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Lessons Learnt from Glycogen Synthase Kinase 3 Inhibitors Development for Alzheimer’s Disease Ana Martinez*, Daniel I. Perez and Carmen Gil Instituto de Quimica Médica-CSIC, Juan de la Cierva 3, 28006 Madrid, Spain Abstract: Glycogen synthase kinase 3 (GSK-3) inhibitors have aroused a great interest for medicinal chemists and pharmaceutical companies in the last years. In fact, some candidates have reached to clinical trials as disease modifying drugs for Alzheimer’s disease. This review will cover the great improvements recently done in the field of GSK-3 inhibitors switching from random discovery to rational drug design, from full GSK-3 inhibition to mild and controlled activity of enzyme reduction, from unknown therapeutic potential to validated efficacy in different animal models of diseases. Moreover some lessons learnt from clinical trials will be described with the aim to improve future designs. Collective results highlight the importance of mild GSK-3 inhibitors as innovative drugs for severe human unmet diseases.
Keywords: GSK-3 inhibitors, neurodegenerative diseases, Alzheimer’s disease, clinical trials, drug design. 1. INTRODUCTION Alzheimer’s disease (AD), the main neurodegenerative pathology of the central nervous system (CNS), is characterized by the progressive loss of hippocampal and cortical neurons [1]. Main clinical symptoms are cognitive impairment and dementia, and the person is completely dependent upon caregivers during the final stage of AD. Emotional, social and health costs are huge [2]. Today, AD affects more than 24 M of human beings. Moreover, as AD incidence increases exponentially with aging, it represents a severe social and health problem to current society where the population worldwide continues to age [3]. As the etiology of AD remains unknown [4], it is very difficult to discover and/or developed effective therapies as the target to be modulated is not known. Today, only palliative drugs have been approved for AD therapy and a significant unmet medical need exists in the pharmacological treatment and/or prevention of this devastating condition [5]. During the last forty years several hypothesis to explain the cause of AD have been proposed, fueling current molecular and pharmacological research. Basically they are focused on the patient’s clinical symptoms and/or on the different histopathological lesions found in AD patient’s brain. Thus, the first one was the cholinergic hypothesis where the cholinergic neurons die progressively with the advance of the disease. This theory considers the pathology as an acetylcholine deficit consequence [6]. This neurotransmitter is physiologically implicated in the cognitive process and low levels of it may produce its decline. In 1993 the first acetylcholinesterase inhibitor (AChEI), tacrine, was approved in US as palliative treatment for AD [7]. Today we *Address correspondence to this author at the Instituto de Quimica MédicaCSIC, Juan de la Cierva 3, 28006 Madrid (Spain); Tel: +34 91 5680010; Fax: +34 91 5644853; E-mail:
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have on the market four drugs approved with the same mechanism of action. AChEIs slight increase short term memory and cognition on patients with the subsequent improvement of their quality of life, but the neurodegenerative disease does not stop [8]. Simultaneously to the start of cholinergic-based pharmaceutical research, the amyloid cascade hypothesis emerged being the most long considered etiological theory for AD [9]. It was postulated that aberrant beta-amyloid overproduction is responsible for the neurotoxicity which leads to the progressive neuronal death and the senile plaque formation. However, it is not clear if beta-amyloid is cause or risk factor for AD pathology [10]. This controversy is enhanced recently because of the lack of efficacy in clinical trials phase III of several drugs specifically designed to interfere within the amyloid cascade. Such is the case of tramiprosate [11], tarenflurbil [12], semagacestat [13], bapineuzumab [14], and intravenous immunoglobulin [15]. However, there is still an active field of research on different immunotherapies based on beta-amyloid hypothesis [16, 17] as a way to decrease AD incidence and severity by decreasing beta-amyloid brain levels. In the nineties, the tau-based hypothesis was postulated [18]. The ethiology of AD following this hypothesis is the aberrant tau protein, a microtubule associated protein that stabilizes the neuronal cytoskeleton. Physiologically, tau protein is able to bind tubulin and thus it can stabilize neuronal cytoskeleton. However, in different pathologies, an aberrant phosphorylation of tau protein appears leading to its aggregation into neurofibrillary tangle and to the neuronal cytoskeleton decline. Intensive research programs lead to the discovery of two kinases, initially called TPK-I and TPK-II, as responsibles for in vivo tau hyperphosphorylation [21]. After cloning, they were identified as glycogen synthase kinase 3 (GSK-3) and cyclin dependent kinase 5 (CDK-5), respectively [22]. Currently there are only two compounds © 2013 Bentham Science Publishers
Lessons Learnt from Glycogen Synthase Kinase 3 Inhibitors
specifically designed to interfere with tau pathogenesis that have reached clinical trials. They are tideglusib, a GSK-3 inhibitor, and methylene blue, a tau antiaggregation compound. Both compounds have shown positive results in AD patients cognition after pivotal phase II clinical trials [19, 20]. Based on the scientific findings described above, in the last years, the GSK-3 hypothesis for AD came out [23]. It locates on this key kinase the link between amyloid and tau cascade [24]. It has been suggested that long-term aberrant Wnt or insulin signaling result in increased GSK-3 function, leading to the AD fatal events [25]. This hypothesis gains relevance today when growing evidence points to a brain insulin signaling deficit as the cause of AD [26]. In fact, there is no doubt about the GSK-3 up regulation on the brains of AD patients, although it is not clear the cause of its exacerbated activity [27]. Nowadays it is well recognized that elevated GSK-3 activity may induce increased betaamyloid formation and toxicity of senile plaques [28], may explain some of the early cognitive deficiencies observed in AD [29], and may be involved in neuroinflammation and finally neuronal death trough microglia activation [30]. All these observations points directly to GSK-3 as an excellent target to effectively treat all the clinical symptoms present on AD and others neurodegenerative diseases [31]. The last decade has witnessed an intensive research on GSK-3 and its inhibitors. Some small candidates have advanced to clinical trials and the scope of GSK-3 as therapeutic target has been broadened to many unmet and severe human diseases. Great advances have been produced in the area, but some shadows remains to be clearer in the next future. These are the points cover in this review where advances on GSK-3 inhibitors development and future points to increase their success in pharmacological therapy will be discussed. 2. ADVANCES ON GSK-3 AS THERAPEUTIC TARGET Although GSK-3 was originally identified thirty years ago as a regulator of glycogen metabolism [32], nowadays it is recognized its key role in the regulation of numerous signaling pathways including cellular processes such as cell cycle, inflammation and cell proliferation [33]. GSK-3 refers to two paralogs. GSK-3alfa and GSK-3beta, that are commonly referred to as different isoforms because of their similar sequences and functions although they are derived from different genes [33]. They are ubiquitously expressed and involved in a large number of cellular functions [34]. GSK3beta is a highly conserved serine/threonine kinase that has highest abundance in the brain during development and is localized primarily in neurons [35]. Consequently, malfunction of this kinase is involved in the pathogenesis of different human diseases, such as nervous system disorders like AD, diabetes, inflammation, cancer and heart failure. Therefore, GSK-3 has arisen as an attractive pharmacological target for the development of new drugs for several prevalent diseases [36-41]. Drug modulation of GSK-3 may represent a hope for AD [31] and many other severe diseases [42]. The number of GSK-3 inhibitors tested with success in different animal models provides additional support for the therapeutic
Current Topics in Medicinal Chemistry, 2013, Vol. 13, No. 15
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potential of this target (Table 1). However, the only way to validate the potential of GSK-3 inhibitors as effective drugs is the human clinical trials. At the same time, insight into the physiological function of GSK-3 has come to light from genetic analysis in different experimental models [33], although the distinct biological functions of the two isoforms remains largely unknown. GSK-3 has shown itself to be completely necessary for life and disruption of the murine GSK-3beta gene results in embryonic lethality caused by severe liver degeneration during mid-gestation [71]. On the other hand, haploinsufficiency on GSK-3beta is compatible with life and mice lacking one copy of the gene encoding GSK-3beta mimics the behavioral and molecular effects of lithium, a drug widely used for mood disorders [72]. In addition, mice lacking GSK-3alpha demonstrated learning in the passive avoidance task equivalently to wild-type mice, but had an impaired ability to form and consolidate memory in a fear conditioning test [73]. Recently, it was discovered that birds are natural GSK-3alpha knockout organisms and may serve as a novel model to study the distinct functions of GSK-3 isozymes revealing their distinct roles in tau phosphorylation during development [74]. Moreover, overexpression of GSK-3beta on the brain during adulthood on conditional transgenic mice induces tau phosphorylation [75], learning deficits [76], dentate gyrus atrophy [77], neuronal death and depletion of neurogenic niches [78]. The role of GSK-3alpha in AD is up-to-date controversial and it has been related to amyloid pathway [79]. Worthwhile is the fact that overexpression of GSK3beta on skeletal muscle resulted in impaired glucose tolerance in mice [80]. Thus, a gain of function on GSK-3beta in different tissues produces different pathologies, such the above recapitulated AD or diabetes type II. For this reason, to modulate GSK-3 as a pharmacological target and recover the physiological state, a mild GSK-3 inhibitor able to restore aberrant activity to normal levels in certain tissues will be of the utmost importance. In fact, as GSK-3 is constitutively active, ubiquitous and essential for life, its basal activity is regulated by diverse mechanism of action including phosphorylation at different residues leading to inactive or super-active enzyme functions [81]. Thus, phosphorylation on the regulatory serine, serine 21 in GSK-3alpha and serine-9 in GSK-3beta, inhibits the activity of GSK-3. By contrast, phosphorylation on a specific tyrosine, tyrosine-279 in GSK-3alpha and tyrosine-216 in GSK-3beta, overactivates the kinase activity [82]. Our organism is prepared to restore, through compensative mechanism of action, a deficit in the expression and/or activity of the enzyme [83]. However, it is not prepare for down regulate GSK-3 endogenously when this enzyme is exacerbated in different pathological conditions such as AD or diabetes type II. Thus, a smooth inhibition of GSK-3 able to restore down levels of activity to physiological ones would be enough to produce an important therapeutic effect. Based on this approach, when a GSK-3 inhibitor is distributed all over the organism, the subtle inhibition of the kinase will restore to normal level the activity of the kinase in the tissues where it is exacerbated, and decrease a little the activity of GSK-3 in the other tissues where compensatory mechanisms will enter
1810 Current Topics in Medicinal Chemistry, 2013, Vol. 13, No. 15
Table 1.
Disease group
Therapeutic In Vivo Effects of GSK-3 Inhibitors in Different Murine Animal Models of Diseases with Exception of AD (See Figure 1).
Pathology
Compound
Chemical structure
name
phic lateral
O
AR-A04418
sclerosis
N H
Fragil X
In vitro
In vivo
GSK-3
GSK-3
admin.
S N H
N
IC50=
not re-
0.1 M
ported
model
G93A-SOD1 mouse model
Treatment time
Chronic 42 days
In vivo effects
Ref.
Prolongued life span Decreased GSK-3
[43]
activity Reverse hippocam-
H N
O
SB216763 N Me
Dosis/ Animal
4 mg/Kg i.p.
MeO
O
CNS
In vitro
NO2
AmyotroCNS
Martinez et al.
Cl
Cl
not re-
IC50=
ported
34 nM
2.0 mg/Kg i.p.
Fmr1 KO mice
Chronic 14 days
pus-dependent learning deficits
[44]
Rescue adult hippocampal neurogenesis
O
CNS
Mood disorders
NP031115
O
S
O
N
N O
IC50=
IC50=
4 M
6.5 M
i.p.
5 mg/Kg
Acute
Reduced immobility
FST
1 dosis
time
9 mg/Kg
Acute
Reduced immobility
FST
1 dosis
time
50 mol/L
Acute
Reduced immobility
FST
3 dosis
time
Chronic
symptoms and Th17
20 days
and Th1 cells in the
[45]
NO2
CNS
Mood disorders
S
O
AR-A04418
N H
N H
N
IC50=
not re-
0.1 M
ported
i.p.
[45]
MeO
CNS
CNS
Mood disorders
Multiple Sclerosis
Peptide
L803-mts
S
O
TDZD-8
N Me
N O
IC50=
not re-
40 M
ported
IC50=
not re-
2 M
ported
i.c.v.
2.5-5.0 i.p.
mg/Kg EAE
[46]
Reduced clinical [47]
spinal cord. Reduced clinical
CNS
Multiple Sclerosis
Peptide
L803-mts
IC50=
not re-
40 M
ported
i.n.
60g
Chronic
symptoms and Th17
EAE
20 days
and Th1 cells in the
[47]
spinal cord. OH
CNS
Multiple Sclerosis
O N H
VP0.7
Reduced clinical
H N 10
O
O
N
IC50=
not re-
2.6 M
ported
i.p.
5.0 mg/Kg
Chronic
symptoms and Th17
EAE
20 days
and Th1 cells in the
[47]
spinal cord. Me O
CNS
CNS
Multiple Sclerosis
Myotonic dystrophy
VP2.51
N H
S O
MeO
S
O
TDZD-8
2.5-5.0
N Me
N H
N Me
N O
IC50=
not re-
0.6 M
ported
IC50=
not re-
2 M
ported
i.p.
mg/Kg EAE
i.p.
Reduced clinical Chronic
symptoms and Th17
20 days
and Th1 cells in the spinal cord.
10 mg/kg
Acute
DM1 mice
2 days
0.01-1 mg/kg NO2
CNS
Neuropathic pain
S
O
AR-A04418
N H
N H
N
IC50=
not re-
0.1 M
ported
Partial ligai.p.
tion of the sciatic nerve
MeO
[47]
Improved muscle strength.
[48]
Reduced myotonia. Reduced mechanical
Semi-Acute
hiperalgesia.
5 days
Reduced proinflam-
[49]
matory cytokines.
model 0.1-0.3 NO2 S
O
CNS
Pain
AR-A04418
N H MeO
N H
N
IC50=
not re-
0.1 M
ported
mg/Kg i.p.
Acetic acid and Formalin models
Acute
Antinociceptive
1 dosis
effect.
[50]
Lessons Learnt from Glycogen Synthase Kinase 3 Inhibitors
Current Topics in Medicinal Chemistry, 2013, Vol. 13, No. 15
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(Table 1) contd….
Disease group
Pathology
Compound
Chemical structure
name
O
CNS
Pain
NP031115
S
O
In vitro
In vitro
In vivo
GSK-3
GSK-3
admin.
IC50=
IC50=
4 M
6.5 M
O
N
N
i.c.v.
Parkinson disease
N
SC001
IC50=
not
3.8 M
reported
Treatment time
In vivo effects
Acute
Antinociceptive
1 dosis
effect.
15 nM
Acute
Dopaminergic
LPS model
1 dosis
protection.
Formalin model
Ph N N
CNS
model 1-3 mg/Kg
i.p.
O
Ph
Dosis/ Animal
Ref.
[50]
Decreased inflammation. i.p
Cl
150 M
Chronic
Dopaminergic
6-OHDPA
10 days
protection.
Chronic
function. Decreased
10 days
inflammation and
[51]
model
CNS
Spinal cord injury
S
O
TDZD-8
N Me
N O
IC50=
not re-
2 M
ported
1 mg/Kg i.p.
NO2
CNS
Spinal cord injury
S
O
AR-A04418
N H
N H
N
IC50=
not re-
0.1 M
ported
4mg/kg i.p.
muscular
O
Br
Spinal CNS
O
N Me
atrophy
H N
O
CNS
Stroke
O
BIP-135
IC50=
7-21 nM
16 nM
75 mg/kg i.p.
7 SMA KO model
[52]
tissue injury. Increased locomotor Acute
abilities. Decreased
1 dosis
inflammation and
[53]
tissue injury.
Chronic 14 days
Prolongued life span.
[54]
O
SB216763 N Me
IC50=
SCI traumatic model
MeO H N
SCI traumatic model
Recovery of limb
Cl
Cl
not re-
IC50=
ported
34 nM
i.p.
1.5 mg/kg
Acute
Decreased inflamma-
pMCAO
1 dosis
tion.
[55]
1 mg/Kg S
O
CNS
Stroke
TDZD-8
N Me
N O
IC50=
not re-
2 M
ported
Transient i.v.
Acute
Reduction brain
1 dosis
infarct volumen.
Chronic
Improved insulin
10 days
sensitivity.
Chronic
Improve glucose
21 days
tolerance
Chronic
Reduction of insulin
20 days
levels
10 mg/Kg
Chronic
weight gain, adipos-
DIO mice
14 days
ity, dyslipidemia, and
cerebral ischemia
[56]
model NH
N
Metabolic
Diabetes type II
N
CHIR98023
N Cl
Metabolic
Metabolic
Diabetes type II
Diabetes type II
H N
N H
N
Cl
Peptide
L803-mts
Ki
