptide is a sequence within the CREB DNA-binding protein. See Wang et al, 
Anal. Biochem 220: 397-402 (1994). The inventors provide a novel 
modification of this peptide substrate. Peptide substrates that are 
phosphorylated by GSK3 have in common the amino acid motif SXXXS (SEQ ID 
NO. 2) where S is serine and X is any amino acid, and where the N terminal 
S is the target of phosphorylation by GSK3, and the C terminal S is 
prephosphorylated. Such peptides are appropriate peptide substrates for 
GSK3 activity assays and can be synthetically made by standard techniques. 
The term "prephosphorylated" refers to phosphorylating a substrate with 
non-radiolabeled phosphate in advance of conducting a kinase assay using 
the substrate. Generally, the substrate that is used for a kinase activity 
assay will contain one or more sites that are phosphorylatable by the 
kinase being tested, and may contain one or more other phosphorylatable 
sites that are not specific for the kinase being tested. These other sites 
need to be phosphorylated in order to create a phosphorylatable motif for 
the kinase being tested. Thus before conducting the kinase assay, it may 
be beneficial or required to phosphorylate a specific phosphorylatable 
site on the substrate, for example, the C terminal serine of the motif 
SXXXS (SEQ ID NO. 2), with non-labeled phosphate in advance of running the 
kinase assay. The prephosphorylation can be performed synthetically. 
An example of a prephosphorylated substrate in the context of an assay of 
the invention is an anchor ligand such as biotin attached to the sequence 
of the CREB peptide SGSGKRREILSRRPSYR (SEQ ID No. 1) where the S near the 
C terminal between P and Y is prephosphorylated and the S between L and R 
is phosphorylatable by GSK3 during a kinase assay. The purpose of the 
prephosphorylation is that GSK3 phosphorylation motif require 
prephosphorylation at the final S of the motif SXXXS (SEQ ID No. 2) 
(reading N terminal to C terminal) in order for the more N-terminal S to 
become phosphorylatable by GSK3. 
In the in vitro kinase assay and in some of the other assays for inhibitors 
of GSK3, recombinant GSK3 or endogenous GSK3 is combined with a peptide 
substrate and a candidate inhibitor. The peptide substrate is selected or 
designed as is appropriate to the particular assay. For example, where 
inhibition of GSK3 activity with respect to phosphorylation of glycogen 
synthase is sought, a glycogen synthase polypeptide, or polypeptide 
derivative of glycogen synthase might be most appropriate. Where 
inhibition of phosphorylation of tau protein is sought, a peptide 
substrate that is the tau polypeptide, or a derivative of the tau 
polypeptide, might be most appropriate. Other potential and exemplary 
peptide substrates of GSK3 can be designed from c-Jun and .beta.-catenin. 
An "anchor ligand" refers to a ligand that is attachable to a peptide 
substrate (for example the peptide substrate of formula SXXXS (SEQ ID NO. 
2)) that can bind a substrate anchor (defined below). The purpose of the 
anchor ligand is to anchor the peptide substrate to an anchor from which 
it is possible to detect whether the peptide substrate has been 
phosphorylated with a labeled phosphate. Thus, the substrate anchor and an 
anchor ligand ensure that the peptide substrate, labeled or not, does not 
get washed from the assay, so that those peptide substrate molecules that 
have been labeled can be detected and quantified. An example of an anchor 
ligand is biotin, where the substrate anchor is streptavidin agarose beads 
impregnated with scintillant or scintillant-lined streptavidin coated 
wells. 
A "substrate anchor" refers to a molecule that is affixed to, for example, 
an agarose bead, or the inside of a microwell of a multiwell plate, and 
that can bind an anchor ligand with high affinity. The anchor ligand will 
generally be covalently or otherwise attached to a peptide substrate. An 
example of a substrate anchor is streptavidin agarose beads impregnated 
with scintillant or scintillant-lined streptavidin coated wells, where the 
anchor ligand is biotin. Typically, the agarose bead or the microwell will 
contain a material that scintillates in the presence of a radiolabeled 
material, so that a substrate anchor that binds an anchor ligand attached 
to a peptide substrate with a radiolabeled phosphorylation site can cause 
the scintillant material to react to the proximity of the radiolabeled 
phosphate. This reaction is quantifiable and indicates the presence of a 
labeled substrate, and so the presence of active GSK3. 
GSK3 isoforms .alpha. and .beta. phosphorylate serine and threonine 
residues in the amino ac motif serine-proline (SP) or threonine-proline 
(TP), as well as at the N-terminal serine in the motif SXXXS (SEQ ID NO. 
2), provided that the C-terminal serine in this sequence is 
prephosphorylated, as described in Wang et al, Anal. Biochem 220: 397-402 
(1994) and Roach, J Biol Chem 266: 14139-42 (1991). The assay published in 
Wang et al, is a low throughput GSK3 assay that makes use of a peptide 
substrate whose sequence is based on that of a GSK3 phosphorylation site 
in the CREB DNA-binding protein. In the published assay, the C-terminal 
serine in the SXXXS (SEQ ID NO. 2) motif is prephosphorylated by casein 
kinase II. The inventors herein have developed a modified peptide of motif 
SXXXS (SEQ ID NO. 2) that can be synthesized with the C-terminal serine 
prephosphorylated (Chiron Mimotopes, Clayton, Australia) and which also 
contains an N-terminal anchor ligand. The novel peptide substrate, so 
designed and constructed with an anchor ligand is then able to accomplish 
binding to a substrate anchor at the N-terminal anchor ligand. This novel 
process eliminates the need to prephosphorylate the C-terminal serine as a 
separate step, and facilitates high throughput screening. A substrate 
anchor is some molecule or mechanism for keeping the substrate present 
during a wash. For example, where the anchor ligand is biotin, especially 
in the case where the peptide substrate is bound at the N-terminus to 
biotin, the anchor can be a molecule that binds biotin, for example, 
streptavidin. 
The in vitro method of identifying an inhibitor of GSK3 activity includes 
constructing peptide substrate. The peptide substrate can be any peptide 
substrate phosphorylatable by GSK3, and may be a peptide substrate 
including the formula: anchor ligand-SXXXS (SEQ ID NO. 2) (where X is any 
amino acid) prephosphorylated at C terminal S, and contacting the 
prephosphorylated substrate with GSK3 in the presence of radiolabeled 
phosphate-.gamma.ATP, a substrate anchor, and a candidate inhibitor. The 
in vitro method of identifying an inhibitor of GSK3 kinase activity 
includes contacting a peptide substrate coupled to an anchor ligand with 
GSK3 polypeptide in the presence of radiolabeled phosphate-.gamma.ATP, a 
substrate anchor, and candidate inhibitor, measuring an incorporation of 
radiolabel into the peptide substrate, then, in a separate assay vessel 
contacting a peptide substrate coupled to an anchor ligand with GSK3 in 
the presence of radiolabeled phosphate-.gamma.ATP, and a substrate anchor, 
and measuring an incorporation of radiolabel into said peptide substrate; 
ultimately an inhibitor of GSK3 kinase activity is identified by a 
reduction of label incorporation in the assay with the candidate inhibitor 
as compared to the assay without the candidate inhibitor. The inhibition 
of GSK3 kinase activity can be identified by a reduction in labeling of 
substrate relative to control. The degree of phosphorylation of the 
peptide substrate is monitored by assaying the incorporation of 
radioactive phosphate into the peptide substrate. Inhibition of this 
incorporation is an indication of inhibition of GSK3, as compared to a 
control reaction conducted in the absence of a candidate inhibitor. 
Alternatively, the GSK3 activity is measured by indirectly observing 
phosphorylation of a coexpressed substrate, for example by mobility shift 
on an SDS gel or by using phosphoform specific antibodies. Another 
alternative is to measure physiological consequence of GSK3 inhibition by, 
for example measuring glycogen synthase activation or .beta.-catenin 
accumulation. 
To conduct the in vitro kinase assay of the invention using microwells, 
scintillant may be present by pre-coating the wells with a scintillant 
material, or by adding it later following wash step. The scintillant can 
be purchased from Packard, Meriden, Conn. Wells coated with scintillant 
are then in addition coated with streptavidin. Where the scintillant is 
added later, the streptavidin can be present on agarose beads. In any 
event, the streptavidin in the wells binds the biotin that contacts it. 
Where the substrate anchor is biotin, the radiolabel on the phosphorylated 
substrate that has been conjugated to the biotin will cause the 
scintillant to emit light. Where the streptavidin is attached to agarose 
beads containing scintillant, binding a biotin-conjugated radiolabeled 
peptide substrate will cause the beads to scintillate and will be an 
indication of the inhibitory activity of the candidate inhibitor, as 
compared to a control. In both the case of the wells lined with the 
scintillant, and the agarose beads containing scintillant, a reduction in 
scintillation as compared to a control amount of scintillation measured 
under non-inhibitory conditions, indicates the presence of a functional 
inhibitor GSK3 activity. If the peptide has been phosphorylated by GSK3 
with .sup.32 P-labeled or .sup.33 P-labeled phosphate, radioactive decay 
will cause the scintillant present in a microwell or mixed in agarose 
beads that are present in the reaction mixture to emit light and the 
measure of the amount of light emitted will be a measure of the activity 
of GSK3 in the assay. Low activity of GSK3 observed in the presence of a 
candidate inhibitor, as compared to the activity of GSK3 in the absence of 
the inhibitor, may indicate that the inhibitor is functional and can 
inhibit GSK3 kinase activity. In any case, an equal amount of streptavidin 
should be loaded into each well or should be affixed to the agarose beads, 
and an equal amount of the beads should be added to each assay. 
B. Cell-Based Assays 
Several cell-based assays can be used to screen for inhibitors that can 
penetrate a cell and can act within the cell at any step in the process of 
expression or activity of GSK3. Thus, a cell-based assay can screen for 
those inhibitors that act during transcription of GSK3 or that can act 
during intracellular post-transcriptional events in the process of making 
mature GSK3, in addition to those that can inhibit GSK3 kinase or binding 
activity. A cell-based assay includes cell that can express GSK3, such as 
a cell transformed with the gene encoding GSK3 including also regulatory 
control sequences for the expression of the gene, or a cell that expresses 
GSK3 endogenously. The cell capable of expressing GSK3 is incubated in the 
presence of a candidate inhibitor. GSK3 from the cell is placed in contact 
with a peptide substrate, and radiolabeled phosphate-ATP. The amount of 
phosphorylation of the peptide substrate is an indication of the degree of 
inhibition accomplished by the candidate inhibitor, as compared to a 
control. 
The cell-based method of identifying a GSK3 inhibitor can include use of a 
peptide substrate, for example a peptide substrate of formula: anchor 
ligand-SXXXS (SEQ ID NO. 2), prephosphorylated at C terminal S, where X is 
any amino acid, and radioactive labeled phosphate-.gamma.ATP, and 
identifying inhibition of GSK3 activity by a reduction in labeling of 
substrate, as compared to a control. 
C. Binding Assays 
A binding assay can be used for identification of molecules that inhibit 
GSK3 binding other molecules. Such identification can act as an initial 
screen of candidate inhibitors of GSK3 kinase activity because GSK3 kinase 
activity may include binding to a phosphorylatable target or other 
molecule. Identification of GSK3 inhibitory molecules can be conducted by 
screening for those molecules that can inhibit binding of GSK3 to a 
potential substrate, including, for example, glycogen synthase or tau 
protein. The term "binding activity" in reference to interaction between 
two molecules indicates a higher affinity binding and a lower dissociation 
constant than non-specific binding, thus distinguishing specific binding 
activity from background binding. An inhibitor functional in a binding 
assay would be expected to inhibit the binding of GSK3 to a substrate, and 
thus might be expected to bind competitively to GSK3 or its substrate. 
Where the peptide substrate is used to conduct a binding assay, the 
presence of an inhibitor is monitored by the interruption of binding of 
the substrate to GSK3 as compared to a control reaction in the absence of 
a candidate inhibitor. 
D. Drosophila Screening Assay 
An alternative or subsequent assay that can be used to screen in vivo for 
inhibitors of GSK3 kinase activity is a Drosophila eye screen for 
inhibitors. The fly eye screen detects inhibitory activity by expressing, 
under control of an eye specific promoter, a polypeptide that can effect a 
reduction in GSK3 activity, either by direct or indirect action on 
endogenous GSK3 in the fly eye cells. For example, such a polypeptide can 
be p110* polypeptide. P110* is effectively inhibitory of GSK3, although 
its method of action is believed to be by activation of the kinase Akt 
that then phosphorylates GSK3, making GSK3 inactive. By reducing the 
activity of endogenous GSK3, expression of a GSK3 inhibitory polypeptide 
or a polypeptide with an indirect inhibitory effect like p110*, sensitizes 
the assay so that lower concentrations of candidate inhibitor can be 
tested. The p110* polypeptide is a fusion mutant derived from 
phosphotidylinositol 3-kinase (PI 3-kinase) and is described in Hu et al, 
Science, 268:100-102 (1995). In addition, the p110* polypeptides can be 
targeted to the cell membrane such as those mutants described in Klippel 
et al, Mol. Cell. Biol. 16(8): 4117-4127 (1996). The eye specific promoter 
can be any promoter specific to expression of proteins in eye tissue, 
including but not limited to, for example, GMR as described in Hay et al, 
Development 120: 2121-9 (1994), and the sevenless promoter, as described 
in Bowtell et al, Genes and Development 2: 620-634 (1988). Expression of 
GSK3 with p110* results in a mutant morphology called roughening or rough 
eye mutant morphology. 
Drosophila embryos are transformed by the method of Karess and Rubin, Cell 
38:135-146 (1984) with a polynucleotide contruct made up of a GSK3 
inhibitory polypeptide coding sequence under the regulatory control of a 
GMR promoter. The preferred polynucleotide encoding is a polynucleotide 
encoding p110*, or a membrane-targeted version of p110*. The flies are 
allowed to develop normally and are selected by eye morphology for 
successful transformants. Successful transformants will have an aberrant 
morphology characterized by rough eye cell morphology that is detectable 
under a dissecting microscope. The transgenic flies are then fed food 
spiked with an appropriate dose of a candidate inhibitor. The amount of 
the inhibitor will depend on the desired potency of the molecule as an 
inhibitor. The flies can be fed different small molecule inhibitors, for 
example, a different inhibitor for each population of tranformants. The 
flies are fed a candidate inhibitor throughout third instar larval 
development during which time they are observed for enhancements of their 
rough eye mutant morphology. Positives are identified. This screening 
method may also be applied as a secondary or tertiary screen using 
candidate inhibitors that have already been found positive in prior 
screens such as the kinase or binding assay screening protocols. 
Variations to the protocol include injecting a candidate inhibitor into 
the third instar larvae of the transformants, which are then observed for 
reversion of the rough eye morphology to normal. 
The advantages of the Drosophila assay are that the assay can give initial 
in vivo data indicating whether the selective GSK3 inhibitor can inhibit 
in vivo, whether the inhibitor is toxic to an insect, whether the 
inhibitor is cell permeable, and whether the inhibitor is stable in an 
animal cell. 
E. Cell-Based Assays Related to GSK3 in NIDDM 
Assays designed for finding inhibitors of GSK3 in the context of NIDDM are 
based on two premises: that GSK3 inhibitors will potentiate insulin 
signaling by activating glycogen synthase (GS) and increasing glycogen 
synthesis and that stimulation of glycogen synthesis by GSK3 inhibitors 
will lead to increased uptake of glucose in NIDDM patients. The following 
assays test the ability of inhibitors of GSK3 to potentiate 
insulin-stimulated GS activity in tissue culture cells, and can ultimately 
be used to investigate the effect of these inhibitors on glucose uptake 
into cells. 
In these assays measurement of the effectiveness of a given inhibitor is 
accomplished by the effect of inhibitors on glycogen synthase activity as 
described in Thomas et al, Anal. Biochem. 25:486-99 (1968), glucose uptake 
as described in Begun and Ragolia Endocrinology 137: 2441-6 (1996), 
Claraldi et al, J. Clin. Invest. 96:2820-27 (1995), Hara et al, PNAS 
91:7415-19 (1994), Robinson and James, Am. J. Physiol. 263:E383-93(1992), 
and Sasoaka et al JBC 270: 10885-92 (1995) and glucose incorporation into 
glycogen as described in Begun and Ragolia Endocrinology 137: 2441-6 
(1996), Sasoaka et al, JBC 270: 10885-92 (1995), and Takata et al, JBC 
267:9065-70 (1992). 
1. Assays using Stable Tissue Culture Cells 
a. Glucose Uptake and Glycogen Synthesis 
Assays for glucose uptake and glycogen synthesis can be conducted as 
described for cell-based assays generally. To measure glucose uptake and 
glycogen synthesis, a variety of stable tissue culture cell lines that 
respond to insulin stimulation by increasing the synthesis of glycogen and 
increasing the rate of glucose uptake from the medium can be used. These 
include HepG2 liver cells, CHO-IR cells, and Hirc cells. Under appropriate 
conditions the addition of insulin and glucose to HepG2 cells stimulates 
glycogen synthase activity and this stimulation of potentiated in the 
presence of the GSK3 inhibitor iodotubercidin. The response of glucose 
uptake in these cells to insulin, glucose and inhibitor can be as much as 
six fold, and consequently this assay is useful to detect the ability of 
inhibitors to potentiate glycogen synthesis in cells. 
Chinese hamster ovary cells over-expressing insulin receptor (CHO-IR) have 
been used to measure insulin-stimulated glucose uptake, as described in 
Hara et al, PNAS 91: 7415-9 (1994), and glycogen synthase activity as 
described in Sakaue et al, JBC 270: 11304-9 (1995). CHO-IR cells show up 
to a 2.5 fold increase in glycogen synthase activity in response to 
insulin and can be used instead as an alternative to HepG2 cells. 
Rat-1 fibroblast cells over-expressing the insulin receptor (Hirc) have 
been used to measure insulin-stimulated glucose incorporation into 
glycogen as described in Sasaoka et al., JBC 270:10885-92 (1995) and 
Takata et al., JBC 267:9065-70 (1992). 
b. Assays Testing Cell Permeability and Intracellular GSK3 Activity 
If compounds fail to stimulate glucose uptake or glycogen synthase 
activity, they may be tested to see if the defect is in their ability to 
enter cells or in their ability to inhibit GSK3 under intracellular 
conditions (i.e. 3 mM ATP). An in vivo kinase assay using tau protein was 
designed for this purpose. The tau assay relies on the ability to observe 
direct consequences of GSK3 activity within a cell, without first having 
to purify the GSK3 from a cell lysate. The tau protein assay uses tau, a 
microtubule-associated protein that is also a GSK3 substrate. See 
Lovestone et al, Current Biology 4(12):1077-86 (1994), Anderton et al, 
Neurobiology of Aging 16(3): 389-97 (1995), Latimer et al, FEBS Let 365: 
42-6 (1995), and Sperber et al, Neurosci Let 197: 149-53 (1995). When GSK3 
is over-expressed in mammalian cells, tau becomes phosphorylated on 
characteristic serine residues and can then be detected by phosphoform 
specific antibodies such as AT8, an antibody specific for phosphorylated 
tau. Thus, in the presence of active GSK3, tau protein can be detected by 
both AT8 and a phosphate-independent antibody such as Tau-1, while if GSK3 
activity is inhibited, only the phosphate-independent Tau-1 antibody will 
detect tau on a western blot; see Stambolic et al, Cur Biol 6(12): 1664-8 
(1996). 
Other assays that test functional GSK3 also provide a further test of 
intracellular GSK3 activity. These assays rely on the ability to observe 
indirect consequences of GSK3 activity within cells. An example of such an 
assay involves .beta.-catenin stabilization. Cellular GSK3 activity leads 
to the destabilization of cytosolic pools of .beta.-catenin in certain 
types of cell. Consequently, inhibition of GSK3 results in .beta.-catenin 
stabilization and accumulation, which be detected on a western blot. For 
example, the addition of iodotubercidin (a kinase inhibitor that is a 
nonspecific nonselective GSK3 inhibitor) to Drosophila cells leads to 
.beta.-catenin accumulation. Similarly, LiCl in the cell culture medium 
causes an increase in intracellular .beta.-catenin levels as described in 
Stambolic et al, Cur. Biol. 6(12): 1664-8 (1996). 
2. Assays Using Differentiated Cell-Lines 
Assays using differentiated cell-lines can be conducted as described 
generally for cell-based assays. Insulin-stimulated uptake of glucose from 
the bloodstream is mostly performed by fat and muscle cells. Because 
different cell lines respond differently to glucose (e.g. liver cells have 
much higher insulin-independent glucose transport) examination of the 
effect of any GSK3 inhibitor in differentiated fat and muscle cells is 
important. These are less convenient cells to work with because they need 
to be exposed to differentiating conditions, sometimes for several weeks, 
before they can be used in assays. Consequently, the assays with 
differentiated cell-lines are to test compounds that potentiate insulin 
signaling in the earlier cell-based assays 
NIH 3T3-L1 adipocytes display the expected properties of a differentiated 
fat cell: they are mitogenically inactive and contain fat globules. They 
are commonly used to measure the insulin dependent stimulation of glycogen 
synthase activity, glucose incorporation into glycogen, and glucose 
transport. See Lin and Lawrence JBC 269(33):21255-61 (1994), Robinson and 
James, Am J. Physiol. 263:E383-93 (1992), and Shepherd et al, Biochem Soc 
Trans 23: 202s(1995). 
L6 myocytes cell line displays the properties of a differentiated muscle 
cell. L6 myocytes have been used to measure the insulin-dependent 
stimulation of glycogen synthase activity, glucose incorporation into 
glycogen, and glucose transport. See Begum and Ragolia, Endocrinology 
137:2441-6 (1996). 
3. Assays Using Human Muscle Primary Myocytes 
Assays using human muscle primary myocytes can be conducted as described 
above for cell-based assays generally. Ultimately, testing the ability of 
inhibitor compounds to stimulate glucose uptake in normal and diabetic 
human cells is important. Procedures for culturing and assaying cells from 
human muscle biopsies (HSMCs) have been developed indicating such an assay 
as the most physiologically relevant cell-based assay of 
insulin-stimulated effects in human cells, and allowing for measurement of 
glucose uptake and glycogen synthesis as described in Claraldi et al, J. 
Clin. Invest. 96:2820-7 (1995), Henry et al, Diabetes 44:936-46 (1995), 
and Henry et al, Diabetes 45:400-7 (1996). Compounds that stimulate 
insulin-dependent effects in differentiated cell lines will be tested in 
the HSMC system. Compounds that are successful in some or all of these 
assays described above are candidates for animal studies. Appropriate 
model systems for NIDDM in mice and rats are well documented as described 
in Leiter, FASEB J 3:2231-41(1989). 
Candidate Inhibitors 
Candidate inhibitors may be derived from almost any source of chemical 
libraries, naturally occurring compounds, or mixtures of compounds. 
Described below are some exemplary and possible sources of candidate 
inhibitors, synthesis of libraries of peptides, peptoids, and small 
organic molecules. The candidate inhibitors can also be polynucleotides, 
example ribozymes or antisense molecules designed based on knowledge of 
GSK3 polynucleotide sequence. 
The term "inhibitor" refers to any inhibitor or antagonist of GSK3 
activity. The inhibitor of GSK3 can be a peptide GSK3 antagonist, a 
peptoid GSK3 antagonist, a small organic molecule GSK3 antagonist or a 
polynucleotide GSK3 antagonist. It is expected that some inhibitors will 
act at transcription, some at translation, and some on the mature protein, 
for example, at the specific site of GSK3 that acts to phosphorylate 
another protein. However, the use and appropriateness of such inhibitors 
of GSK3 for the purposes of the invention are not limited to any theories 
of mechanism of action of the inhibitor. It is sufficient for purposes of 
the invention that an inhibitor inhibit the activity of GSK3, for example, 
and most particularly, the kinase activity of GSK3. 
Analogs of peptides as used herein include peptides having one or more 
peptide mimics, for example peptoids that possess protein-like activity. 
Included within the definition are, for example, peptides containing one 
or more analogs of an amino acid (including, for example, unnatural amino 
acids), peptides with substituted linkages, as well as other modifications 
known in the art, both naturally occurring and not naturally occurring. 
The term "small molecule" includes any chemical or other moiety that can 
act to affect biological processes. Small molecules can include any number 
of therapeutic agents presently known and used, or can be small molecules 
synthesized in a library of such molecules for the purpose of screening 
for function. Small molecules are distinguished from polymers and 
macromolecules by size and lack of polymerization. Small molecules can 
include peptides, peptoids and small organic molecules. 
The candidate inhibitors and libraries of candidate inhibitors for 
screening by the methods of the invention can be derived from any of the 
various possible sources of candidate inhibitors, such as for example, 
libraries of peptides, peptoids, small molecules, and polynucleotides. The 
polynucleotide libraries can include antisense molecules or ribozymes. The 
inhibitor could be a polypeptide presented by phage display, provided 
mechanisms are designed to get the polypeptide inhibitor into the cell, or 
the polypeptide inhibitor was used to construct an intrabody or 
intracellular antibody. In general a GSK3 inhibitor can be any molecule 
that may be capable of inhibiting GSK3 activity. Some libraries for 
screening can be subdivided into library pools for assaying inhibition of 
GSK3 activity by the method of the invention. Some of each pool is assayed 
and some is saved for reassay, or to further subdivide into subpools, 
should a positive be identified. Generation of some of the possible 
libraries suitable for assay by the methods of the invention is described 
herein. 
Libraries that are peptide and peptoid inhibitors of GSK3 are made as 
follows. A "library" of peptides may be synthesized and used following the 
methods disclosed in U.S. Pat. No. 5,010,175, (the '175 patent) and in PCT 
WO91/17823. In the method of the '175 patent, a suitable peptide synthesis 
support, for example, a resin, is coupled to a mixture of appropriately 
protected, activated amino acids. The method described in W091/17823 is 
similar but simplifies the process of determining which peptides are 
responsible for any observed alteration of gene expression in a responsive 
cell. The methods described in WO91/17823 and U.S. Pat. No. 5,194,392 
enable the preparation of such pools and subpools by automated techniques 
in parallel, such that all synthesis and resynthesis may be performed in a 
matter of days. 
Further alternative agents include peptide analogs and derivatives that can 
act as stimulators or inhibitors of gene expression, or as ligands or 
antagonists. Some general means contemplated for the production of 
peptides, analogs or derivatives are outlined in CHEMISTRY AND 
BIOCHEMISTRY OF AMINO ACIDS, PEPTIDES, AND PROTEINS----A SURVEY OF RECENT 
DEVELOPMENTS, Weinstein, B. ed., Marcell Dekker, Inc., publ. New York 
(1983). Moreover, substitution of D-amino acids for the normal 
L-stereoisomers can be carried out to increase the half-lives of the 
molecules. 
Peptoids, polymers comprised of monomer units of at least some 
N-substituted moieties, can act as small molecule stimulators or 
inhibitors herein and can be synthesized as described in PCT 91/19735. 
Presently preferred amino acid substitutes are N-alkylated derivatives of 
glycine, which are easily synthesized and incorporated into polypeptide 
chains However, any monomer units that allow for the sequence specific 
synthesis of pools of diverse molecules are appropriate for use in 
producing peptoid molecules. The benefits of these molecules for the 
purpose of the invention is that they occupy different conformational 
space than a peptide and are more resistant to the action of proteases 
because their amide linkages are N-substituted. 
Peptoids are easily synthesized by standard chemical methods. The preferred 
method of synthesis is the "submonomer" technique described by R. 
Zuckermann et al., J. Am. Chem. Soc. 114:10646-7 (1992). Synthesis by 
solid phase techniques of heterocyclic organic compounds in which 
N-substituted glycine monomer units forms a backbone is described in 
copending application entitled "Synthesis of N-Substituted Oligomers" 
filed on Jun. 7, 1995 and is herein incorporated by reference in full. 
Combinatorial libraries of mixtures of such heterocyclic organic compounds 
can then be assayed for the ability to alter gene expression. 
Synthesis by solid phase of other heterocyclic organic compounds in 
combinatorial libraries is also described in copending application U.S. 
Ser. No. 08/485,006 entitled "Combinatorial Libraries of Substrate-Bound 
Cyclic Organic Compounds" filed on Jun. 7, 1995, herein incorporated by 
reference in full. Highly substituted cyclic structures can be synthesized 
on a solid support by combining the submonomer method with powerful 
solution phase chemistry. Cyclic compounds containing one, two, three or 
more fused rings are formed by the submonomer method by first synthesizing 
a linear backbone followed by subsequent intramolecular or intermolecular 
cyclization as described in the same application. 
Where the selected inhibitor of GSK3 is a ribozyme, for example, a ribozyme 
targeting a GSK3 gene, the ribozyme can be chemically synthesized or 
prepared in a vector for a gene therapy protocol including preparation of 
DNA encoding the ribozyme sequence. The synthetic ribozymes or a vector 
for gene therapy delivery can be encased in liposomes for delivery, or the 
synthetic ribozyme can be administered with a pharmaceutically acceptable 
carrier. A ribozyme is a polynucleotide that has the ability to catalyze 
the cleavage of a polynucleotide substrate. Ribozymes for inactivating a 
gene can be prepared and used as described in Long et al., FASEB J. 7:25 
(1993), and Symons, Ann. Rev. Biochem. 61:641 (1992), Perrotta et al., 
Biochem. 31:16, 17 (1992); and U.S. Pat. Nos. 5,225,337, 5,168,053, 
5,168,053 and 5,116,742, Ojwang et al., Proc. Natl. Acad. Sci. USA 
89:10802-10806 (1992), U.S. Pat. No. 5,254,678 and in U.S. Pat. Nos. 
5,144,019, U.S. Pat. Nos. 5,225,337, 5,116,742, 5,168,053. Preparation and 
use of such ribozyme fragments in a hammerhead structure are described by 
Koizumi et al., Nucleic Acids Res. 17:7059-7071 (1989). Preparation and 
use of ribozyme fragments in a hairpin structure are described by Chowrira 
and Burke, Nucleic Acids Research 20:2835 (1992). 
The hybridizing region of the ribozyme or of an antisense polynucleotide 
may be modified by linking the displacement arm in a linear arrangement, 
or alternatively, may be prepared as a branched structure as described in 
Horn and Urdea, Nucleic Acids Res. 17:6959-67 (1989). The basic structure 
of the ribozymes or antisense polynucleotides may also be chemically 
altered in ways quite familiar to those skilled in the art. Chemically 
synthesized ribozymes and antisense molecules can be administered as 
synthetic oligonucleotide derivatives modified by monomeric units. 
Ribozymes and antisense molecules can also be placed in a vector and 
expressed intracellularly in a gene therapy protocol. 
The invention includes generating cRNA and cDNA libraries for screening for 
inhibition of GSK3 activity, can require overexpression of recombinant 
GSK3, and can also involve transforming a cell with the gene for GSK3 for 
expression in the assay. However, it is not necessary to overexpress GSK3 
in all the assays as GSK3 is endogenously expressed in almost all cells. 
For example the tau phosphorylation assay prescribes overexpression of 
GSK3, whereas the assays including the CHO-IR or HEPG2, 3T3-L1 or L6, or 
human muscle myoctye assays employ GSK3 endogenously expressed in the cell 
system being used. Exemplary systems for generating polypeptides or 
libraries useful for the method of the invention would include, for 
example, any standard or useful mammalian, bacterial, yeast or insect 
expression system, many of which are described in WO 96/35787. Thus any 
polypeptide or peptide useful in the invention can be made by these or 
other standard methods. 
Other items not specifically exemplified, such as plasmids, can be 
constructed and purified using standard recombinant DNA techniques 
described in, for example, Sambrook et al. (1989), MOLECULAR CLONING, A 
LABORATORY MANUAL, 2d edition (Cold Spring Harbor Press, Cold Spring 
Harbor, N.Y.), and Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 
(1994), (Greene Publishing Associates and John Wiley & Sons, New York, 
N.Y.) under the current regulations described in United States Dept. of 
HHS, NATIONAL INSTITUTE OF HEALTH (NLH) GUIDELINES FOR RECOMBINANT DNA 
RESEARCH. These references include procedures for the following standard 
methods: cloning procedures with plasmids, transformation of host cells, 
cell culture, plasmid DNA purification, phenol extraction of DNA, ethanol 
precipitation of DNA, agarose gel electrophoresis, purification of DNA 
fragments from agarose gels, and restriction endonuclease and other 
DNA-modifying enzyme reactions. 
Pharmaceutical Compositions and Methods of Treatment 
A GSK3 inhibitor identified from a library of candidates using one of the 
several methods of identification of selective GSK3 inhibitors of the 
invention described herein can be prepared as a pharmaceutical composition 
using a pharmaceutically acceptable carrier. The pharmaceutical 
composition including the selective GSK3 inhibitor can be administered to 
a subject having NIDDM, Alzheimer's, the potential for either disease, or 
having any other disorder mediated by GSK3 activity. Once a selective GSK3 
inhibitor is shown to work in the stable tissue culture cells, appropriate 
formulations can be devised. In addition, preliminary pharmacokinetic and 
absorption studies can be done in preparation for the animal studies, 
which will ensue if the compounds are effective in the assays using human 
muscle primary myocytes. 
"Therapeutically effective amount" as used herein refers to that amount 
that is effective to obtain the desired therapeutic result. The term "an 
effective amount" of an inhibitor of GSK3 refers to an amount that is 
effective to induce an inhibition of GSK3 activity. That activity can be 
GSK3 kinase activity. The inhibitory amount may be determined directly by 
measuring the inhibition of a GSK3 activity, or, for example, where the 
desired effect is an effect on an activity downstream of GSK3 activity in 
a pathway that includes GSK3, the inhibition may be measured by measuring 
a downstream effect. Thus, for example where inhibition of GSK3 results in 
the arrest of phosphorylation of glycogen synthase, the effects the 
inhibitor may be effects on an insulin-dependent or insulin-related 
pathway, and the inhibitor may be administered to the point where glucose 
uptake is increased to optimal levels. Also, where the inhibition of GSK3 
results in the absence of phosphorylation of a protein that is required 
for further biological activity, for example, the tau protein, then the 
inhibitor may be administered until polymerization of phosphorylated tau 
protein is substantially arrested. Therefore, the inhibition of GSK3 
activity will depend in part on the nature of the inhibited pathway or 
process that involves GSK3 activity, and on the effects that inhibition of 
GSK3 activity has in a given biological context. 
The amount of the inhibitor that will constitute an inhibitory amount will 
vary depending on such parameters as the inhibitor and its potency, the 
half-life of the inhibitor in the body, the rate of progression of the 
disease or biological condition being treated, the responsiveness of the 
condition to the dose of treatment or pattern of administration, the 
formulation, the attending physician's assessment of the medical 
situation, and other relevant factors, and in general the health of the 
patient, and other considerations such as prior administration of other 
therapeutics, or co-administration of any therapeutic that will have an 
effect on the inhibitory activity of the inhibitor or that will have an 
effect on GSK3 activity, or a pathway mediated by GSK3 activity. It is 
expected that the inhibitory amount will fall in a relatively broad range 
that can be determined through routine trials. 
"Co-administration" as used herein means administration of an inhibitor of 
GSK3 according to the method of the invention in combination with a second 
therapeutic agent. The second therapeutic agent can be any therapeutic 
agent useful for treatment of the patient's condition. For example, 
inhibition of GSK3 with lithium as a second therapeutic agent used in 
conjunction with a therapeutic agent inhibitor of GSK3 is contemplated. 
Additionally, for example, a first therapeutic agent can be a small 
molecule inhibitor of GSK3 activity, and a second therapeutic agent can be 
an antisense or ribozyme molecule against GSK3 that, when administered in 
a viral or nonviral vector, will facilitate a transcriptional inhibition 
of GSK3 that will complement the inhibitory activity of the small 
molecule. The second therapeutic agent can also be lithium ion. 
Co-administration may be simultaneous, for example, by administering a 
mixture of the therapeutic agents, or may be accomplished by 
administration of the agents separately, such as within a short time 
period. Co-administration also includes successive administration of an 
inhibitor of GSK3 and one or more of another therapeutic agent. The second 
therapeutic agent or agents may be administered before or after the 
inhibitor of GSK3 The second therapeutic agent may also be an inhibitor of 
GSK3, which has particular advantages when administered with the first 
inhibitor. Dosage treatment may be a single dose schedule or a multiple 
dose schedule. 
Administration of small molecule therapeutic agents will vary depending on 
the potency of the small molecule. For a very potent small molecule 
inhibitor, nanogram (ng) amounts kilogram of patient, or microgram (.mu.g) 
amounts per kilogram of patient may be sufficient. Thus, for small organic 
molecules, peptides, or peptoids, the dosage range can be for example, 
from about 100 ng/kg to about 500 mg/kg of patient weight, or the dosage 
range can be a range within this broad range, for example, about 100 ng/kg 
to 400 ng/kg, from about 500 ng/kg to about 1 .mu.g/kg, from about 5 
.mu.g/kg to about 100 .mu.g/kg, from about 150 .mu.g/kg to about 500 
.mu.g/kg, from about 600 .mu.g/kg to about 1 mg/kg, or from about 25 
mg/kg, to about 500 mg/kg of patient weight. 
The individual doses for viral gene delivery vehicles for delivery of 
polynucleotide inhibitors normally used are 10.sup.7 to 10.sup.9 c.f.u. 
(colony forming units of neomycin resistance titered on HT1080 cells) per 
body. Dosages for adeno-associated virus (AAV) containing delivery systems 
are in the range of about 10.sup.9 to about 10.sup.11 particles per body. 
Dosages for nonviral gene delivery vehicles for delivering polynucleotide 
inhibitors of GSK3 are described for example in U.S. Pat. Nos. 5,589,466 
and 5,580,859. Dosage of nonviral gene delivery vehicles can be 1 .mu.g, 
preferably at least 5 or 10 .mu.g, and more preferably at least 50 or 100 
.mu.g of polynucleotide, providing one or more dosages. 
Non-coding sequences that act by a catalytic mechanism, for example, 
catalytically active ribozymes, may require lower doses than non-coding 
sequences that are held to the restrictions of stoichometry, for example, 
antisense molecules, although expression limitations of the ribozymes may 
again raise the dosage requirements of ribozymes being expressed in vivo 
in order to achieve efficacy in the patient. Factors such as method of 
action and efficacy of transformation and expression are therefore 
considerations that will effect the dosage required for ultimate efficacy 
for polynucleotides. Where greater expression is desired, over a larger 
area of tissue, larger amounts of DNA or the same amounts readministered 
in a protocol of successive administrations, or several administrations to 
different adjacent or close tissue portions may be required to effect a 
positive therapeutic outcome. 
In all cases, routine experimentation in clinical trials will determine 
specific ranges for optimal therapeutic effect, for each therapeutic and 
each administrative protocol, and administration to specific patients will 
also be adjusted to within effective and safe ranges depending on the 
patients' condition and responsiveness to initial administrations. 
All of the therapeutic agents discovered by the methods of the invention 
can be incorporated into an appropriate pharmaceutical composition that 
includes a pharmaceutically acceptable carrier for the agent. The 
pharmaceutical carrier for the agents may be the same or different for 
each agent. Suitable carriers may be large, slowly metabolized 
macromolecules such as proteins, polysaccharides, polylactic acids, 
polyglycolic acids, polymeric amino acids, amino acid copolymers, and 
inactive viruses in particles. Such carriers are well known to those of 
ordinary skill in the art. Pharmaceutically acceptable salts can be used 
therein, for example, mineral acid salts such as hydrochlorides, 
hydrobromides, phosphates, sulfates, and the like; an the salts of organic 
acids such as acetates, propionates, malonates, benzoates, and the like. A 
thorough discussion of pharmaceutically acceptable excipients is available 
in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991). 
Pharmaceutically acceptable carriers in therapeutic compositions may 
contain liquids such as water, saline, glycerol and ethanol. Additionally, 
auxiliary substances, such as wetting or emulsifying agents, pH buffering 
substances, and the like, may be present in such vehicles. Typically, the 
therapeutic compositions are prepared as injectables, either as liquid 
solutions or suspensions; solid forms suitable for solution in, or 
suspension in, liquid vehicles prior to injection may also be prepared. 
Liposomes are included within the definition of a pharmaceutically 
acceptable carrier. Liposomes are described in U.S. Pat. Nos. 5,422,120 
and 4,762,915, WO 95/13796, WO 94/23697, WO 91/144445 and EP 524,968, and 
in Starrier, Biochemistry, pages 236-240 (1975) W. H. Freeman, San 
Francisco, Shokai, Biochem. Biophys. Acct. 600:1 (1980); Bayer, Biochem 
Biophys Acct 550:464 (1979); Rivet, Meth. Enzyme. 149:119 (1987); Wang, 
Proc. Natl. Acad. Sci. 84:785: (1987); and Plant, Anal. Biochem 176:420 
(1989). 
The pharmaceutically acceptable carrier or diluent may be combined with 
other agents to provide a composition either as a liquid solution, or as a 
solid form (e.g., lyophilized) which can be resuspended in a solution 
prior to administration. The composition can be administered by parenteral 
or nonparenteral routes. Parenteral routes can include local injection 
into an organ or space of the body or systemic injection including 
intravenous, intraarterial injections or other systemic routes of 
administration. Nonparenteral routes can include oral administration. 
The subject to be treated by the method of the invention should be 
diagnosed with a biological condition mediated by GSK3 activity. The term 
"biological condition" as used herein refers to a particular state of 
molecular and cellular systems in a biological context. A biological 
context includes any organism considered to have life, and for the 
purposes of this invention includes but is not limited to the following 
organisms: animals, mammals, humans, invertebrates and vertebrates. A 
biological condition can include, for example, a disease or a medical 
condition that may or may not be characterized by identifiable symptoms or 
indicators. The term "biological condition mediated by GSK3 activity" as 
used herein refers to any biological or medical condition or disorder in 
which GSK3 activity is identified, whether at normal or abnormal levels. 
The GSK3 activity mediates activity causing or related to causes of the 
biological or medical condition that causes the patient to seek medical 
attention. For example, such activity of GSK3 can be phosphorylation of 
glycogen synthase in the case of NIDDM, or hyperphosphorylation of the tau 
protein in the case of Alzheimer's disease. The condition or disorder may 
be caused by the GSK3 activity or may simply be characterized by GSK3 
activity. That the condition is mediated by GSK3 activity means that some 
aspect of the condition can be traced to the GSK3 activity. By using the 
methods of treatment of the invention, inhibiting the GSK3 activity will 
then prevent, ameliorate or treat the condition so characterized. The term 
"susceptible to such condition" as used herein refers to prophylactic 
administration of a GSK3 inhibitor to a patient who is at risk for 
developing a condition mediated by GSK3 activity. Such a subject might be 
a person experiencing some presymptomatic indications of NIDDM or 
Alzheimer's.

EXAMPLES 
The following examples are exemplary only, and are not intended to limit 
the invention. 
Example 1 
Assay of Phosphorylation of GSK3 Peptide Substrate 
One possible method for screening for an inhibitor of GSK3 involves a 
kinase assay in which the kinase activity of GSK3 is measured in the 
presence of a candidate inhibitor. The assay relies on the ability of GSK3 
to phosphorylate a substrate in the absence of an inhibitor. This example 
demonstrates the success of the claimed substrate in accomplishing this 
function. 
A GSK3.beta. gene was created in which a haemagluttinin (HA) epitope was 
fused to the N-terminal end of the GSK3.beta. open reading frame in 
plasmid vector pCG, a pEVRF derivative, described in Giese et al. Genes & 
Development (1995) 9:995-1008, and in Matthias et al., Nucleic Acids Res. 
(1989) 17: 6418. pCG has a modified polylinker, and directs expression in 
mammalian cells from the human cytomegalovirus promoter/enhancer region. 
The resulting plasmid is pCG-HA-GSK3.beta.. pCG-HA-GSK3.beta. was 
transiently transfected into COS cells on 10 cm tissue culture plates 
using DEAE-Dextran, as described in Ausubel et al (1994) CURRENT PROTOCOLS 
IN MOLECULAR BIOLOGY, (Greene Publishing Associates and John Wiley & Sons, 
New York, N.Y.). 
The final density of cells was 70% confluent and these cells were lysed in 
700 .mu.l Triton lysis buffer (20 mM TrisHCl pH7.9, 137mM NaCl, 1.0% 
Triton X-100, 10% glycerol, 1 mM NaVO.sub.3, 20 mM NaF, 30 mM pNpp, 15 mM 
PP.sub.i). Anti-HA antibody (12CA5 monoclonal antibody purchased from 
Boehringer Mannheim, Indianapolis, Ind.) was added to 300 .mu.l of this 
lysate to a final concentration of 4 .mu.g/ml and incubated for 1 h at 
4.degree. C. 100 .mu. of a 50% slurry of protein A-Sepharose.RTM. beads 
was added for 2 h at 4.degree. C. 
The beads were pelleted by centrifugation for 10 seconds in a 
microcentrifuge and washed with 0.5 M LiCl, 0.5% Triton X-100, twice with 
phosphate buffered saline (PBS) and once with 1 mM TrisHCl pH 7.5, 5 mM 
MgCl.sub.2, 1 mM DTT. All wash buffers contained 1 mM NaVO.sub.3 and 25 mM 
.beta.-glycerolphosphate. 33 .mu.l of the beads were analysed by SDS-PAGE 
an western blotting with anti-HA antibody (12CA5 monoclonal antibody 
purchased from Boehringer Mannheim, Indianapolis, Ind.) to quantitate the 
amount of HA-GSK3.beta. present. 
The remaining 17 .mu.l of beads was assayed according to the protocol of 
Wang et al, Anal Biochem 220: 397-402 (1994). To each 17 .mu.l of pellet 
was added 3 .mu.l 10.times. GSK buffer (100 mM MgCl.sub.2, 20 mM DTT, 3M 
TrisHCl pH7.5), 0.7 .mu.l CREB peptide (either prephosphorylated or 
non-prephosphorylated, 5 mg/ml, Chiron Mimotopes Peptide Systems, San 
Diego, Calif.), 0.3 .mu.l 10 mM rATP, 1 .mu.l g.sup.32 P-ATP (6000 
Ci/mmol), 0.06 .mu.l 5mg/ml of protein kinase inhibitor (a protein 
kinase-A inhibitor or PKI), and 25 .mu.l H.sub.2 O. The reaction was 
allowed to proceed for 20 minutes at 22.degree. C. and then was stopped 
with 8 .mu.l 500 mM EDTA. 22 .mu.l of each reaction was spotted onto P81 
phosphocellulose filter paper (purchased from Gibco-BRL Life Technologies 
Gaithersburg, Md.) and washed 4 times for 5 minutes in 75 mM H.sub.3 
PO.sub.4. The filter papers were then assayed in a scintillation counter. 
Filter papers from experiments using the prephosphorylated CREB peptide 
substrate yielded counts of 85,000.+-.5,000 cpm/min, where filter papers 
from experiments using the non-prephosphorylated CREB peptide substrate 
yield counts of 5,000.+-.1,000 cpm/min. The results indicated that the 
substrate was phosphorylated by GSK3 in the absence of an inhibitor, 
although the control substrate was not phosphorylated. This experiment 
demonstrates the specificity of the claimed peptide as a GSK3 substrate. 
Example 2 
Screening a Peptide Library of Random Hexamers for An Inhibitor of GSK3 
A library of random hexamers can be purchased from Chiron Mimotopes, 
Clayton, Australia, or, alternatively a set of mixtures of random hexamers 
can be prepared by the met of either U.S. Pat. Nos. 5,010,175 or 
5,194,392. For example, hexamers of the formula D1D2-XXXX, where D1 can 
equal any one of the 20 amino acids, and D2 can equal any on the 20 amino 
acids, to yield a library of 400 possible hexamers are used; X is any 
amino acid, and the amino acid character of X is not controlled. Pools of 
this library are created and screened for an inhibitor by a kinase assay 
using purified GSK3 and the GSK3 phosphoryla substrate the CREB peptide 
prephosphorylated at the C terminal serine. Positives are identified and 
the identity of D1 and D2 are determined for those positives. Further 
hexamers are resynthesized with the formula D1D2D3D4-XX, where D3 and D4 
are one of the 20 amino acids, and D1 and D2 are established amino acids. 
Thus, another 400 random hexamers are screened and the positives 
identified and the identity of D3 and D4 determined. One more round of the 
screening ensues to identify a D5 and D6 amino acid for the positive 
hexamer. Once the positive hexamer sequence is determined, the inhibitor 
hexamer is tested in a fly eye in vivo screen for its ability to enhance 
the rough cell morphology of the eyes of a GSK3 inhibitor protein 
expressing transgenic fly, indicating a functional non-toxic inhibitor 
that crosses the cell surface. 
Example 3 
Cell-Based Assay for GSK3 Inhibitor 
A small molecule GSK3 inhibitor is synthesized in sufficient quantities for 
cell-based assays. In a cell-based assay in Drosophila cells, the compound 
is tested for inhibition of GSK3 at an inhibitor concentration up to that 
at which it remains soluble in the culture medium. A positive result in 
this assay indicates that the inhibitor can enter cells and inhibit 
intracellular GSK3. 
The inhibitor is then tested in a stable tissue culture cell assay in 
CHO-IR cells. An inhibitor that can function and exhibit a positive result 
in a stable tissue culture cell assay is then tested in a differentiated 
cell line using NIH 3T3-L1 adipocytes. An assay using this cell line will 
determine whether the GSK3 inhibitor can potentiate an insulin independent 
stimulation of glycogen synthase activity and glucose incorporation into 
glycogen, and glucose transport. Tests in human muscle primary myocytes 
are then conducted to determine whether the GSK3 inhibitor stimulates 
glucose uptake in normal and diabetic human cells. 
Example 4 
Treating a Patient with NIDDM 
A patient is diagnosed in the early stages of non-insulin dependent 
diabetes mellitus. A small molecule inhibitor of GSK3 is formulated in an 
enteric capsule. The patient is directed to take one tablet after each 
meal for the purpose of stimulating the insulin signaling pathway, and 
thereby controlling glucose metabolism to levels that obviate the need for 
administration of exogenous insulin. 
Example 5 
Treating a Patient with Alzheimer's Disease 
A patient is diagnosed with Alzheimer's disease. The patient is 
administered a selective small molecule inhibitor of GSK3-mediated tau 
hyperphosphorylation prepared in a formulation that crosses the 
blood/brain barrier. The patient is monitored for tau phosphorylated 
polymers by periodic analysis of proteins isolated from the patient's 
brain cells for the presence of phosphorylated forms of tau on an SDS-PAGE 
gel known to characterize the presence of and progression of the disease. 
The dosage of the inhibitor is adjusted as necessary to reduce the 
presence of the phosphorylated forms of tau protein. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- - - - (1) GENERAL INFORMATION: 
- - (iii) NUMBER OF SEQUENCES: 2 
- - - - (2) INFORMATION FOR SEQ ID NO:1: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- - Ser Gly Ser Gly Lys Arg Arg Glu Ile Leu Se - #r Arg Arg Pro Ser 
Tyr 
1 5 - # 10 - # 15 
- - Arg 
- - - - (2) INFORMATION FOR SEQ ID NO:2: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
- - Ser Xaa Xaa Xaa Ser 
1 5 
__________________________________________________________________________