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Current Pharmaceutical Design, 2004, 10, 1105-1137
1105
Discovery and Development of GSK3 Inhibitors for the Treatment of Type
2 Diabetes
Allan S. Wagman†,*, Kirk W. Johnson‡ and Dirksen E. Bussiere¥
†
Medicinal Chemistry, Chiron Corporation, 4560 Horton Street, M/S 4.5, Emeryville, CA 94608-2916, ‡Pharmacology
and Preclinical Development, Genesoft Pharmaceuticals, Inc., 7300 Shoreline Ct., South San Francisco, CA 94080,
¥
Computational Chemistry and Structural Biology, Chiron Corporation, 4560 Horton Street, M/S 4.5, Emeryville, CA
94608-2916
Abstract: Originally identified as a modulator of glycogen metabolism, glycogen synthase kinase-3 (GSK3) is now
understood to play an important regulatory role in a variety of pathways including initiation of protein synthesis, cell
proliferation, cell differentiation, apoptosis, and is essential for embryonic development as a component of the Wnt
signaling cascade. GSK3 can be considered as a target for both metabolic and neurological disorders. GSK3's association
with neuronal apoptosis and hyper-phosphorylation of tau make this kinase an attractive therapeutic target for
neurodegenerative conditions such as head trauma, stroke and Alzheimer's disease. While noting GSK3's many associated
functions, this review will focus on GSK3 as a central negative regulator in the insulin signaling pathway, its role in
insulin resistance, and the utility of GSK3 inhibitors for intervention and control of metabolic diseases including type 2
diabetes. Recent crystal structures, including the active (phosphorylated Tyr-216) form of GSK3β, provide a wealth of
structural information and greater understanding of GSK3's unique regulation and substrate specificity. Many potent and
selective small molecule inhibitors of GSK3 have now been identified, and used in vitro to modulate glycogen
metabolism and gene transcription, increase glycogen synthase activity and enhance insulin-stimulated glucose transport.
The pharmacology of potent and selective GSK3 inhibitors (CT 99021 and CT 20026) is described in a number of in vitro
and in vivo models following acute or chronic exposure. The efficacy of clinical candidates in diabetic primates and the
implications for clinical development are discussed. The profile of activity is consistent with a unique form of insulin
sensitization which is well suited for indications such as metabolic syndrome X and type 2 diabetes.
INTRODUCTION
In recent years there has been a dramatic increase in the
global prevalence of diabetes. Incidence is steadily
increasing, creating a new burden on health care systems and
a huge unmet need for new methods of treatment [1]. The
central role of GSK3 in glucose metabolism makes it an
exciting target for controlling hyperglycemia, and inhibition
of GSK3 activity may represent a novel mechanism for
improving glucose disposal in an insulin-conserving manner.
GSK3 is one of many signaling components downstream
from the insulin receptor (IR), but this kinase has several
unique features that make it an attractive target for drug
discovery [2]. Unlike other known intracellular protein
kinases, GSK3 is constitutively active in resting cells and is
inhibited through the action of extracellular signals, such as
insulin, and activation of cell signaling pathways. Insulin
modulates glycogen accumulation in sensitive tissues by
increasing glucose transport and increasing glycogen
synthesis. In diabetics, both of these pathways may be
defective resulting in diminished glycogen storage. Through
the action of insulin, glycogen synthase (GS) is
dephosphorylated and activated by an increase in protein
phosphatase 1G (PP1G) activity and the inhibition of kinases
such as PKA and GSK3 [3]. In response to insulin, IRS-1
*Address correspondence to this author at the Medicinal Chemistry, Chiron
Corporation, 4560 Horton Street, M/S 4.5, Emeryville, CA 94608-2916;
Tel: (510) 923-7796; Fax: (510) 923-3360;
E-mail: [email protected]
1381-6128/04 $45.00+.00
activates akt/PKB which in turn inhibits GSK3. The negative
regulation of GSK3 by insulin occurs in the key cell types
important for glycogen metabolism: hepatocytes, myocytes,
and adipocytes [4-6]. Disregulation or overexpression of
GSK3 in these cells could lead to insulin resistance. Thus,
inhibiting GSK3 in concert with insulin-induced signaling
should increase the activity of GS and improve glycogen
deposition in critical glucose-controlling tissues.
GSK3 is a cytosolic serine/threonine protein kinase found
in two closely related isoforms, GSK3α and GSK3β, which
are expressed ubiquitously in mammalian tissues. Both
isoforms have nearly identical biochemical functions and
substrate affinities [7], and have been recently reviewed in
detail [8-10]. The genes of GSK3α and GSK3 β show a high
degree of sequence identity (~85%), while the active sites
share 93% identity [11]. GSK3 can be considered an
essential gene in that mouse GSK3β knockouts exhibit
normal development in utero until liver failure leads to death
between E13.5 and E14.5. The lethal phenotype suggested
that the GSK3α isoform can not fully compensate for
GSK3β deficiency and would indicate certain independent
regulatory roles for each isoform [12]. Some differences in
gene expression and protein concentration have been found
in mammalian tissues that might represent differential
regulation, but no strong correlations have emerged [8].
Thus, while some differential expression and effects of the
two isozymes have been reported, clear discrimination of
functional roles has not been apparent.
© 2004 Bentham Science Publishers Ltd.
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Current Pharmaceutical Design, 2004, Vol. 10, No. 10
Abnormal overexpression of GSK3 in peripheral tissues
could potentially contribute to the pathology of diabetes.
Studies in vitro show that hyperactive mutants of GSK3
expressed in cells lead to a reduction in GS activity [13, 14].
Compounds known to attenuate insulin signaling, such as
okadaic acid and TNFα, increase the serine/threonine phosphorylation of IRS-1 [15, 16]. GSK3 also phosphorylates
IRS-1 on serine residues and likewise inhibits insulin signaling in cells [17]. Thus, if serine phosphorylation of IRS-1
contributes to insulin resistance, abnormally high amounts of
GSK3 in tissue might lead to impaired insulin signaling and
GSK3 would be further enhanced by the impairment
signaling through IRS-1. Recently, two studies indicate a
potential mechanism by which GSK3 may have a direct
effect on glucose transport. In the first study, GSK3 was
shown to phosphorylate and inactivate kinesin light chains
[18]. The second study links kinesin to the regulation of
membrane trafficking of glucose transport protein-4 (GLUT4) vesicles to the plasma membrane [19]. Overall, GSK3 has
the potential to interfere with normal insulin signaling through
hyperactivity or overexpression in muscle, adipose or liver.
Insulin resistance could arise through GSK3-dependent phosphorylation of GS, IRS-1 or kinesins involved in GLUT-4
translocation. To further define GSK3's role in insulin resistance and other pathways, highly potent, selective and cellpermeable GSK3 inhibitors have been useful biochemical
probes. Compounds such as CT 99021 and CT 20026 have
provided a roadmap for better understanding the role of
GSK3 in glucose metabolism and the potential of GSK3
inhibitors as metabolic disease therapeutics.
BIOLOGY, ENZYMOLOGY AND STRUCTURE OF
GSK3
I. Human Isoforms of GSK3 and Homologs in Other
Organisms
GSK3 was first cloned in 1990 and is known to exist in
eukaryotes in two isoforms, α and β [20], which are encoded
by two independent genes. These genes encode two proteins
with molecular weights of 51 and 47 kDa, respectively. The
two genes are located on separate chromosomes in humans:
the cytological location of GKS3α is 19q13.2, whereas
GSK3β is located on 3q13.3 [21]. GSK3α and GSK3β share
an overall sequence identity of approximately 85%, but are
significantly more homologous within the catalytic domain,
where they exhibit a sequence identity of 93%. The predominant difference between the two isoforms is the attachment of a long 83-residue, mostly poly-glycine, tail to the Nterminus of the α-isoform. Despite this unusual structural
variation, in prior published studies, purified recombinant
GSK3α and GSK3β are stated to exhibit similar biochemical
properties as well as similar substrate specificities [7]. In our
experience, however, the β-isoform, expressed in bacteria or
in insect cells, is far more stable for in vitro studies; the αisoform is much more difficult to over-express, purify, and
store, and has a shorter half-life when enzymatic activity is
monitored. Studies have shown that the two genes are
variably expressed in different mammalian tissues [22]. This
differential expression may result from differential levels of
transcriptional and translational regulation for the two
isoforms. The biological significance of these differences in
isoform sequence and structure, as well as the differences in
Wagman et al.
isoform expression, is unknown. Recently, a splicing variant
of the β-isoform has been isolated from the brains of mice,
rats, and humans [23]. This variant has a 14 amino acid
insert in the C-terminal domain of the protein. The nature
and effect of this insert on protein structure and function is
not currently known.
Like many other kinases, GSK3 activity is both activated
and repressed by various phosphorylation events. In other
kinases, these phosphorylation events act in an almost Boolean manner, and GSK3 is no exception. Phosphorylation of
an N-terminal serine in GSK3 (Ser21 in the α-isoform; Ser9
in the β-isoform), which, in insulin signaling, occurs via by
the upstream protein kinase B/Akt, results in inhibition of the
enzyme. This inhibitory event can be reversed by protein
phosphatase 2A, which has been shown to return GSK3 to an
active state [24]. In addition to this inhibition event, GSK3
can also be activated via phosphorylation. Auto-phosphorylation of a tyrosine in the activation segment (Tyr279 in the
α-isoform; Tyr216 in the β-isoform) increases enzymatic
activity moderately. Dephosphorylation of this tyrosine by
tyrosine phosphatase reduces its enzymatic activity [25, 26].
The molecular basis for these inhibition and activation
events will be discussed in a following section.
There are numerous GSK3 homologs in other organisms
and GSK3 has been shown to be widely conserved throughout evolution. Each of these homologs typically segregates
towards showing stronger identity to one of the two human
isoforms, α or β. GSK3 homologs have been identified in
mammals (rats, mice, and others), fish (zebrafish), invertebrates (nematodes, fruit flies, sea urchins, and others), parasites (Plasmodium malariae), plants (Arabidopsis, rice,
tobacco, and others), fungi (baker’s yeast and fission yeast),
and slime molds [27]. The homology of each of these
homologs towards one of the two human isoforms varies
significantly from organism to organism, but the sequence
identity within the catalytic domain does not fall below 54%,
while the overall sequence identity does not fall below 45%.
The role of these homologs is also different between species.
While the role of GSK3 in mammals and other higher
organisms such as fish is strongly conserved and centers
around the Wnt signaling and the modulation of glycogen
metabolism, as one moves down the evolutionary tree, the
role of GSK3 in the cellular milieu changes. This can be seen
in sea urchin, where GSK3 is implicated in the establishment
of the animal-vegetal (A-V) axis during the early development of the urchin [28, 29]. Further down the evolutionary
tree, in the slime mold Dictyostelium discoideum, which
possesses a single GSK3 homolog sharing a 70% sequence
identity with human GSK3β, the GSK3 homolog plays a
critical role in specifying cell fate and controls the
differentiation of slime mold cells into either spore or stalk
cells [30, 31]. An exhaustive listing of the variation of each
homolog’s role in its organism’s biochemistry is beyond the
scope of this review, but it is clear that the role of GSK3
homologs shows significant inter-species variation as one
moves beyond mammals.
II. Enzymology of GSK3
Both isoforms of GSK3 possess a similar enzymatic
mechanism: the transfer of the γ-phosphate from ATP to
either a serine or threonine, preferentially four residues N-
Discovery and Development of GSK3 Inhibitors
terminal to a previously phosphorylated serine or threonine
(Fig. 1). The preference of GSK3 for substrates with a
‘priming’ phosphorylation event is rare among characterized
kinases. Many GSK3 substrates, such as glycogen synthase,
often have repeats of -S/T-XXX-S/T- that are phosphorylated in rapid succession [41, 78] once a priming phosphorylation event occurs at the C-terminal serine or threonine within
the repeats. This priming event is triggered by another
kinase, such as casein kinase 2 (CK2) in the case of glycogen
synthase [32]. It should be noted that not all GSK3 substrates
require this priming phosphorylation: some proteins involved
in the Wnt signaling may not require prior priming to be
GSK3 substrates. In such circumstances, the overall structure
and local amino acid sequence of the substrate molecule may
overcome the need for a priming phosphate (see, for
example, the phosphorylation of APC shown in Fig. 1C).
The structural basis for the enzymatic mechanism and the
need for a priming phosphorylation event will be discussed
in the section that follows. GSK3-mediated phosphorylation
appears to always lead to inhibition of the substrate: phosphorylation of a substrate by GSK3 has never been shown to
be an activating event.
III. Overall Architecture of GSK3 and the Structural
Basis for Catalysis, Activation, and Inhibition
There is currently only structural information for human
GSK3β in the form of five crystal structures, four of which
Current Pharmaceutical Design, 2004, Vol. 10, No. 10 1107
have been published in the literature, and one of which has
been published as a patent application [33-37]. Representative structures are shown in Fig. 2. The overall structure
and fold of the human α-isoform and other homologs of this
enzyme family should be extremely similar given the strong
sequence identity. These crystal structures reveal that GSK3
shares the canonical fold typically seen in serine/threonine
kinases and is comprised of an N-terminal β-sheet lobe and a
C-terminal α-helical domain [38]. The N-terminal domain
forms an incomplete β-barrel and is comprised mainly of
seven anti-parallel β-strands as well as one α-helix, the ‘αC’
helix that is responsible for maintaining the nucleotide
binding site and activation segment in a catalytically active
state. The C-terminal domain is comprised completely of
helices and associated loops. The activation segment, which
contains Tyr-216, is part of the C-terminal α-helical domain,
as is a majority of the substrate-binding site. The ATPbinding site, the catalytic site that encompasses the catalytic
machinery, and the substrate-binding site are formed at the
interface of the N- and C-terminal domains. This active site
‘channel’ between the two domains measures approximately
22 Å by 13 Å by 15 Å (giving a total approximate volume of
4290 Å3 for the entire active site). By its very nature, the
active site of GSK3 is not subdivided into convenient
component pockets, but rather is contiguous, with each area
melding into the other. An exception is the structural feature
responsible for binding the C-terminal priming phosphate
group: the oxyanion-binding pocket. This is a structural
Fig. (1). For most substrates, GSK3 (either α- or β- isoform) preferentially binds to-and phosphorylates-substrates with a ‘priming’
phosphate four residues to the C-terminus of the serine or threonine residue to be phosphorylated. Many GSK3 substrates have several
instances of appropriately placed serine or threonine repeats that are progressively phosphorylated once the far C-terminal residue is
phosphorylated (or ‘primed’) by another kinase. Examples of this type of site are shown in (A) and (B). (C) illustrates the GSK3β
phosphorylation sites for APC protein. Three more homologous repeats of this type exist on APC protein. Experiments have shown that a
priming phosphorylation is not required for subsequent GSK3β phosphorylation [63]. The numbers to the right and left of each particular
sequence denote the starting and ending amino acid number of that sequence within the protein in question.
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Current Pharmaceutical Design, 2004, Vol. 10, No. 10
Wagman et al.
Fig. (2). GSK3β structures. (A) Crystal structure of active, phosphorylated Tyr-216, GSK3β. The structure shown is done by Bussiere and
colleagues, and is similar to the structures deposited in the Protein Data Bank under accession codes 1H8F and 1I09, which are structures of
the less-active, unphosphorylated kinase. The N- and C-terminal ends of the protein are identified, as is the active site, which is denoted by
an ‘A’. (B) Crystal structure of active GSK3-β in complex with an axin fragment (PDB accession code 1O9U). (C) Crystal structure of active
GSK3β in complex with FRATtide, a fragment of the FRAT protein (PDB accession code 1GNG). Note that axin and FRATtide share the
same binding site.
feature common to all GSK3β structures and is formed from
two arginine residues (Arg-96 and Arg-180) and two lysine
residues (Lys-94 and Lys-205) which are arranged in such a
way that, in conjunction with local secondary structure, they
create a small, highly electropositive, 125 Å 3 pocket ideal for
binding a phosphate group. A model of the possible binding
mode of primed substrate is shown in (Fig. 3). In support of
this structural evidence, mutation of residues within this
region greatly reduces the selectivity of GSK3β for substrates with a C-terminal phosphate at the N+4 position [48].
This pocket has been proposed as being suitable for targeting
in drug discovery, as it is unique to GSK3 and would enable
one to target specific biological responses and disease states
[48, 78]. For example, in glucose homeostasis GSK3 is
required to recognize ‘primed’ substrates such as glycogen
synthase, while in other pathways (such as the Wnt pathway,
which will be discussed below), both primed and un-primed
substrates are recognized by GSK3 and the primary point of
control is the activation of another distinct multi-protein
complex [39]. However, recent data has shown that GSK3
mediated phosphorylation of β-catenin is dependent on a
priming event by casein kinase-1 α-isoform [40, 41]. Therefore, it is unlikely that compounds that target this site will be
directed against a specific disease state. Another drawback is
that any molecule designed to bind tightly within this site
would most certainly need to be highly negatively-charged,
which might lead to undesirable ADME properties for the
compound.
Proceeding from the oxyanion binding pocket towards
the active site (Fig. 3), molecular modeling would predict
that the substrate makes a majority of its interactions with the
C-terminal domain, although there is the distinct possibility
that GSK3 is capable of recognizing structural information at
the tertiary level and, therefore, that recognition of substrate
is dispersed throughout the molecule and would include
recognition by the N-terminal domain as well. Based on
structural work with other serine/threonine kinases, as well
as the crystal structure of GSK3β, a model of ATP bound in
the GSK3β active site can be constructed which predicts that
the bound ATP would be stabilized by multiple interactions.
First, the adenine ring would form the canonical ‘donoracceptor’ series of interactions, with the extracyclic amino
group donating a hydrogen bond to the carbonyl of Asp-133
and the N7 nitrogen of the adenine accepting a hydrogen
bond from the amine of Val-135. Additionally, the α- and
Discovery and Development of GSK3 Inhibitors
Current Pharmaceutical Design, 2004, Vol. 10, No. 10 1109
Fig. (3). Structural features of GSK3β. (Α) The active site has several discrete areas: the oxyanion binding pocket, responsible for
recognizing the priming phosphate; the phosphotyrosine recognition site, composed of two arginines; and, finally, the active site, responsible
for binding substrate and ATP, as well as catalyzing the reaction. (B) The oxyanion binding pocket is made up of two arginines and two
lysines, appropriately placed to create a small pocket with a strong electropositive potential. (C) The phosphotyrosine recognition site is
composed of two arginines that interact with the negatively-charged phosphotyrosine (Tyr216 in GSK3β), thereby preventing the tyrosine
from swinging into the active site and blocking the binding of substrate. Binding of substrate places a serine or threonine adjacent to both the
γ-phosphate of ATP (here modeled as AMPPNP) and the catalytic base (Asp-181). Activation of the catalytic base leads to Sn-2 type
nucleophilic attack on the γ-phosphate. The transition state is stabilized by Lys-183.
γ-phosphates of the ATP would be positioned properly to be
stabilized by the Mg2+ ion bound to the ATP itself. The
Mg2+ ion is, in turn, predicted to be stabilized by interactions
with the side-chains of Asn-186 and Asp-200. With the
substrate bound, first at the oxyanion binding pocket and
subsequently at various other contact points, the model
would predict that the reaction proceeds via activation of the
substrate serine adjacent to the ATP through the abstraction
of a proton via the catalytic base, in this case, Asp-200. The
activated serine or threonine at the N+4 position relative to
the priming phosphate would then execute an Sn-2 attack on
the γ-phosphate resulting in a phosphorylated serine or
threonine and ADP molecule. The transition state of such a
reaction is negatively-charged, hence the positioning of the
positively charged Lys-85 directly above the site of attack to
stabilize the transition state [42]. The proper positioning of
Lys-85 is ensured by a stabilizing electrostatic interaction between this lysine and Glu-97, which resides on the αC helix.
As mentioned previously, GSK3β can be activated by
phosphorylating Tyr-216, thereby increasing its enzymatic
activity several hundred-fold. How does this activation work?
Current structural information shows that phosphorylation of
Tyr-216 and subsequent electrostatic interaction of the negatively-charged phosphotyrosine with the positively-charged
Arg-220 and Arg-223 residues swings the phosphotyrosine
out of its ‘default’ position to a location where it is no longer
blocking the protein substrate’s access to the active site. The
default position for the un-phosphorylated tyrosine, on the
other hand, places the tyrosine in a position to prevent the
binding of protein substrate in the active site. Other than this
major structural rearrangement, the remaining structural
differences between these two states are relatively minor.
This mode of inhibition is contrary to the typical mode of
inhibition seen in many closely related kinases where, as in
GSK3, phosphorylation of an amenable residue within the
activation segment activates the kinase. In other kinases of
this type, the activating phosphorylation and subsequent
interactions between the phosphorylated residue(s) and the
protein results in an ordering of the activation segment
leading to optimal positioning of both catalytic residues and
residues responsible for binding of substrate in the active site
for maximal activity [43]. In GSK3, however, phosphorylation of Tyr-216 reorients this single side-chain relative to the
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Current Pharmaceutical Design, 2004, Vol. 10, No. 10
active site, but causes no major changes in the conformation
of residues within the activation segment [44].
The mode of phosphorylation-based inactivation of
GSK3 by the upstream kinase protein kinase B/Akt at the Nterminal serine (Ser-21 in the α-isoform and Ser-9 in the βisoform) is also hypothesized to occur by a conformational
change, but one of a more dramatic nature [36, 48]. In the
GSK3β crystal structures, the immediate N-terminus
containing the inactivating phosphorylation site is disordered
and is not represented in the electron density. Experimental
evidence based on competition experiments with peptides
derived from the N-terminus, as well as molecular modeling,
supports the model that phosphorylation of this N-terminal
serine transforms the N-terminus into a pseudosubstrate for
the kinase [33, 45]. The model further suggests that in the
inhibited form of GSK3, the phosphorylated N-terminus is
positioned into the active site with the phosphoserine positioned in the oxyanion binding pocket and a non-reactive
amino acid (alanine the case of the α-isoform; proline in the
β-isoform) positioned at the N+4 residue position adjacent to
the γ-phosphate of ATP. Such positioning of the N-terminus
would readily block access to the active site. A crystal structure of the inactivated form of either the α- or β- isoform has
yet to be published or disclosed, but a model is presented in
Fig. 4.
Wagman et al.
ANTI-TARGETS OF GSK3
The true challenge of kinase drug discovery and design
lies in developing bioavailable, selective, and potent inhibitors [46, 47]. A high degree of selectivity may be required to
prevent any given potent compound from having underlying
toxicities in vivo. The high degree of three-dimensional
structural fold and sequence homology conservation seen
between serine/threonine kinases makes attaining this high
degree of selectivity a difficult task. To aid in the design of
compound specificity in a kinase drug discovery project, one
must consider which kinases are ‘anti-targets’ of the kinase
of interest. In most cases, an anti-target will almost always
be a kinase with significant sequence and structural homology to which lead compounds will also bind. Some project
teams initially identify potential anti-targets based on
sequence homology, molecular modeling and threading, and
by their roles within a cell [48]. Other project teams rely
solely on an experimental approach, where initial lead
compounds are screened against a broad panel of diverse
kinases and cross-reactivity patterns are noted and analyzed
for each particular chemical scaffold. It is our opinion that a
combined approach is the most appropriate. Any suspected
anti-targets identified through bioinformatics or molecular
modeling must be confirmed experimentally. Of course, this
is not always possible due to the difficulty in overexpressing,
Fig. (4). Model of inactivated GSK3β (phosphorylated at Ser-9). Current experimental data supports a model wherein the phosphorylated Nterminus (Ser-9 in GSK3β) acts as a pseudo-substrate for the kinase. The phosphoserine at this position inserts into the oxyanion-binding
pocket, thereby positioning a non-reactive residue (a proline in the case of the β-isoform) into the active site, thereby blocking the active site
and inhibiting the kinase. This model was constructed using the crystal structure of active GSK3β and structural information from various
other kinase crystal structures.
Discovery and Development of GSK3 Inhibitors
purifying, and storing some kinases, particularly ones which,
while identified as part of the kinome, have yet to be
expressed, purified, and characterized. Once a panel of significant anti-targets has been confirmed experimentally, additional possible anti-targets can be extrapolated and identified
using computational methods. One must take note that an
anti-target will be determined predominantly by the particular chemical scaffold(s) under investigation and secondly
by the degree of homology between the target of interest and
potential anti-targets. Differences in the chemical structure
of the compound will combine with the differences in the
structural and chemical makeup of the kinase active site to
which the compound binds, leading to a different selectivity
profile for each chemical scaffold. In addition, one must be
aware that, in most cases, the ATP binding pocket will share
a higher homology between kinases than the substratebinding pocket(s) or other available pockets, making these
other pockets of strategic importance for kinase drug
discovery. With this said, however, it should be further noted
that currently drug discovery and design directed outside the
ATP-binding pocket of a kinase is far more challenging.
However, there have been some successes in this area, most
notably the development of Gleevec, which was revealed to
have a novel binding mode to the inactive form of Abl
kinase via an allosteric mechanism [49]. There are reports of
other successes using this particular approach, including
development of an allosteric P38 MAPK inhibitor [50-52].
Finally, an interesting approach has been taken by several
groups who have developed chemical scaffolds known as
‘bisubstrate’ inhibitors. These bisubstrate inhibitors access
both the ATP and substrate binding pockets; the drawback to
such compounds, despite their potency, is their relatively
large size when compared to inhibitors which access only
one binding pocket within the active site [54, 55]. The bioavailability and ADMET properties of such scaffolds remain
to be determined.
What selectivity against anti-targets should be achieved
by a kinase therapeutic? This is a difficult question. To some
extent it depends both on the anti-target and its cellular role
and on the therapeutic goal of the drug discovery project. For
example, if one were developing kinase inhibitor as a cancer
therapeutic, one might be more tolerant of compound crossreactivity and potential toxicities. However, as the potential
indications for a GSK3 inhibitor would most likely be as a
therapeutic for the treatment of diabetes or for the treatment
of Alzheimer’s disease or a related neuropathology, one
would be seeking a more stringent selectivity profile and a
compound with no toxicity. For our GSK3 project, we sought
a minimum of several hundred-fold selectivity between our
GSK3 lead compounds and confirmed anti-targets. By combining synthetic efforts with molecular modeling and broad
kinase screening, we were able to develop over a thousandfold selectivity between a compound’s GSK3 activity and its
activity in anti-targets. Of course, the guidelines and approaches followed to identify anti-targets and the desirable
specificity to be achieved will vary from effort to effort.
What are the potential anti-targets for a GSK3 drug
discovery project? Again, as discussed above, to a large
extent this question is dependent on the chemical scaffold of
a lead compound. It is possible, however, to identify broad
classes of anti-targets based on structural and sequence
Current Pharmaceutical Design, 2004, Vol. 10, No. 10 1111
homologies. The crystal structures of GSK3β showed that
there is a structural relationship between GSK3, the various
cyclin-dependent kinases (CDKs), and the mitogen-activated
protein kinases (MAPKs). Because of this homology, any
kinases in these two classes are possible anti-targets for any
chemical scaffold. In particular, sequence homology and
threading of the active site region suggest that the active sites
of cyclin-dependent protein kinases 2 (CDK2/p33) and 3
(CDK3) are most homologous to the GSK3β active site [56,
57]. However, these broad classes do not represent the only
possible anti-targets. For example, our GSK3 project team
empirically identified CHK1 as an anti-target for one of our
second-generation chemical scaffolds, yet it was not a significant anti-target for our first-generation compound (Data to
be published elsewhere). This identification would not have
been made on the basis of structure and sequence homologies; instead it had to be identified experimentally. This
cross-reactivity arose to due an electronegative patch present
in both the GSK3 active site and the CHK1 active site that
interacts with a positively charged moiety within the chemical scaffold. The electronegative patch in the active site
arises not only from the primary amino acid sequence, but
also from main-chain carbonyl orientations that cannot be
predicted on the basis of amino-acid sequence comparisons,
thereby illustrating the often-arcane mechanism of crossreactivity. By using structure-based drug design, the project
team was able to design in over one-thousand fold selectivity
against CHK1 into this chemical scaffold, thereby illustrating the importance of molecular modeling and structural
biology in kinase drug discovery.
GSK3, MULTI-PROTEIN COMPLEXES, AND THE
WNT SIGNALING PATHWAY
Aside from GSK3 roles in the insulin signaling pathway,
tau protein hyperphosphorylation, and other physiological
events where it acts alone by catalyzing the phosphorylation
of select serine and threonine residues, a portion of the cellular GSK3 forms a component of a multi-protein complex
involved in the Wnt signaling pathway. GSK3 is able to
perform its role in both of these pathways with no apparent
cross talk between the pathways [58]. This multi-protein
complex is comprised of GSK3β, axin, β-catenin, and APC
(adenomatous polyposis coli) protein [59]. GSK3α has not
been identified as being involved in this multi-protein
complex. Wnt signaling pathways are mediated by secreted
glycoproteins known as Wnts. These Wnts are paracrine
signaling molecules and activate numerous signaling cascades
inside target cells [59, 60]. In the absence of an active Wnt
signal, the GSK3β in the complex is active and phosphorylates each member of the complex at various positions in the
constituent proteins [61, 62]. These phosphorylations potentiate the action of two of the proteins within the complex: the
phosphorylation stabilizes axin and strengthens the interaction between β-catenin and APC protein [63]. At the same
time, however, the phosphorylation targets β-catenin for
ubiquitin-mediated proteolytic degradation by the proteasome (Fig. 5) [64, 65]. In the presence of an active Wnt
signal, the activity of GSK3β in the multi-protein complex is
inhibited by the disruption of the GSK3β:axin interaction by
another multi-protein complex, which contains at least two
proteins. These proteins, known as DVL (disheveled) protein
1112
Current Pharmaceutical Design, 2004, Vol. 10, No. 10
Wagman et al.
Fig. (5). GSK3β’s role in the Wnt pathway. In the absence of Wnt signaling via the binding of Wnt to its receptor, a multi-protein complex
scaffolded by APC and Axin promotes phosphorylation of β-catenin by GSK3β. APC and Axin are also phosphorylated by GSK3β in this
complex; some models also propose that casein kinase 1 (α− or ε−isoform) and other proteins are also members of this complex. In this
schematic, they have been omitted to simplify the model. The phosphorylated β-catenin is degraded by ubiquitin-mediated proteolysis by the
proteasome. It should be noted that this pathway, which leads to degradation of b-catenin is the basal state. Wnt binding to the 7-TM
‘frizzled’ receptor activates another multi-protein complex comprised of DVL protein, FRAT protein, and perhaps other as-of-yet
unidentified protein members, which then bind to GSK3β and inactivate it, displacing the other three proteins, APC, Axin, and β-catenin.
This prevents phosphorylation of β-catenin by GSK3β and, thus, prevents subsequent degradation of the β-catenin by the proteasome. βcatenin accumulates both in the cytosol and the nucleus, where it serves as a trans-activator in complex with Lef/TCF DNA-binding proteins
leading to activation of various developmentally critical target genes.
and FRAT (an acronym for ‘frequently rearranged in advanced T-cell lymphomas’) protein, bind directly to GSK3β and
inhibit its function as well as its binding interaction with axin
[66]. The molecular mechanism that regulates binding of this
complex directly to GSK3β is unknown. The resulting inhibition of GSK3β stabilizes the β-catenin in the cell, which
leads to its steady accumulation and eventual translocation to
the nucleus, where it interacts with transcription factors
within the T-cell factor (TCF) family [67]. This interaction
triggers the transcription of genes that are required for
determination of cell fate during embryogenesis. This event
is also likely to be involved in regulating the expression of
other genes involved in regulating events in adult tissues.
The formation of the GSK3β:axin:β-catenin:APC complex
would explain the dichotomy between the roles of GSK3β in
the insulin and Wnt signaling pathways. Sequestration of
GSK3 into the complex facilitates access to the primary
substrate in the Wnt pathway: β-catenin. The formation of
the complex might also prevent protein kinase B/Akt from
accessing the N-terminal inactivating serine, thereby
preventing GSK3β from being inactivated while it is in the
multi-protein complex.
Direct structural determination of the complete GSK3β:
axin:β-catenin:APC protein complex has yet to be achieved,
but components of this multi-protein complex have been
determined by X-ray crystallography. These include the multiple structures of GSK3β (discussed previously), structures
of β-catenin in complex with other members of the Wnt
pathway, and finally, a structure of a fragment of the APC
protein in complex with β-catenin [68-70]. There are currently
only two protein complex structures involving GSK3β and
another member of the Wnt-pathway, the first being a
structure of active GSK3β (Tyr216 is phosphorylated) with a
peptide fragment of the FRAT protein known as the
‘FRATtide’, which has been shown experimentally to inhibit
GSK3 in manner equivalent to the intact FRAT protein [71].
Discovery and Development of GSK3 Inhibitors
This structure shows that the fragment of FRAT, of which
residues 188-226 are visible in the electron density maps
(comprising approximately 17% of the total protein), forms a
helix-turn-helix that interacts with GSK3β at the C-terminal
α-helical domain of the protein. The interaction between
GSK3β and the FRATtide is primarily mediated by
hydrophobic side-chain contacts along the face of the Cterminal FRATTide α-helix that are inserted into a
hydrophobic patch between a loop (residues 285-299) and an
α-helix (residues 262-273) on the C-terminal domain of
GSK3β. Structural and biochemical details of FRAT
activation, which may involve an activating event such as
phosphorylation, are not currently known. The second
crystal structure is a structure of GSK3β in complex with a
19 amino acid minimal binding segment of human axin [37,
72]. This structure reveals that the interaction between
GSK3β and the minimal axin binding segment, which forms
an α-helix in the crystal structure, is very similar to the
interaction between GSK3β and the FRATTide: the
interaction is mainly through hydrophobic contacts at the
same hydrophobic patch as is seen in the previous structure.
Based on this data, the binding of FRAT or axin to GSK3β
would be mutually exclusive, as both share the same binding
region. This is in agreement with the current model. These
two structures are presented in Fig. 2.
GSK3 SMALL MOLECULE INHIBITORS AND DRUG
DISCOVERY PROGRAMS
While our understanding of the prominent regulatory role
of kinases in cell signal transduction has grown remarkably
over the last 20 years, our understanding of the intricate and
often interdependent cellular kinase control mechanisms has
recently undergone a renaissance with the increasing
availability of potent and selective small molecule kinase
inhibitors. Pharmaceutical research that has recently yielded
kinase inhibitors of clinical interest in the treatment of cancer
and chronic inflammatory diseases, among others, has also
created a bounty of kinase-specific molecular probes which
have been successfully exploited in the study of cell
signaling [73]. Encouraged by a growing number of kinase
drug targets and an increasingly sophisticated knowledge of
kinase activity and modulation, drug discovery research has
taken up the challenge of finding safe and efficacious kinase
inhibitors for the treatment of chronic conditions such as
diabetes [74]. A promising drug target, GSK3 has been the
focus of intensive medicinal chemistry efforts not only for
insulin resistance, but also for Alzheimer's disease, stroke
and bipolar disorders [2, 9, 75]. In this section, research
which has yielded potent and selective inhibitors of GSK3
will be highlighted along with a summary of the classes of
small molecule inhibitors which appear in the patent
literature. For recent reviews see [74, 76, 77]. Emphasis will
be placed on structurally novel compounds and small
molecule research programs that have generated in vitro
SAR, cellular potency and biological studies.
Before the availability of selective inhibitors, initial
studies of GSK3 were conducted using lithium. The effects
of lithium on developing organisms were traced to GSK3
inhibition and shown to mimic the effects of Wnt signaling
[78-80]. Since lithium is used in the treatment of bipolar
disease and depression, a connection was made between
Current Pharmaceutical Design, 2004, Vol. 10, No. 10 1113
GSK3 and possible molecular mechanisms leading to these
mood disorders [81-83]. The potential association between
GSK3 and mood-stabilization was further enhanced by the
discovery that valproic acid, used in the treatment of bipolar
disorder, also activated the Wnt signaling pathway [84]. The
research surrounding lithium and valproate as modulators of
the Wnt pathway, their mechanistic and biological
characteristics and utility in bipolar disorder has been
accounted in detail [85]. Recently, however, histone
deacetylase has been identified as the target for valproate and
the basis of this compound's potent anticonvulsant and
mood-stabilizing action [86]. Further research will be needed
to evaluate the involvement of GSK3 in valproate mediated
physiological responses [87].
LITHIUM
Lithium has been shown to reduce the phosphorylation of
tau protein in cells at therapeutic concentrations in a manner
consistent with inhibition of GSK3 [88]. Lithium has also
been shown to protect neuronal cells from apoptosis caused
by the accumulation of fibrillary β-amyloid which can be
explained via lithium's action as an inhibitor of GSK3 and
provides evidence that GSK3 might be a viable target for the
prevention of neurodegenerative disorders such as
Alzheimer's disease. The implication of GSK3's involvement
in the pathology of Alzheimer's disease through interactions
with protein tau, presenilin 1, the amyloid-β peptide, the
amyloid precursor protein, and acetylcholine and
proapoptotic mechanisms has prompted a great deal of
interest in these pathways and the possible use of GSK3
inhibitors for the treatment of many neurodegenerative
diseases including stroke and acute brain trauma [89-92]. As
an inhibitor, lithium exerts a direct effect on GSK3 having
an IC 50 of 2mM under conditions thought to approximate the
intracelluar environment i.e. a free concentration of Mg2+
(0.5mM) and isotonic KCl 150mM [93]. Lithium does affect
GSK3 activity to a greater extent then other kinases tested
under the same conditions, however lithium is not highly
potent or selective. At several multiples (3-7 times) of the
IC50 of GSK3, lithium inhibited casein kinase 2 (CK2), p38regulated/activated kinase (PRAK) and MAPK-activated
protein kinase 2 (MAPKAP-K2). It is possible that some of
the effects attributed to GSK3 from studies using lithium
may in part be from alternate kinase targets. For example,
before glycogen synthase (GS) can be inhibited through the
kinase action of GSK3, it must first be primed by
phosphorylation by CK2. The ambiguity associated with
lithium's cellular activity underscores the need for selective,
potent and cell-permeable probes of kinases (and GSK3) to
resolve their independent contributions to cellular regulation.
GENERAL KINASE INHIBITOR POTENCY AND
SELECTIVITY SURVEY
As part of a greater effort to characterize the mechanism
of action and specificity of 42 commercially available kinase
inhibitors against a large panel of some 30 different protein
kinases, several small molecules were found to significantly
inhibit GSK3 [93, 94]. While this study was instrumental in
identifying GSK3 inhibitors, it also defined the selectivity
patterns of a variety of ATP competitive kinase inhibitor
compounds and classes of molecule, including some natural
1114
Current Pharmaceutical Design, 2004, Vol. 10, No. 10
products. In general, this selectivity and potency information
helps guide the application and interpretation of studies
undertaken with these inhibitors. Many compounds inhibited
more kinases than their expected target and even the most
selective molecules inhibited at least one additional kinase
along with their target. It is, therefore, critical to take these
findings into consideration when designing new experiments
or interpreting existing whole cell or in vivo studies with
these classes of kinase inhibitor. In the first phase of the
study, Ro 318220 (1), which was designed and used as a
PKC inhibitor, was found to be the most potent inhibitor of
GSK3 (IC50 = 38nM) among a group of bisindolylmaleimides. All assays were run at ATP concentrations of 0.1mM,
and inhibition is expressed as the percent activity as
compared to 100% activity using the appropriate substrate.
Other bisindolylmaleimides including Bis-1, Bis-3, Bis-4,
Bis-5, Bis-8, Bis-10, Go6976, K252c, KT 5720 and UCN1
were assayed. Of these only Bis-1, Bis-3, Bis-4, Bis-8, Bis10 and Go6976 had activities between 21 and 50% of normal
full activity at 1uM. None were highly selective for GSK3.
Three additional compounds (Rottlerin - 13% activity at
20uM, KN62 - 38% activity at 10uM and Quercetin - 30%
activity at 20uM) were found to weakly inhibit GSK3 among
other kinases [93]. SP600125 and KT5823 were also found
to have weak activity [94].
O
H
N
N
Wagman et al.
inactive [97]. The higher IC50 reported in these studies may
be explained by the lower ATP concentrations used in the
assays, as IC50 increases with ATP concentration for ATPcompetetive inhibitors such as these. While in most cases
potent CDK inhibitors are active against GSK3, selectivity
was found with two examples from the indirubin class; 5, 5'dibromoindirubin (3) was selective for GSK and 5-SO3Naindirubin-3'-monoxime (4) was more active against the
CDKs. The combination of known selectivity in this scaffold
and the X-ray crystal structure of indirubin-3'-monoxime (2)
bound in the active site of CDK2 may indicate productive
areas where these molecules might be further modified to
enhance their selectivity toward GSK3. Since the parent
compound indirubin (5) has poor bioavailability and has
shown some gastrointestinal effects, the scaffold would also
have to be chemically modified to improve PK and
tolerability for use in a chronic application [95].
R3
R2
R1
N
H
NH
O
2: R1 = R3 = H; R2 = NOH
3: R1 = R3 = Br; R2 = O
4: R1 = H; R2 = NOH; R 3 = SO 3Na
5: Indirubin: R1 = R3 = H; R2 = O
O
N
Me
NH
S
NH 2
1: Ro 31-8220
INDIRUBINS
In the second part of the study, traditional CDK inhibitors reported to have GSK3 activity were confirmed. Indirubin-3'-monoxime (2), which is a component of the traditional
Chinese medicine Danggui Longhui Wan, has been used in
the treatment of chronic diseases such as leukemia [95]. It is
known as a potent (IC50 = 50-100nm) ATP competitive
inhibitor of CDK1, CDK2 and CDK5 and has been found to
initiate cell-cycle arrest as expected from a CDK inhibitor
[96]. In the survey study, indirubin-3'-monoxime (2) was
active against GSK3 (IC 50 = 190nM), CDK2 (IC50 = 590nM)
and several other kinases, such as AMP-activated protein
kinase (AMPK) and serum- and glucocorticoid-induced
kinase (SGK) [94]. It is not surprising that an ATP competitive inhibitor of the CDK family would also be an inhibitor
of GSK3 since these kinases are closely related and have
similar active sites (see above). In a separate study, a variety
of indirubins, including indirubin-3'-monoxime (2), were
identified as potent inhibitors of GSK3 (IC50 = 5-50nM) and
CDKs, while other structurally related indigoids were
PAULLONES
Members of the naturally occurring family of benzazepinones called paullones (6: paullone) were found to be
potent inhibitors of GSK3 with fairly good selectivity against
the panel of kinases assayed. Kenpaullone (7) (IC50 = 230nM)
and alsterpaullone (8) (IC50 = 110nM) were both potent ATP
competitive inhibitors of GSK3, but also inhibited LCK and
CDK2/cyclin A. Alsterpaullone (8) was actually more potent
against CDK2/cyclin A (IC50 = 80nM) than GSK3. Although,
the IC50's for both compounds were within 3 fold for all three
of these kinases [94]. Similar to previous well known kinase
inhibitors, paullones were found to have anti-cancer activity
which was linked to their potency against CDK1/cyclin B
[98, 99], and later to their ability to inhibit GSK3, CDK2 and
CDK5 [100]. While not inhibiting any isoform of PKC,
MAPKK or c-raf, alsterpaullone (8) did inhibit CDK1/cyclin
B, CDK2 cyclinA, CDK2 /cyclin E, CDK5/p35 and GSK3α
and β. The anti-tumor effects of alsterpaullone (8) are most
likely due to inhibition of CDK1/cyclin B. The suppression
of tau phosphorylation by either alsterpaullone (8) or kenpaullone (7) may be due to GSK3 and/or CDK5 inhibition.
Alsterpaullone (8) also suppresses the phosphorylation of
DARPP-32 by CDK5/p25 which may reduce apoptosis
signals in neurons and would be helpful in the treatment of
Alzheimer's disease. SAR from a diverse set of 56 paullones
showed that substitution of small groups at the C-9 position
increased potency. It was hypothesized that incorporating a
hydrogen bond acceptor at C-9 would increase the binding
Discovery and Development of GSK3 Inhibitors
Current Pharmaceutical Design, 2004, Vol. 10, No. 10 1115
pocket affinity by interaction with a proximal molecule of
water identified in the crystal structure of CDK2. The addition of a nitro group at C-9 did improve the IC50 of alsterpaullone (8) (IC 50 CDK1 = 35nM) over the parent paullone
(IC50 CDK1 = 3uM) [99], however the selectivity between
GSK3, CDK1 and CDK5 was lost [100]. A small lipophilic
group at C-9, such as a chloro or bromo group, increased the
potency and selectivity for GSK3. The pyrrole-like NH at
position 12 could be alkylated with many small groups while
still maintaining potency. Interestingly, a methyloxycarbonylmethyl group on position 12 nearly obliterates CDK5
binding and improves GSK3 selectivity (IC 50 GSK3 = 75nM,
CDK1 = 1.4uM, CDK5 = 350uM). Methoxy groups at C-2
and 3 are also tolerated, but do not improve the kinase
selectivity.
5
H
N
O
3
2
12
HN
9
R
11
6: R = H Paullone
7: R = Br Kenpaullone
8: R = NO2 Alsterpaullone
HYMENIALDISINE
Hymenialdisine (9) and related metabolites produced by
several species of marine sponge represent a novel family of
ATP competitive kinase inhibitors [101, 102]. While screening natural products for possible anti-cancer activity, hymenialdisine (9) was found to be a potent inhibitor of CDK1/
cyclin B. Other than cancer applications, interest in these
compounds has also been raised due to their anti-inflammatory activity in U937 cells through inhibition of nuclear
factor-kB [103]. Hymenialdisine (9) shows potent activity
against CDK1/cyclin B (IC 50 = 22nM), CDK2/cyclin A (IC50
= 70nM), CDK2/cyclin E (IC50 = 40nM), CDK3/cyclin E
(IC50 = 100nM), CDK5/p25 (IC50 = 28nM), CK1 (IC50 =
35nM) and GSK3 (IC50 = 10nM) with good selectivity
versus other kinases such as Erk2, c-raf, MAPKK and the
PKCs [104], and has been filed in a patent application [105].
The chemically related diacetylhymenialdisine (10) and
diacetyldebromohymenialdisine (11) are fairly active inhibitors of GSK3 (IC50 = 130 and 160nM), while the close
analogues hydantoin (axinohydantoin, 12) and 2-aminoimidazole (Stevensine/odiline, 13) had poor or no activity
respectively. Clues to the selectivity of hymenialdisine (9)
can be gleaned from the crystal structure of the inhibitor
bound to the ATP binding site of CDK2. The guanidine ring
of hymenialdisine (9) which seems to be a key component of
potency and selectivity is in H-bond proximity to three
crystal bound waters in the ATP pocket near Gln131 and
Asp145. The hydrophobic bromopyrroloazepine bicyclic
ring system of hymenialdisine (9) fits snugly in a hydrophobic pocket making key backbone H-bonds to Leu83 and
Glu81. Structure based approaches for increasing the selectivity of these compounds might be possible by studying the
differences between the CDK2 and GSK3 crystal structures
which are now available. Experimental studies using hymenialdisine (9) show promise for treatment of neurodegenerative diseases. Phosphorylation by GSK3 and CDK5/p35 was
suppressed in a dose dependent manner. At high doses,
hymenialdisine (9) blocked the phosphorylation of MAP-1B
and had an affect on axonal remodeling. Consistent with
GSK3's activity against other substrates, hymenialdisine (9)
inhibited the phosphorylation of expressed human tau with
an IC 50 of ~33nM giving an indication that potent inhibitors
of GSK3 and/or CDK5/p35 may find utility in the treatment
of Alzheimer's disease [104].
SMALL MOLECULE PHARMACEUTICAL
RESEARCH PROGRAMS
Highly Substituted Purines, Aminopyrimidines and
Aminopyridines
Early collaborative research at Chiron in 1996 identified
substituted purines (14) as novel synthetic, ATP competitive
inhibitors of GSK3 (63% inhibition at 1uM), validating the
tractability of the kinase as a target for medicinal chemistry
[106, 107], and helping to improve and refine the optimal
methods for screening potent small molecules [108, 109].
Further medicinal chemistry research yielded a very potent
series of GSK3 inhibitors based on highly substituted aminopyrimidine 15 (W = N) or aminopyridine 15 (W = CH) core
O
H 2N
O
NH
N
N
HN
O
H2N
NH
HN
HN
O
Br
R1
O
HN
NH
NH
O
O
9: Hymenialdisine
Br
HN
N
NH
Br
O
Br
HN
NH
N
10: R1 = Br
11: R1 = H
O
NH
O
12
13
1116
Current Pharmaceutical Design, 2004, Vol. 10, No. 10
Wagman et al.
rings [110, 111]. Later it was revealed that the core ring
could also be an aminopyrazine 16 [112] or a fused bicyclic
system 17 wherein a ring was formed between the pictured
C-5 and C-6 positions of the core ring [113]. A common
feature of this family of analogues is the arenes linked by a
bridging ethylenediamine. On one side of the four atom
bridge are the substituted pyrimidines and pyridines having a
decorated phenyl group in the C-4 position (see structure
15). Substitution at the C-5 position was highly varied, but
many groups were capable of making productive first and
second shell water contacts on the surface of the protein, as
well as possible H-bonding interactions with amino acids at
the entrance of the ATP binding pocket. This position also
provided a convenient chemical handle to optimize the
physicochemical and PK properties of the compounds. The
C-4 phenyl ring was typically substituted with small groups,
such as halo, methyl and methoxy moieties at the C-2' and 4' positions, although much larger groups were tolerated the
4' carbon which was directed back toward the substrate
binding area of the active site. The C-2' substituents were
optimally placed in an accommodating, small hydrophobic
pocket which greatly enhanced the kinase selectivity of the
series [114]. Linked through the C-2 position of the pyrimidine core, the distal nitrogen of the ethylenediamine was
typically coupled via SnAr reaction to an activated nitrogencontaining heteroaromatic ring such as, a 5-nitropyridine, 5cyanopyridine or 4-nitrothiazole.
Some of the details of the evolution of the aminopyrimidine and aminopyridine series of compounds in the context
of a program directed toward novel anti-hyperglycemics
have been disclosed [74, 115-117]. This series found it's
genesis in a kinase-directed library of 80 pools of 3, 4dihydropyrimidines 18. Each pool contained a mixture of 18
compounds. After extensive GSK3 screening, only one pool
furnished modest inhibition activity in the 3-5 uM range.
This mixture of 18 molecules continued to be used in
biochemical studies where it was noted that over time, the
activity of the compounds seemed to be improving. Aware
that the 3, 4-dihydropyrimidines could undergo oxidation
when exposed to chemical oxidants or air and heat, it was
assumed that the fully aromatic 2-aminopyrimidines were
responsible for the improved activity of the pool. Upon
investigation of the pool using HPLC and MS, it was found
that not only had the core ring aromatized, but the residual
Rink-resin linker amide on the C-4 phenyl group had
dehydrated to a cyano group as represented by the transition
from CT 98018 (19) to CT 98016 (20). Thus, when the pool
constituents were deconvoluted and individually assayed, CT
98016 (20) was found to inhibit GSK3 with an IC50 of 50nM
and had activity in an insulin-responsive CHO cell line. With
this serendipitous discovery, the most potent leads in this
medicinal chemistry program transitioned from low uM
IC50s to nM potencies and provided a highly attractive
scaffold for optimization.
CF3
HN
O
O
N
N
N
N
N
H2N
X
14
O
Y
X
Y
X
Heterocycle
Heterocycle
4
R1
4
R1
NH
N 3
5
5
W 2 N
H
1
HN
5
2 N
H
1
O
NH2
NH2
NH
N 3
N 2 N
H
1
16
15: W = CH or N
O
Heterocycle
4
NH
N 3
N
R2
Y
17
CN
NO2
NO2
N
O
R2
O
R1
NH
NH
N
18
N
H
N
O
O
NH
NH
N
N
H
19: CT 98018
IC50 = 3-5 µM
EC50 = >10 µM
O
O
NH
N
N
N
H
20: CT 98016
IC50 = 50 nM
EC50 = 1.6 µM
Discovery and Development of GSK3 Inhibitors
Current Pharmaceutical Design, 2004, Vol. 10, No. 10 1117
Traditional medicinal chemistry surveys of allowable
substitution demonstrated that a linear four atom linker was
needed, and an ethylenediamine linker was optimal between
the aryl groups. Substitution on the NH proximal to the
pyrimidine was tolerated, but an H-bond donor was needed
on the distal side to make backbone amide bonds in the ATP
pocket. A four atom linker also provided the best alignment
between the C-4 phenyl, core pyrimidine and 5-nitropyridine
in the binding pocket. Larger or smaller linkers were stericly
disfavored, presumably causing collisions between the linker
and main chain residues and misaligned the C-4 phenyl in a
channel leading to the substrate binding area. Groups such as
the C-6 ethyl were removed to lower molecular weight and
decrease lipophilicity. The C-5 ethyl ester of CT 98016 (20)
was utilized to improve solubility. Unfortunately, while the
free carboxylate, basic ester or basic amides at C-5 were
potent in the cell free assay, they did not show GSK3 activity
in the IR-CHO cell assay which is most likely due to poor
membrane permeability (21) [115]. Substituents on the C-4
phenyl group were best tolerated in the ortho and para
CN
NO2
positions. Smaller groups, such as F, Cl, Br, CF3, OMe, and
OCF3, were preferred in the ortho position, while larger
groups, such as ethyl, propyl and imidazole, were permitted
at the para position. However, the series was optimized using
a 2, 4-dichlorophenyl at C-4 which seemed to give improved
IC50's. Switching from linear moieties to heterocycles, an Nlinked imidazole, such as CT 98014 (22), gave a notable
improvement in the whole cell activity. A second refinement
which enhanced the IR-CHO cell EC50 was the discovery a
new 6-amino-5-nitropyridine group at the distal end of the
ethylenediamine linker. A survey of several hundred
heterocycles at the distal linker position yielded only a few
compounds with sufficiently potent IC50's (e.g. <100 nM)
(Scheme 1). 2-Aminopyridines with electron withdrawing
groups at the para position gave the best results. It became
apparent that an electron withdrawing group was needed in
the para position of the pyridine for optimal binding, for
example CT 98018 and CT 98224 in Scheme 1. Crystal
structures and modeling show a close approach of this NH to
the backbone in the ATP binding pocket [115]. This suggests
Cl
NO2
Cl
NO2
NH2
N
O
N
Cl
X
N
N
NH
N
N
N
H
21: X = -OH, -OCH2CH2NMe2
-NHCH2CH2NMe2
N
22: CT 98014
N
H
23: CT 98024
IC50 = 560 pM
EC50 = 387 nM
IC50 = 580 pM
EC50 = 83 nM
IC50 = 30-160 nM
EC50 = 3-10 mM
NH
N
N
H
N
H
N
Cl
N
NH
N
N
NH2
Cl
Cl
N
Heterocycle
N
NH
N
N
NO2
CN
NO2
N
CT 98018
IC50 = 6 nM
N
H
CT 98014
IC50 = 1 nM
CT 98224
IC50 = 10 nM
N
N
N
N
Cl
CT 98022
IC50 =201 nM
Scheme 1.
CT 98032
IC50 = 24 nM
NH2
N
N
N
CT 98124
IC50 = 223 nM
CF3
CN
N
Cl
N
N
N
CT 98028
IC50 = 87 nM
NO2
S
N
NH2
CF3
N
CT 98030
IC50 = 236 nM
N
CT 98026
IC50 = 381 nM
S
CT 98132
IC50 = 596 nM
1118
Current Pharmaceutical Design, 2004, Vol. 10, No. 10
Wagman et al.
that modulation of the pKa of the NH of the amino pyridine
group adjusts the strength of an important backbone Hbonding interaction. Application of these modifications lead
to highly potent compounds, and prompted the initiation of
animal studies.
While not very soluble, CT 98014 (22) was used to examine the potential of the series to improve the insulin sensitivity and glucose disposal in a mouse model of diabetes.
Following oral dosing at 30 mg/kg (20% Captisol) in db/db
diabetic mice, the compound elicited a 25% reduction in
blood glucose AUC in an oral glucose tolerance test (oGTT)
with a rapid (~30min) onset of action. To improve the
solubility and formulatability of the series, the C-5 imidazole
was rotated in CT 98024 (23) to provide a H-bond donor, an
accessible pKa and sub-nM potency (IC50 = 560pM). CT
98024 (23) was easily formulated in citrate buffered water
and was orally efficacious in an oGTT model in db/db mice
(30% reduction in glucose AUC at 30mg/kg dose) and ZDF
rats (29% reduction glucose AUC at 8mg/kg dose). Unfortunately, CT 98024 (23) suffered from primary and secondary
metabolism resulting in a poor half life (t1/2) in rodents (46
min. oral t1/2 in rat). The metabolism issues were address by
methylating the easily oxidized imidazole and by replacing
the nitropyridine with a 5-cyanopyridine generating an early
clinical candidate, CT 99021 (24). The oral half life doubled
in rats to 92min. The oGTT showed a 32% reduction in
glucose AUC at 8mg/kg dose and an ED 50 of 6mg/kg in ZDF
rats with twice-daily dosing. Further iterative cycles of medicinal chemistry and improvements of in vivo characteristics
lead to CT 20026 (25), one of Chiron's most advanced predevelopment compounds. Replacement of the metabolically
unstable imidazole with a monoketopiperazine improved the
solubility and stability of CT 20026 (25) over previous leads.
Use of a 2-aminopyridine core in place of a pyrimidine gave
a different metabolic profile, as well as improving cell
permeability as evidenced in the IR-CHO cell activity (EC50
= 66nM). The PK of CT 20026 (25) was also improved
showing longer half-lives in rats (205min), beagle dogs
(163min) and cynomolgus monkeys (173min). The oGTT in
ZDF rats provided an ED 50 of 10 mg/kg after single administration with an oral bioavailability of ~30%. Despite the
documented mutagenic potential of nitroarenes, no
geneotoxicity was observed in a full Ames study.
Extensive evaluation of this family of GSK3 inhibitors in
vitro has established that they are exquisitely selective for
Cl
the GSK3 ATP binding site even when assayed against
structurally similar kinases with high sequence identity like
CDK1 and ERK2, the closest homologs with 30% amino
acid identity within their catalytic domains [115]. The results
of the selectivity panel for CT 98014 (22) and CT 99021
(24) assayed against 20 kinases show 500 to >10, 000 fold
selectivity, which is representative of the whole class of
inhibitors (Table 1). There was virtually no difference in IC 50
between the isoforms of GSK3α and β, and little difference
in inhibition between human, mouse and rat derived proteins.
CT 99021 was also evaluated against a panel of 22 standard
screening receptors and 23 non-kinase enzymes. The only
off-target activity (Ki = 8.3 umol/l) was against phosphodiesterase III. Having established the high degree of selectivity of these compounds, they were used with confidence in
studies designed to probe GSK3's role in cellular glucose
metabolism. In insulin receptor-expressing CHO-IR cells,
GS activity was increased several fold above basal in a dose
dependent manner in response to either CT 98014 (22) or CT
99021 (24) (EC 50's of 106 and 763 nmol/l respectively). The
higher EC 50 for CT 99021 (24) is consistent with it's higher
Ki. Some variation in EC50 between compounds may also be
due to differences in cell membrane permeability or protein
binding. To demonstrate that these GSK3 inhibitors could
function in a more natural cellular environment, CT 98014
(22) was added to primary rat hepatocytes which responded
as the CHO-IR cells did with a two- to threefold stimulation
of the GS activity. The EC50 of CT 98014 (23) in the rat
hepatocytes was 107 nmol/l which is very similar to the EC 50
in CHO cells [115]. It is interesting to note that the GSK3
inhibitors were able to increase the activity of GS to the
same or greater extent as insulin might accomplish through
suppression of GSK3 activity. The effect of these compounds
on insulin sensitivity and improved glucose uptake will be
discussed below.
MALEIMIDES
Staurosporine (25) and structurally related maleimidecontaining molecules, such as Ro 318220 (1) and GF
109203x (25), have consistently demonstrated their utility as
kinase inhibitors, especially inhibitors of PKC. As early as
1999, Ro 318220 (1) and GF 109203x (25) were identified
as inhibitors of GSK3 β with IC 50's in the low nanomolar and
hundreds of nanomolar range respectively [119]. Unfortunately, staurosporine and Ro 318220 (1) (as noted above) are
CN
Cl
NO 2
NH 2
H3 C
N
Cl
N
N
NH
N
N
N
H
N
N
H
N
Cl
NH
N
O
N
N
H
24: CT 99021
25: CT 20026
IC50 = 5 nM
EC50 = 1 uM
IC50 = 4 nM
EC50 = 66 nM
Discovery and Development of GSK3 Inhibitors
Table 1.
Current Pharmaceutical Design, 2004, Vol. 10, No. 10 1119
Selectivity of Kinase Inhibition by CT 98014 and CT 99021
Kinase
species
group
identity
CT 98014 IC50 (nM)
CT 99021 IC50 (nM)
GSK3-β
human
CMGC3
100%
0.58
Ki = 0.87nM
6.7
Ki = 9.8nM
GSK2-α
human
CMGC3
91%
0.65
10
cdc2
human
CMGC1
30%
3,700
8,800
erk2
human
CMGC2
31%
>10,000
>10,000
PKC-α
human
AGC2
22%
>10,000
>10,000
PKC-ς
human
AGC2
19%
akt1/PKB
human
AGC3
21%
>5,000
>10,000
p70S6K
rat
AGC6
22%
>1,000
>10,000
p90RSK2
rabbit
C6,CAMK6%,
22%
>10,000
>10,000
PTK01
19%
>1,000
c-src
>10,000
tie2
human
PTK13
18%
>5,000
>5,000
fit1
human
PTK14
17%
>5,000
>5,000
KDR
human
PTK14
18%
>2,000
>5,000
bFGFRTK
human
PTK15
20%
>1,000
>5,000
IGF1RTK
human
PTK16
16%
>2,000
>10,000
InsulinRTK
human
PTK16
16%
>2,000
>10,000
AMPkinase
rat
OPK
22%
>10,000
pdk1
human
OPK
23%
>10,000
chk1
human
OPK
21%
>10,000
>10,000
CK1-ε
human
OPK
17%
>5,000
>5,000
DNAprotein
human
P13K
Low
>10,000
PI3kinase
human
P13K
Low
>2,000
>10,000
Percentage identity refers to amino acid identity versus human GSK3β within the protein kinase catalytic domain. Abbreviations: RTK, Receptor Tyrosine kinase; CK1ε, Casein
Kinase 1 epsilon. The group classification represents the class of kinase from which the particular enzyme derives [118].
not highly specific kinase inhibitors [93], although staurosporine analogues have been filed as GSK3 inhibitors [120].
Indeed, Ro 318220 (1) may have biological activity against
targets other than kinases, such as inhibition of sodium
channels [121]. While interesting as biochemical probes,
these compounds do not have sufficient selectivity to be used
as anti-hyperglycemics. However maleimides are potent
kinase inhibitors which might be a good starting point for
optimization. Having identified this scaffold by high throughput screening of rabbit GSK3α, SmithKline Beecham generated a matrix of compounds to explore the SAR of GSK3α
binding [122]. Maleimide analogues were quickly established with low nanomolar activity against human GSK3α
(IC50s 20-50nM) [123-125]. The 3 most potent compounds
reported had acidic phenols (27) or carboxylates (28, 29) on
the aniline moiety and a nitro group on the aryl ring.
Methylation on either the aniline nitrogen (30) or the
maleimide nitrogen (31) resulted in a significant loss of
potency. Interestingly, the binding pocket did tolerate the use
of an indoline (32) in place of an aniline with minimal loss
of activity. In the indoline series, the aniline H-bond is not
critical, suggesting an alternate binding mode. Five top
compounds (including SB-415286 (33)) demonstrated good
selectivity when screened against a panel of 20 kinases. As
expected from the similarity of the ATP binding pocket,
these molecules were equipotent against GSK3α and β. SB415286 (33) was modestly active against RSK-2 and AMPK.
The biological activity of the arylindolemaleimide SB216763 (34) and anilinomaleimide SB-415286 (33) (IC50's of
34nM and 78nM respectively) were reported in detail [126]
(arylindolemaleimides were also filed in applications
independently [127]). Both were shown to be highly potent
ATP competitive inhibitors of human GSK3α. They were
also able to stimulate glycogen synthesis in Chang human
liver cells and HEK293 human kidney cells at levels higher
than the maximal rate of insulin alone. To investigate the
1120
Current Pharmaceutical Design, 2004, Vol. 10, No. 10
Wagman et al.
NH
H
N
O
O
H
N
O
O
R2
N
Me
HN
O
N
N
H
MeO
N
R1
H
MeHN
26: GF 109203x
R1
H
N
O
N
27: R1 = 3-NO2; R 2 = 3,5diCl-4-OH
28: R1 = 2-NO2; R 2 = 4-Cl-3-CO2 H
29: R1 = 3-NO2; R 2 = 4-Cl-3-CO2 H
N
25: Staurosporine
O
O
O
O
O
H
N
O
Cl
O2N
N
N
O2N
HN
OH
R2
32
30: R 1 = H; R2 = Me
31: R 1 = Me; R2 = H
ability of these GSK3-specific inhibitors to induce β-catenin
stabilization and accumulation in the cytosol, compounds
were assayed in HEK293 cells with a β-catenin-LEF/TCF
regulated reporter gene. Both compounds gave a dosedependent response indicating a direct inhibition of GSK3
and increased β-catenin accumulation in this in vitro system.
Additional studies show that these compounds can mimic the
action of insulin by suppressing gene transcription of key
enzymes (glucose-6-phosphatase and phosphoenolpyruvate
carboxykinase) in the gluconeogenic pathway [128]. SB216763 (34) and SB-415286 (33) are also able to protect
both central and peripheral nervous system neurons from
death induced by abrogation of PI3 Kinase activity using the
PI3 Kinase inhibitor LY-294002 (35) in culture [129]. The
neuronal protection correlated with the inhibition of GSK3
and the modulation of tau phosphorylation and β-catenin
protein levels [130]. These results, combined with recent
studies demonstrating that FRAT1 overexpression is
neuroprotective, emphasize the potential value of selective
and potent small molecule GSK3 inhibitors, not just for
chronic diseases such as diabetes and Alzheimer's, but also
for the acute prevention of neural damage due to stroke or
injury [131].
33: SB-415286
In a separate program at Johnson&Johnson, research
focused on bisindolylmaleimides with PKC activity [132,
133]. The maleimide H-bond was important for potency as
methylation (36) abolished activity as a GSK3 inhibitor.
Some selectivity was gained by varying the size of the
macrocycle formed by tethering the indoles together. Where
n = 2 or 3 (37), GSK3 IC50's were 22 nM and 26 nM
respectively. While a smaller ring (n = 1) reduced potency, a
larger ring size (n = 4) only doubled the IC50 to 51nM. In 37,
bisindoles (A = B = CH) or mixed indole/azaindole (A =
CH, B = N) compounds showed dual activity inhibiting both
PKCγ (1.2-5.2 uM) and GSK3β (17-136 nM). Much greater
selectivity toward GSK3β was gained from incorporating a
nitrogen into both of the indole benzyl rings (38). Not only
did the 7-azaindoles 38 impart selectivity for PKC (IC50 =
>10uM), they were also much more selective against CDK1,
CDK2 and VEGF-R. Computational modeling of these
structures into the active stie of GSK3 helps to explainthe
selectivity. One of the azaindoles makes a potential third Hbond with the Arg-141 in GSK3 which is unique to this ATP
binding pocket. These 7-azaindoles exhibited good selectivity against a 50 kinase panel. Both optimal compounds (38)
were able to stimulate GS activity in HEK293 cells.
R
O
H
N
O
O
Cl
Ph
O
N
34: SB-216763
O
N
A
Cl
N
O
B
N
N
O
35: LY-294002
O
O
n
36: R = Me, A = B = CH, n = 2
37: R = H, A = B = CH, n = 2, 3
38: R = H, A = B = N, n = 2, 3
Discovery and Development of GSK3 Inhibitors
Current Pharmaceutical Design, 2004, Vol. 10, No. 10 1121
5-ARYL-PYRAZOLOPYRIDAZINES AND PYRIDINES
IC50 = 600nM and CDK5/p25 with an IC50 of 400nM. With
the similarity between their ATP binding sites, it is not
unexpected that these compounds also inhibited GSK3β at
comparable concentrations, IC 50 = 1 uM. Only compound 44
was tested against GSK3β, and none of the other compounds
was more potent against the CDK's than 44.
Further work at GlaxoSmithKline identified pyridazine
39 (IC 50 = 250nM) as a lead in a new series of GSK3 inhibitors [134]. Related compounds have been published in patent
applications by GSK [135-137] and Vertex [138]. The C-4
phenyl and N at the 6 position could be removed without significant loss of activity. The IC50 of these compounds could
be increased considerably by introducing a small lipophilic
amide on the C-3 amino group. Fluoro and chloro groups on
the ortho position of the C-5 arene also lowered the IC50s to
the 20nM range. Compounds 40 and 41 were the most potent
of this series with IC50's of 5nM and 7nM respectively.
Computational modeling helps to explain the SAR with the
major H-bonding occurring from the amino-pyrazole of the
compounds to GSK3's backbone Asp 133 and Val 135. In a
kinase selectivity panel, excellent selecti-vity was observed
except for CDK2 which has a closely related homology at
the ATP binding site. Further refine-ments based on the Xray crystal structure-based design led to the highly potent
(GSK3α IC 50 = 22nM) compound 42 which takes advantage
of a H-bond to a structural water in the active site [139]. The
introduction of a dimethylamine solublizing group to the C-3
amide lead to vastly improved CDK2 selectivity. Compound
42 was nearly 700 fold more selective for GSK3 over CDK2
while maintaining it's excellent selectivity in the kinase
panel. Interestingly, a series of 4, 7-dihydro-2H-pyrazolo[3,
4-b]pyridines (43) was filed in an application by Sanofi-Synthelabo, Fr./Mitsubishi-Tokyo Pharmaceuticals, Inc [140].
These compounds while structurally comparable to 40 could
conceivably undergo oxidative aromatization to produce 1Hpyrazolo[3, 4-b]pyridines.
1, 3, 4- AND 1, 2, 5-OXADIAZOLES
Novo Nordisk has published two approaches to discovery
of novel GSK3 inhibitors. Using a virtual screening approach,
a novel class of 1, 3, 4-oxadiazoles was identified with IC50's
below 1uM. The method described was derived from the
CATS (Chemically Advanced Template Search) molecular
descriptor, which is a technique used to compare the topological pattern of atoms and functional groups in a pharmacophore. Through iterative refinement, libraries of compounds
made on solid-support defined the SAR and lead to the most
potent compound 45, IC 50 = 390nM [142]. In a second program, optimization was based on an HTS-derived screening
hit. A series of potent 1, 2, 5-oxadiazoles was explored [143,
144] and medicinal chemistry quickly established the requirement for a para-substituted pyridine to ensure low IC 50s. This
group was attached to the main scaffold through a hydrazide
which was subsequently replaced with an amide bond. Later
improvements included the substitution of a triazole for the
amide which improved potency and could potentially
increase solubility. Compound 46 was found to be highly
potent (IC 50 = 280nM) and selective (~100 fold) for GSK3β
over CDK2. Compound 46 and 2 other lead compounds were
used to characterize the GSK3 activity of the series. All were
proficient at stimulating glycogen synthase activity and βcatenin protein expression in CHO-IR cells. Furthermore
these compounds were able to increase glycogen synthase
activity in isolated rat soleus muscle. This series exhibited
good selectivity against a panel of 31 kinases.
CYCLIN-DEPENDENT KINASE INHIBITORS WITH
GSK3 ACTIVITY
A series of pyrazolo[3, 4-b]quinoxalines were developed
as inhibitors of cyclin-dependent kinases, such as CDK1/
cyclin B and CDK5/p25 [141]. These compounds would be
expected to exhibit similar H-bond donor and acceptor interactions to the pyrazolopyridines (above). The most potent
compound in this class 44 inhibited CDK1/cyclin B with an
NON-ATP COMPETITIVE THIADIAZOLIDINONES
GSK3 INHIBITORS
A family of thiadiazolidinones has been described as
having potential for treating Alzheimer's disease pathology.
O
O
HN
4
5
N
6
F
N
3
N2
N
H
N
41
40
N
39
N
F
N
H
N
N
H1
N
7
HN
NH 2
NH
O
HN
Cl
NH 2
F
F
HN
N
N
N
H
N
42
N
N
F
NMe2
O
N
H
N
O
43
44
1122
Current Pharmaceutical Design, 2004, Vol. 10, No. 10
Wagman et al.
These compounds show non-competitive inhibitor kinetics in
relation to varied ATP or inhibitor concentrations in contrast
to the ATP competitive comparator, Ro 318220 (1) [145,
146]. While the inhibition of GSK3β was modest (IC50's in
the low uM range see 47 below, IC50 = 2uM), non-competitive inhibitors may show marked differences in activity
between substrates and might also effect GSK3's ability to
form protein-protein associations in complexes necessary to
initiate tau phosphorylation or the Wnt pathway through
presenilin 1. These compounds did show excellent selectivity
with no detectable inhibition of CDK1/cyclin B, CK-II, PKA
or PKC at 100uM. It will be interesting to determine if these
thiadiazolidinones suppress tau hyperphosphorylation in
whole cell studies.
MISCELLANEOUS GSK3 INHIBITORS
Several patent applications from Vertex have published
describing small molecule GSK3 inhibitors. A recent application discloses a scaffold which incorporates a pyrimidine /
pyridine moiety [149]. Compound 50 was reported to have a
Ki <100nM. Also disclosed were pyrazolone 51 [150],
pyrazolamine derivatives 52 as inhibitors of GSK3, Aurora-2
and CDK-2 [151-158] and triazoles such as 53 with Ki's
<100nM for GSK3β [159].
Janssen Pharmaceutica has filed patent applications on
several series of GSK3 inhibitor all based on substituted
amino pyrimidines 54 [160], 55 [161] and 56 [162].
Sanofi-Synthelabo, Fr./Mitsubishi-Tokyo Pharmaceuticals, Inc [163] most recently published a patent application
describing a rotationally restricted analog 57 of their earlier
series 59. 2-pyrimidinyl- 6, 7, 8, 9- tetrahydropyrimido [1, 2-a]
pyrimidin- 4-ones 57 and 7-pyrimidinyl- 2, 3-dihydroimidazo
[1, 2-a]pyrimidin- 5(1H)- ones were specifically disclosed to
be useful for the treatment of neurodegenerative diseases. In
this case, the C-7 pyridinyl group was replaced with a pyrimidinyl group. The C-7 puridinyl 2, 3-dihydroimidazo analogues 58 were also specifically mentioned for their potential
in the treatment of neurodegenerative diseases [164]. The
remainder of the published intellectual property consists of
MUSCARINIC AGONISTS AS INHIBITORS OF GSK3
M1 muscarinic agonists are used as therapeutics to
enhance cognition, but also have an affect on decreasing tau
hyperphosphorylation in Alzheimer's disease [147]. Two of
this class of agonist AF102B (48) and AF150 (49) have been
shown to reduce tau hyperphosphorylation through a GSK3
inhibition mechanism in cell studies [148]. There may be a
potential for synergy between classic GSK3 inhibitors along
with muscarinic agonists or the non-competitive thiadiazolidinones for the treatment of Alzheimer's disease.
S
N
N
N
N
N
N
O
N
I
N
O
N
N
N
N
N
45
O
O
F3 C
46
48: AF102B
47
49: AF150
50
N
Me
H
N
H
N
O
N
CN
N
N
N
NH 2
N
N
S
S
N
O
O
NH
H
N
HN
N
NH
N
HN
N
HN
Me
HN
N
F3 C
N
Cl
O
O
O
Me
N
N
53
52
51
N
O
O
N
N
O
N
H 2N
HN
NH
H
N
O
NH
O
N
N
N
HN
54
N
55
O-
HN
N+
O
56
N
Discovery and Development of GSK3 Inhibitors
Current Pharmaceutical Design, 2004, Vol. 10, No. 10 1123
the parent pyrimidones 59 claimed as potent GSK3β
inhibitors for use as medicaments for prevention and/or
treatment of diabetes, diabetic complications and neurodegenerative diseases or as immunopotentiators [165-171].
5-a]-1, 3, 5-triazine derivatives 67 with activity as CDK and
GSK3 inhibitors [179]. These compounds were reported to
inhibit the phosphorylation of histone H1 by CDK1/cyclin
B1 in vitro. In a test for anti-proliferative activity against
cancer cells, all compounds tested had activity against
human pancreatic cancer cells Mia-PaCa2, and some had
similar activity against human prostate cancer cells DU-145.
Other applications claiming methods of treatment of
conditions involving GSK3 include a method of antisense
modulation of GSK3 α expression by Isis Pharmaceuticals,
Inc. [180] and peptide inhibitors specific for GSK3 by an
early researcher in the field, Prof. H. Eldar-Finkleman [181,
182].
Pfizer has filed patent applications on acylaminoimidazoles, for example 60, as inhibitors of CDK5, CDK2, and
GSK3 [172]. Also published are thiazole derivatives, for
example 61, as inhibitors of CDKs and GSK3 for treating
cancer, neurodegenerative diseases and conditions affected
by dopamine neurotransmission. [173].
Thiazoles were also the basis for two GSK3 patent
applications by Novo Nordisk, although the thiazoles were
more substituted than those of Pfizer, for example 62 and 63
[174, 175].
PHARMACOLOGY OF GSK3 INHIBITORS
Other than the maleimides and pyrazolo[3, 4-c]pyridines,
GSK has an application claiming triarylimidazoles for the
treatment of diabetes [176]. The compound 64 is reported to
show an IC50 value in the range of 50 nM against GSK3β.
Abnormal Glucose Metabolism in Type 2 Diabetes and
Obesity
Diabetes mellitus afflicts an estimated 17 million Americans and can lead to blindness, renal failure, limb amputations, and cardiovascular disease. The majority (>90%) of
diabetics have type 2 diabetes which is characterized by
resistance to insulin-mediated glucose metabolism and
defective insulin secretion, see reviews [183-187]. Insulin
resistance, which is also observed in obesity and metabolic
syndrome (syndrome X) [188, 189], results in impaired
Astrazeneca has filed patent applications on two series of
structures for use in conditions involving the inhibition of
GSK3, pyrazine-2-carboxamides 65 [177] and anilinopyrimidines 66 [178].
The Societe De Conseils De Recherches Et D'applications Scientifiques (S.C.R.A.S.), Fr. has disclosed pyrazolo[1,
N
N
R
N
N
N
N
N
O
N
N
58
R
NH
N
N
O
57
AcHN
N
R
NH
NH
O
N
O
59
60
O
O
N
O
O
Boc
S
S
N
N
H
N
61
O
N
H
S
NH 2
N
N
H
63
N
NH 2
N
F
N
N
O
S
O2
O
N
H
N
N
HN
NH 2
N
62
N
N
N
Ph
N
N
65
N
HN
Br
64
Br
S
O
N
N
66
N
HN
N
N
N
67
1124
Current Pharmaceutical Design, 2004, Vol. 10, No. 10
glucose uptake and/or metabolism by the key regulatory
peripheral tissues (muscle, adipose, and liver). One manifestation of impaired glucoregulation in these individuals is
glucose intolerance which may progress to overt hyperglycemia. Mechanisms for impairment of glucose transport and
glycogenesis in type 2 diabetics has been the subject of keen
investigation for many years. A key concept of the diabetic
phenotype, which is pertinent to studies of GSK3, is that
muscle glycogen synthesis is the major pathway for glucose
metabolism in the body and is defective in type 2 diabetics
[186]. While the primary basis for sub-optimal glycogenesis
in type 2 diabetics may be linked to impaired glucose transport [190], it is apparent that rates of both glucose uptake
and glycogen synthase (GS) can be limiting for glycogen
accumulation [191]. In the review of pharmacological data
supporting the use of GSK3 inhibitors in the treatment of
type 2 diabetes, we will focus on studies carried out at Chiron
corporation using compounds from the pyridine / pyrimidide
class.
DISREGULATED GSK3 AND GS IN HUMAN TYPE 2
DIABETES
Aside from the work described above with various models
systems, a compelling set of studies from the laboratories of
Drs. Shulman and Henry provided strong rationale for considering GSK3 as a target for novel therapeutic development.
The investigators first showed that muscle tissue from type 2
diabetics, in comparison to normal tissue, had reduced
glycogen content that correlated with decreased glycogen
synthase activity and impaired GS responsiveness to insulin
[192, 193]. A myocyte culture system was established which
retained the GS characteristics of normal vs diabetic subjects
and which exhibited appropriate coupling of glucose
transport and glycogenesis [194, 195]. While the researchers
had postulated early on that the reduced GS activity might be
related to downregulation by kinase-mediated phosphorylation, the link was not solidified until a strong association
with GSK3 was established [196]. They showed that in
obese, glucose-intolerant subjects, as well as type 2 diabetics, the reduced GS activity, and not protein level, correlated
with an increase in total GSK3 activity that was related to
increased α and β isoform levels.
The link between GSK3 and attenuated GS activity was
further supported by a series of reports using lithium [78].
For example, it has been shown that lithium reduced hyperglycemia in a diabetic patient [197] and improved insulin
sensitivity in diabetic rats [198]. Lithium was also shown to
increase hepatic glycogen synthesis in diabetic rats [199].
More recently, inhibition of GSK3 by lithium was shown to
increase both glucose transport and glycogen synthase
activities in 3T3-L1 adipocytes [200].
PHARMACOLOGICAL PROOF-OF-CONCEPT
STRATEGY
When discovery and optimization of GSK3 inhibitors
was initiated at Chiron, there was no validation that potent
and selective GSK3 inhibitors might represent effective
therapeutics for type 2 diabetes. Hence, a proof-of-concept
program was assembled involving both internal and external
efforts. In vitro and in vivo models of glucose metabolism
Wagman et al.
were established internally and collaborations were secured
with a number of laboratories as follows: Drs. Gary Cline
and Gerald Shulman applied quantitative NMR methodology
to analyze glucose metabolism in intact ZDF Fa/Fa rats, Drs.
Robert Henry and Ted Ciaraldi investigated effects of inhibitors in human muscle cell cultures, Dr. Erik Henricksen
studied muscle biopsies from normal or diabetic rats, Dr.
Ormond MacDougald studied adipocyte regulation, and supplementary animal pharmacology studies were outsourced to
GMI (aka Preclinomics, Indianapolis). All approaches proved
productive and insightful and are described below.
GSK3 INHIBITOR REGULATION OF GLUCOSE
UPTAKE IN VITRO AND EX VIVO
The potent and selective inhibitors CT 98014 and CT
98023 were tested in human skeletal muscle cell cultures
after both acute and chronic exposure [201]. The compounds
rapidly increased glycogen synthase activity in normal and
diabetic tissue of a magnitude greater than either lithium or
insulin. Importantly, glucose transport was likewise upregulated, but in a manner that differed from that of insulin.
Whereas insulin treatment yielded upregulated glucose
transport over a time course of 1-12 hr, Chiron inhibitor
treatment increased glucose uptake to a magnitude slightly
greater than insulin (or lithium), but with delayed kinetics
(i.e., a plateau extending from 24-96 hr of treatment).
Interestingly, such upregulation was associated with a
decrease in GSK3 protein levels and increase in IRS-1
protein levels - both of which would be desirable events
should such a response be observed in the treatment of
human diabetics. As a control, the inhibitor responses were
shown to be specific for GSK3 inhibition as a structurallyrelated, but inactive compound had no effect.
Similar studies were also performed with isolated muscles
from lean and obese, diabetic ZDF fa/fa GMI rats, the latter
of which are insulin-resistant and well-established as perhaps
the premier animal model for type 2 diabetes [202, 203],
reviewed in [204, 205]. Consistent with the human muscle
cell cultures, ZDF rat muscle (following treatment ex vivo or
after oral administration with ex vivo analysis) exhibited an
increase in GS activity of a magnitude greater than insulin
alone, and also showed an enhanced response upon exposure
to an inhibitor/insulin combination [115, 201, 206]. Moreover, an upregulation of glucose transport in the presence of
insulin following GSK3 inhibitor treatment was observed,
although, the kinetics were not delayed in the same fashion
as observed in the human muscle cultures. Notably, unlike
the human muscle cell cultures wherein similar upregulation
of glucose transport was apparent in normal and diabetic
tissue, GSK3 inhibitor modulation of ZDF rat glucose uptake
was stimulated in diabetic, but not lean rat muscle [206].
Hence, the overall results with validated models in vitro and
ex vivo supported the potential use of GSK3 inhibitors for
the improvement of glucose metabolism in type 2 diabetes.
ANIMAL PHARMACOLOGY APPROACH
Given the novelty of GSK3 as a target for treating
diabetes as well as the unique chemical structures identified
and optimized, we reasoned that a broad range of animal
models should be investigated for at least two reasons: First,
Discovery and Development of GSK3 Inhibitors
to provide confidence in the target and leads, and second, to
guide clinical development through thorough characterization
of the pharmacological profile. Hence, we utilized several
mouse models of obesity and type 2 diabetes including ob/ob
mice, db/db mice, and a “nongenetic” model of STZ/dietinduced type 2 diabetes [207, 208]. Such models were
appropriate as they have been well validated for predictive
value of other therapeutics. Furthermore, disregulation of a
similar nature to that in the animals models was reported in
diabetic humans. In particular, it was shown that C57BL/6J
mice which are susceptible to diet-induced obesity and
diabetes exhibited a two-fold increase in adipose GSK3
activity [209]. Additionally, data presented at a Keystone
symposium demonstrated that ob/ob mice exhibited an
increase in muscle GSK3 activity which correlated with
reduced GS activity and glucose intolerance [210].
Chiron studies principally employed diabetic ZDF fa/fa
rats. This model is well-established with defined and relevant biochemical and physiological endpoints [202-204, 207,
211]. A potential link to GSK3 stems from a report that the
ZDF fatty rats exhibit downregulated IRS-1 and IRS-2 in
liver and muscle [212]. Additionally, Brozinick et al. have
presented that obese, but not lean, ZDF fa/fa rats exhibited
an increase in basal GSK3 activity in skeletal muscle [213].
Additionally, we performed limited studies in obese, glucoseintolerant SHHF rats (GMI) and even lean STZ-treated rats;
a model of type 1 diabetes with glucose intolerance.
Current Pharmaceutical Design, 2004, Vol. 10, No. 10 1125
6A Plasma exposure profile
Plasma Insulin (ng/ml)
Time
Control
30 mg/kg
-300
7.7
8.2
-10
7.3
7.8
25
9.4
8.4
6B Improved glucose tolerance with insulin conservation .
To further validate both the concept and lead molecules,
we performed a series of studies in obese, diabetic rhesus
monkeys in collaboration with Dr. Barbara Hansen for the
evaluation of pharmacokinetics and pharmacodynamics in
primates [214].
ORAL EFFICACY IN MOUSE MODELS OF TYPE 2
DIABETES
Initial evaluations of the oral pharmacokinetics of CT
99021 in db/db mice showed oral bioavailability of ~25%
with plasma concentrations at the tested dose in a predicted
efficacious range based on in vitro potency (Fig. 6A). Impressive in vitro and ex vivo data led us to reason that it might
be feasible to observe acute efficacy upon oral administration
of the compounds. We also identified early on that the oral
glucose tolerance test (oGTT), a common screen in animals
and man for impaired glucose tolerance, showed responsiveness to GSK3 inhibitors after acute exposure. However, as
the plasma half-life of CT 99021, and other similar molecules being screened, often ranged 1-2 hr in rodents we
chose to dose twice prior to the oGTT (at 4.5 and 0.5 hr prior
to glucose administration) to improve the sensitivity of the
assay as well as to allow responsiveness to compounds of
varying pharmacokinetics.
As indicated in (Fig. 6B), oral administration of CT
99021 to db/db mice prior to an oral glucose challenge
yielded a dose-related improvement in glucose disposal. At
the higher dose, there was also a trend for reduced hyperglycemia prior to the glucose administration. As plasma
insulin levels in the CT 99021-treated animals were similar,
or even possibly conserved, relative to vehicle controls and
as glucose levels in the urine were not elevated in treated
rodents (not shown), it was concluded that there was an
Fig. (6). Oral exposure and efficacy of CT 99021 administered
to db/db mice. A) Plasma concentration vs time profile. 50 mg/kg
CT 99021 formulated in 15% captisol administered once orally to
8-9 week old female db/db mice. Quantitation by LC/MS/MS.
Results are mean + sem from 6 animals. B) Improved glucose
tolerance with insulin conservation. Treatment with vehicle control
(10% captisol) or indicated doses of CT 99021 at -4.5 and -0.5 hr
relative to t = 0 oral glucose. Groups of 6-8 mice per treatment
fasted for ~6 hr prior to a oGTT. SEM < 15%. Experimental
methods as described in [115].
improvement in whole body glucose disposal. Subcutaneous
administration of another potent, but poorly orally bioavailable, compound (CT 98014) also exhibited improved glucose
tolerance [115]. The magnitude of efficacy in such studies
1126
Current Pharmaceutical Design, 2004, Vol. 10, No. 10
could be quantified as the reduced glucose AUC for comparison between GSK3 inhibitors and reference therapeutics
such as troglitazone, rosiglitazone, and metformin. The
glucose AUC reduction of the 30 mg/kg regimen in the
oGTT shown is of a magnitude similar to that observed
following a metformin or troglitazone regimen. Notably,
such efficacy by those therapeutics can not be observed
within the first day of administration and typically require 12 weeks of daily dosing.
Employing the same test compound and regimen, ob/ob
mice were likewise tested in an oGTT model and, as indicated in (Fig. 7), improved glucose tolerance in an insulinconserving manner was observed. Within the age ranges
tested, the ob/ob mice are less hyperglycemic and insulin
7A Improved glucose tolerance
Wagman et al.
resistant than the db/db mice and generally exhibited slightly
greater efficacy upon comparison.
Finally, we chose to extend the demonstration of acute
oGTT efficacy to a mouse model not linked to a leptin or
leptin receptor deficiency characteristic of ob/ob or db/db
mice respectively [207]. Consequently we applied an alternative model based on a high fat diet and intermittent
streptozotocin treatment wherein animals progress to glucose
intolerance and then overt diabetes [208] as is observed in
man. Oral administration of CT 99021 twice prior to oral
glucose challenge showed both a reduction in hyperglycemia
and an impressive (53%) reduction in post-challenge glucose
AUC (Fig. 8). The improved glucose disposal in the GSK3treated mice occurred without an increase in peripheral
7B Insulin conservation
Fig. (7). Improved glucose tolerance with insulin sparing in ob/ob mice treated orally with CT 99021. A) Improved glucose tolerance.
oGTT as described in figure 1 with 9-10 week old ob/ob mice. n = 6-8 animals/group, mean + sem. B) Insulin conservation. Plasma insulin
levels (n = 6-8 animals/group, mean + sem).
Fig. (8). Reduced hyperglycemia and improved glucose tolerance in nongenetic diabetic mice. Male C57BL/6J mice exposed to high fat
diet and treated with STZ as described in [208]. Vehicle control (15% captisol) or CT 99021 administered orally at 4.5 and 0.5 hr prior to
oGTT. n = 8 animals/group, mean + sem.
Discovery and Development of GSK3 Inhibitors
Current Pharmaceutical Design, 2004, Vol. 10, No. 10 1127
insulin levels (not shown) consistent with the ob/ob and
db/db mouse oGTT results. Furthermore, the monitoring of
blood glucose levels for an extended (up to 20 hr) period of
time following vehicle control vs GSK3 inhibitor treatment
in some of these models indicated that there was no compensatory rebound hyperglycemia in the treated animals (not
shown).
[115]. We addressed the specificity of the GSK3 inhibitor
oGTT response to insulin-resistant, glucose-intolerant animals
rather than to normal animals using lean littermates of 10week-old obese, mildly diabetic animals. As seen in (Fig. 9A
and 9B), the metabolically impaired animals showed normalization of their oral glucose tolerance following CT 99021
administration, whereas the lean littermates showed little
change. [201, 206].
IMPROVED GLUCOSE TOLERANCE AND REDUCED
HYPERGLYCEMIA IN RATS
A more recently derived rat model of obesity and glucoseintolerance uses SHHF rats (Preclinomics/Charles River,
Indianapolis IN) which also exhibit congestive heart failure.
As the metabolic abnormality in SHHF rats is not linked to a
leptin defect, it represented an additional element of concept
validation. SHHF rats, like the nongenetic diabetic mice,
showed an impressive reduction in hyperglycemia and
improved glucose tolerance upon CT 99021 treatment (Fig.
10).
We extended the pharmacological evaluation to appropriate rat models of obesity and diabetes. CT 99021 was
assessed in 12-week-old ZDF fa/fa obese, diabetic rats for
alteration of glucose disposal in a oGTT model as described
above. At dose levels of 30-48 mg/kg administered orally, a
significant improvement of glucose disposal accompanied by
insulin sparing relative to controls was observed (Fig. 9A)
9A Obese ZDF rats
Plasma Insulin (ng/ml + SEM)
Time (min)
Control
99021
-250
7.2+0.6
6.3+0.3
-45
6.9+0.3
5.3+0.3
25
8.5+0.7
4.8+0.2
60
7.8+0.8
5.2+0.5
9B Lean littermates
Plasma Insulin (ng/ml + SEM)
Time (min)
Control
99021
-250
0.2+0.1
0.3+0.1
-45
0.2+0.1
0.3+0.1
25
0.4+0.1
0.6+0.1
60
0.3+0.1
0.4+0.1
Fig. (9). Differential responsiveness of obese vs lean ZDF rats to oral treatment with CT 99021. A) Obese ZDF rats. 10 week-old ZDF
fa/fa rats fasted overnight and treated with vehicle control (captisol solution) or 30 mg/kg CT 99021 4.5 and 0.5 hr prior to oGTT. Methods
as described above, n = 8/group, results are mean + sem. B) Lean littermates. Same as in A except age-matched lean littermates.
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Current Pharmaceutical Design, 2004, Vol. 10, No. 10
Wagman et al.
11A Reduced hyperglycemia
Fig. (10). Improved glucose disposal in obese SHHF rats
treated orally with CT 99021. Same study design as
described in figure 4 except with 12 week-old obese and
glucose-intolerant GMI SHHF rats. n = 8/group, results are
mean + sem. Note reduced hyperglycemia after a single oral
exposure prior to 2nd oral dose and subsequent oGTT.
To better focus upon the regulation of glucose metabolism independent of glucose challenges, a series of studies
were performed in ZDF rats following either acute or multiday sustained exposure to CT 99021. In acute studies, the
GSK3 inhibitor was orally administered once to ZDF fa/fa
rats with subsequent monitoring of plasma glucose and
insulin levels (Fig. 11) [17]. A dose-related and significant
(at 30 mg/kg) reduction in hyperglycemia was observed
which initiated within 60 min of administration and yielded a
maximum reduction of >150 mg/dL. CT 99021-treated
animals exhibited a trend for insulin lowering relative to the
vehicle controls (Fig. 11B). Notably, the pharmacodynamics
correlated well with the pharmacokinetics as the onset of
glucose lowering coincided with plasma CT 99021 levels
approaching maximal levels (Cmax) and with attenuation of
the response coinciding with compound elimination (Fig. 1
and data not shown). In the longer-term studies, CT 99021
was continuously infused intravenously over a 4-day period
at a total dose of ~92 mg/kg/d in order to ascertain the impact
of high, sustained GSK3 inhibitor exposure on circulating
glucose and insulin levels. Interestingly, a significant reduction in fasting hyperglycemia to nearly normoglycemic was
observed without a significant change in plasma insulin
(Table 2).
11B Insulin conservation
Fig. (11). Oral CT 99021 reduces hyperglycemia with insulin
conservation in ZDF Fa/Fa rats. A) Reduced hyperglycemia. 12
week old male ZDF fa/fa rats with food removal 3-4 hr prior to
single oral administration of vehicle control (15% captisol) or
indicated doses of CT 99021. Serial tail-snip bleeds from n =
8/group. B) Insulin conservation. Plasma insulin levels in same
animals. Results are mean + sem.
Table 2.
QUANTITATIVE ANALYSIS OF GLUCOSE METABOLISM IN GSK3 INHIBITOR-TREATED ZDF RATS
To provide insights into the mechanisms for glucoregulation by GSK3 inhibitor treatment in vivo 13C-NMR spectroscopy was applied. Glucose metabolism was studied in ZDF
rats treated orally with CT 98023 prior to a 13C-oGTT and in
hyperinsulinemic-euglycemic clamps following either acute
(2-4 hr) or more chronic (20-24 hr) intravenous infusion of
CT 99021. Regardless of the compound or regimen, the
results suggested that hepatic glycogen formation was a
primary disposal mechanism concomitant with reduced
Effect of Sustained CT 99021 Exposure1 on ZDF
Fa/Fa Rat Glycemia and Insulinemia
Parameter
Time
Vehicle
CT 99021
Glucose 2
(mg/dL)
Pretreatment
Day 4
188 + 18
181 + 20
223 + 18
116 + 6
Insulin 2
(ng/ml)
Pretreatment
Day 4
3.5 + 0.4
4.0 + 0.4
3.9 + 0.6
3.1 + 0.4
1
Intravenous prime (5 mg/kg) with continuous infusion at 64 ug/kg/min for 4 days to
12-13 week old ZDF Fa/Fa rats.
2
Plasma samples from overnight-fasted animals.
endogenous glucose production [205]. Specifically, in the
oGTT studies, CT 98023 mediated a 41% reduced glucose
Discovery and Development of GSK3 Inhibitors
AUC with insulin sparing (26% reduced AUC) accompanied
by a ~2-fold increase in hepatic glycogen formation and no
significant increase in muscle glycogen content despite a ~3fold increase in GS activity. Likewise, in glucose clamp
studies, a 2-3 fold increase in liver glycogen formation was
observed, with a concomitant ~40% reduction in basal endogenous glucose output. Muscle GS activation was observed,
although increases in glycogen formation were minimal.
Hence, under these conditions, GSK3 inhibitor administration was shown for the first time to exhibit significant antidiabetic efficacy in vivo with a predominant effect on liver
glucose metabolism. Glucose push in concert with activated
GS likely accounted for increased glycogen synthesis [205].
Reduced hepatic glucose output may likewise be direct as
other investigators demonstrated downregulation of expression of the gluconeogenic enzymes (see above) [215].
Acute ex vivo glucose uptake studies with obese, diabetic
ZDF fa/fa muscle [115, 206] and human cells [201] would
support the use of GSK3 inhibitors for the treatment of human
diabetes. On the contrary the glucose clamp studies described
above argue against human efficacy. This issue therefore
remains an open question and awaits testing in man.
Current Pharmaceutical Design, 2004, Vol. 10, No. 10 1129
12A Improved glucose tolerance
12B Insulin sparing
PHARMACOLOGICAL PROFILE IN RATS AND
PRIMATES OF THE POTENT GSK3 INHIBITOR, CT
20026
The GSK3 inhibitor optimization cycle yielded a compound, CT 20026, with very good in vitro potency and selectivity. In initial animal screens it proved orally bioavailable
and potent in vivo. Administration once via oral gavage to
mildly diabetic ZDF rats 30 or 60 min prior to an oGTT
yielded dose-related, significantly improved glucose disposal
in an insulin-sparing manner (Fig. 12A, 12B), at a dose as
low as 16 mg/kg. At a high dose (64 mg/kg), glucose tolerance was completely normalized. The compound showed
similar potency and efficacy in mouse oGTT and reduced
fasting hyperglycemia in ZDF rats and ob/ob mice.
A number of studies were performed to evaluate the
pharmacokinetics and pharmacodynamics of CT 20026 in
rats and the best relationship arose from the correlation of
plasma exposure (CT 20026 AUC) with oGTT efficacy
(reduced glucose AUC). As depicted in (Fig. 13), a linear
relationship (r2 = 0.71) was evident and higher exposure
efficacy equaled that achievable by any therapeutic regimen
including metformin (e.g., 100 mg/kg BID 1 week) or
rosiglitazone (e.g., 5 mg/kg QD 2 weeks).
Applying pharmacological relationships established in
rodents, we advanced CT 20026 into acute efficacy studies
in obese, diabetic rhesus monkeys. The physiology of these
animals is well described the similarities of rhesus type 2
diabetes to that of man have been documented [214]. We
selected two mildly diabetic subjects and two markedly
diabetic subjects. Preliminary pharmacokinetics demonstrated
that the orally dosed compound showed a delayed plasma
Tmax relative to rodents and an increased AUC per given
dose as compared to rodents: a 5 mg/kg oral dose in the
primates approximated the AUC of a 15 mg/kg oral dose in
rats. We selected a relatively low dose (5 mg/kg) for
assessing potential improvements in glucose tolerance. Due
to the substantial variability in fasting glucose and insulin
Fig. (12). Single administration oral efficacy of CT 20026 in
ZDF Fa/Fa rats. A) Improved glucose tolerance. 10 week old male
rats fasted overnight and administered a single oral dose of vehicle
control (10% captisol) or increasing doses of CT 20026 30 min
prior to oGTT. B) Insulin sparing. Plasma insulin levels monitored
during oGTT study in same animals as in A. Results are from n =
8/group, mean + sem.
levels between animals, we expressed the results as the
percent change relative to pre-dose (and pre-oGTT) baselines. As shown in (Fig. 14), single oral administration of CT
20026 prior to an oGTT improved glucose disposal in an
insulin-conserving manner relative to vehicle control responses in the same animals. Importantly, such efficacy was
observed in the mildly diabetic primates, but not in the severely diabetic subjects which exhibited essentially controllevel glucose and insulin responses (data not shown).
The differential magnitudes of glucose-lowering efficacy
of GSK3-inhibitor treatment between mild and advanced
diabetic animals – whether in acute or 1-2 week studies - has
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Current Pharmaceutical Design, 2004, Vol. 10, No. 10
Wagman et al.
14A Improved glucose tolerance
Fig. (13). Oral PK/PD of CT 20026 in the ZDF fa/fa rat oGTT
model. Results shown represent the summary of at least 6 separate
studies in 9-11 week old, fasted male rats (n = 6-8 animals/group).
Animals were treated with different oral dose levels of CT 20026 at
30-60 min prior to a standard oGTT. Plasma samples were analyzed
for glucose and CT 20026 levels in order to compare the
pharmacokinetics (PK) and pharmacodynamics (PD, e.g., glucoselowering). The zone of maximum achievable efficacy pertains to
the range of maximal reduced glucose AUC (trapezoidal) that can
be obtained in this animal model with any treatment. (GSK3
inhibitor or optimal treatment regimens with troglitazone,
rosiglitazone, or metformin).
14B Insulin conservation
also been observed in our mouse and rat models and may be
a general phenomenon. Whether the basis is simply related
to greater insulin resistance in the advanced diabetics or
whether there is a quantal limit of glucose “disposal” which
is proportionally less in the markedly hyperglycemic
subjects is currently unclear.
CHRONIC CT 20026 ADMINISTRATION
METABOLIC REGULATION
AND
To ascertain the therapeutic potential of GSK3 inhibitor
treatment for type 2 diabetes we evaluated long-term
efficacy in ZDF fa/fa rats. We began treating animals at 8-9
weeks for 30 days with CT 20026 at 16 mg/kg each morning
and evening. The regimen was well-tolerated and, as
indicated in (Fig. 15), diabetic progression (as measured by
changes in HbA1c and insulinemia) was prevented in the
treated animals. While the terminal HbA1c was significantly
reduced in the CT 20026-treated animals relative to the
vehicle controls, it is worth noting that treatment with 5
mg/kg rosiglitazone over a similar period lowers glucose
more profoundly (data not shown). Also, in separate studies
with older, more hyperglycemic and insulin-resistant ZDF
rats, the same regimen of CT 20026 failed to significantly
lower HbA1c levels (data not shown).
Paradoxically, insulin levels in the CT 20026-treated
animals trended higher than the vehicle controls over the last
2 weeks of exposure. However, this insulin profile is
consistent with reduced diabetic progression by GSK3inhibitor treatment as it is well-established that ZDF fa/fa
Fig. (14). Oral efficacy of CT 20026 in mildly diabetic obese
rhesus monkeys. A) Improved glucose tolerance. Two obese male
rhesus rhesus monkeys with fasting plasma glucose levels 101 and
129 mg/dL (normoglycemic rhesus = 65-70 mg/dL) were given a
single oral gavage of vehicle control (5% HPBCD solution) or 5
mg/kg CT 20026 1-2hr prior to an oGTT. Results are the average
of the two animals from the pre-dose baseline level. B) Insulin
conservation. Results are the average plasma insulin levels of the
two animals from the pre-dose baseline level (139 and 58 ng/ml,
respectively).
rats, like humans, become insulinopenic with advancing
diabetes. Additional support for such an interpretation of
insulin dynamics comes from the observation that chronic
rosiglitazone treatment of ZDF fa/fa rats prevents the loss in
beta-cell mass thereby sustaining insulin levels [216]. Hence,
GSK3-inhibitor treatment prevents both the increase in
hyperglycemia and reduction of insulinemia observed in
developing diabetes in ZDF rats.
Additional endpoints were followed in the chronic ZDF
rat study. At termination, there was no significant change in
lipid or cholesterol profiles nor other clinical chemistries
relative to the vehicle controls. Muscle glycogen content
Discovery and Development of GSK3 Inhibitors
increased slightly and liver glycogen content was significantly
increased by 49%. This is a physiologically meaningful
increased and likely to be beneficial. Finally, there was a
trend for increased body weight in the CT 20026-treated rats
(Fig. 15C) that is also consistent with improved metabolism.
Similar increases in weight gain can be observed in chronic
rodent (and human) studies with other effective diabetes
therapeutics.
15A HbA1c change
Current Pharmaceutical Design, 2004, Vol. 10, No. 10 1131
ALTERNATIVE POTENTIAL EFFECTS OF GSK3INHIBITOR TREATMENT
As GSK3 may participate in a number of regulatory
processes (for review see: [9, 10, 27, 76]), it is possible that
sustained treatment with a GSK3 inhibitor may have
therapeutic utility in other diseases, as well as potentially
undesirable side effects. Other therapeutic utilities may
include the treatment of neurodegenerative diseases like
Alzheimers or ischemia-reperfusion injury [217].
Alternatively, potential upregulation of β-catenin may
predispose recipients to certain types of cancer (for review
see: [9, 10, 27, 76]). We have chosen to focus upon potential
applications of GSK3 inhibitors for metabolic disorders in
this review. Nonetheless, we note that chronic lithium
treatment has not been associated with tumorigenesis in man.
POSITIONING GSK3 INHIBITORS IN THE CLINIC
AS METABOLIC DISEASE THERAPEUTICS
15B Plasma insulin
15C Body weight change
As presented here, GSK3 inhibitors clearly have potential
as novel therapeutics for the treatment of certain metabolic
diseases. The pharmacological profile and non-clinical
validation supports their use as a insulin-mimetics or insulinsensitizers. Despite the advances in diabetes therapeutics in
recent years, there is still a tremendous unmet medical need
– particularly for agents with novel modes of action. While
GSK3 inhibitors may act alone, they would likely be
beneficial in combination with insulin or other “sensitizers”
such as thiazolidinediones or metformin. In fact,
combination studies in rodent models, enhanced glucose
lowering efficacy observed in the combination groups (data
not shown).
A strategic requirement for potential clinical
development is identification of the patient type likely to be
most responsive to the drug candidate. As described above in
multiple animal species and in either acute or chronic
settings, the glucose lowering efficacy of the Chiron GSK3
inhibitors was most evident in glucose-intolerant animals
ranging from the pre-diabetic to moderately diabetic stage.
Aside from regulation of glucose metabolism, GSK3
inhibitors may also have direct effects on obesity via
regulation of adipocyte function [10, 218, 219]. Accordingly,
we would propose that GSK3 inhibitor treatment is best
suited for pre-diabetic obese and/or metabolic syndrome
patients and early-stage type 2 diabetics. Moreover, given
the efficacy of GSK3 inhibitors in improving post-prandial
glucose disposal and potentially reducing hepatic glucose
output, we anticipate that an optimal dosing regimen would
provide effects throughout the day and night.
ACKNOWLEDGEMENTS
Fig. (15). Efficacy of oral CT 20026 administered for 1 month to
ZDF fa/fa rats. Treatment of 8 animals/group beginning at 8-9
weeks of age with control (0.5% CMC suspension) or 16 mg/kg CT
20026 every morning and evening for 30 days. A) HbA1c change.
B) Plasma insulin. C) Body weight change. Results are mean + sem.
We are grateful for the contributions from our:
Collaborators: R. Henry, T. Ciaraldi, G. Shulman, G. Cline,
B. Hansen, E. Henricksen. Advisors: G. Reaven, R. Sherwin.
Colleagues: S. Harrison, D. Ring, R. Johnson, E. Tozzo, L.
Seely, J. Plattner, M. Desai, I. Samuels, G. Dollinger, K.
Wang. Special thanks to Dr. Gary Cline, Yale, for critical
review of the pharmacology section of this manuscript, and
to Dr. Steven Harrison, for critical review of the completed
manuscript and for useful discussions.
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Current Pharmaceutical Design, 2004, Vol. 10, No. 10
Wagman et al.
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