Maleimide-based Glycogen Synthase
Kinase-3 Inhibitors: From Bipolar
Disorder to Anxiety
Dr. Hendra Gunosewoyo
Lecturer, School of Pharmacy, Curtin University (current)
Postdoctoral Res .Associate, Dept. of Med. Chem. & Pharmacognosy, UIC, Chicago (2010-2013)
Postdoctoral RA, U of KwaZulu Natal, Durban, S Africa (2011)
PhD, School of Chemistry, U of Sydney (2006-2010)
Neurotrauma Research Program Seminar
2 April 2014
Current Projects
1) GSK-3β inhibitors as stimulators of steroidogenesis
J. Med. Chem. 2013, 56 (12): 5115-5129
2) Antitubercular drugs for tuberculosis-HIV co-infection
J. Med. Chem. 2013, 56 (10): 4093-4103
Nature Comm. 2013, 4: 2907
Vassilios Papadopoulos
McGill U Health Centre
William Bishai
JHU, HHMI
Alan Kozikowski
UIC
Bipolar Disorder
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A.k.a. manic-depressive disorder
CNS disorder characterized by dramatic up&downs in mood, energy
& activity levels
Severe, intense emotional states alternating between mania phase
and depression (“mood episodes”)
Symptoms of mania or a manic Symptoms of depression or a
episode include:
depressive episode include:
50% of the onset before age 25
•Mood Changes
•Mood Changes
A long period of feeling "high," A long period of feeling worried
2.6% prevalence of US adults
or an overly happy or outgoing or empty
•Loss of interest in activities
mood
•Extremely irritable mood,
once enjoyed, including sex.
Prone to suicide
•Behavioral
http://www.nimh.nih.gov
agitation, feeling "jumpy" or
"wired."
•Behavioral Changes
Talking very fast, jumping from
one idea to another, having
racing thoughts
•Being easily distracted
•Increasing goal-directed
activities, such as taking on new
projects
•Being restless
•Sleeping little
•Having an unrealistic belief in
one's abilities
•Behaving impulsively and
taking part in a lot of
pleasurable,
high-risk behaviors, such as
spending sprees, impulsive sex,
and impulsive business
investments.
Changes
Feeling tired or "slowed down"
•Having problems concentrating,
remembering, and making
decisions
•Being restless or irritable
•Changing eating, sleeping, or
other habits
•Thinking of death or suicide, or
attempting suicide.
Classification & Current Treatment
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According to Diagnostic & Statistical Manual of Mental Disorders
(DSM):
– Bipolar I Disorder: alternating, severe manic & depressive
episodes lasting for >7 days
– Bipolar II Disorder: cycle of depression & hypomania, no full
mania
– BD Not Otherwise Specified: not type I or II
– Cyclothymia: mild form of BD, hypomania/mild depression cycles
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Current treatment:
Mood stabilizers: lithium, valproic acid, lamotrigine
Atypical antipsychotics & antidepressants: Zyprexa, Abilify,
Prozac, etc.
Electroconvulsive therapy
Sleep medication, psychotherapy
http://www.nimh.nih.gov
Mood Stabilizers
N
H2N
N
OH
Li2CO3
O
lithium carbonate
valproic acid
NH2
N
Cl
lamotrigine
Cl
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Antimanic effect of lithium was discovered by John Cade in 1949
Anticonvulsants VPA and lamotrigine
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Temporal effect of lithium in bipolar disorder prophylaxis (weeks)
Major thyroid problems associated with lithium
Direct targets of lithium include:
GSK-3, phosphoglucomutase, inositol monophosphatase, other
inositol polyphosphatases, and monophosphoesterases
Other kinases targeted include:
CK2, PRAK, MAPKAPK2
Animal Models for Bipolar Disorder
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Progressive & cyclical nature of BD between mania and depression
Diagnosis of mood disorder is based on diverse symptoms
No fully validated biomarkers for mood disorder
Many features can only be seen in humans
Some animal models of depression:
– Porsolt forced swim test
– Learned helplessness
– Social defeat, chronic NSF
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Some animal models of mania:
Barbara Caldarone
Harvard NeuroDiscovery Center
Psychogenics
– Psychostimulant-induced hyperactivity model
– Open field
– Sleep deprivation
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Known models that simulate the progressive phenomenon of BD:
– Amygdala kindling
– Behavioral sensitization to psychostimulants, e.g. cocaine
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The circadian rhythm genes mPer1 and mPer2 influence the cocaine sensitization & reward
– Extreme sensitization
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When animals reach extreme sensitization to cocaine, oscillations in behavioral & biochemical
responses occur, e.g. efflux of Nac dopamine, hippocampal 5HT, plasma corticosterone, and shockinduced hypoalgesia.
Goodwin & Jamison. Manic-Depressive Illness.
Glycogen Synthase Kinase-3
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Two subtypes:
GSK-3α and GSK-3β
Widely distributed in tissues
GSK-3β abundant in CNS
Constitutively active
Cell cycle, development,
survival, apoptosis, circadian
rhythm, cellular plasticity and
resilience neurogenesis
Involved in Alzheimer’s
Demonstrated both antimanicand antidepressant-like
phenotypes in vivoa unique
target profile of GSK-3.
Many known small-molecule
ATP-competitive inhibitors
www.sabiosciences.com
Known Kinase Inhibitors
Nature Chemical Biology 2, 689 - 700 (2006)
The Beginning
Br
Total Distance (cm/5min)
4500
4000
3500
3000
2500
2000
1500
1000
4000
Total Distance (cm/5min)
5000
1%Tween-Water
ING-135-Water
1%Tween-Amph
1%Tween-Amph+CDP
Valproate-Amph+CDP
ING-135-Amph+CDP
3500
3000
O
H
N
O
N
O
Me
Lead Compound ING-135
1%Tween-Water
LiCl (4mEq/kg)-Water
1%Tween-Amphetamine
1%Tween-Amphetamine+CDP
LiCl (4mEq/kg)-Amphetamine
LiCl(4mEq/kg)-Amphetamine+CDP
2500
2000
1500
1000
500
500
0
0
5 10 15 20 25 30 35 40 45 50 55 60
5 10 15 20 25 30 35 40 45 50 55 60
Time (min)
Time (min)
Figure 4. Effect of ING-135 and the known mood stabilizers lithium and valproate on the locomotor
hyperactivity induced by
the amphetamine (Amph) and Amph + chlordiazepoxide (CDP) mixture (Mix). Mice were pretreated with either compound
ING-135 (50 mg/kg), valproate (400 mg/kg), lithium (4 mEq/kg) or 1% Tween vehicle. After 5 min or 30 min (valproate
group) mice were injected with either water vehicle, amphetamine (4 mg/kg) alone (Amph), or a mixture of amphetamine (4
mg/kg) and chlordiazepoxide (2.5 mg/kg) (Amph + CDP). Locomotor activity was automatically recorded every 5 min for a
60 min period in a square locomotor activity chamber surrounded by infrared beams. The mixture of Amph + CDP produced
greater locomotor activity than Amph alone (p <0.05 Veh–Mix vs Veh–Amph; Fisher’s PLSD). Pretreatment with ING-135,
valproate and lithium normalized the locomotor activity produced by the Amph + CDP mixture (p < 0.05 ING135/Lithium/Valproate-Amph + CDP vs 1% Tween-Amph + CDP (Fisher’s PLSD, n=8–9 per group)).
JACS 2007, 8328-8332
Lead ING-135
Time Course of Brain Concentrations of Fluoxetine and ING-135
Confidential data
Peak level of ING-135 was observed in the brain after 15 min
A: Concentrations of Fluoxetine were measured following subcutaneous administration of 3 or 10 mg/kg of the drug in male
Sprague-Dawley rats (Ying Qu, et al. 2009 Pharm. Biochem. Behavior) B: Concentrations of ING-135 were measured following
iv administration of 10 mg/kg in male Sprague-Dawley rats (SRI, studies B360-09).
Confidential data
Pharmacokinetic parameters and preliminary
ADME/Tox data for ING-135 including Cmax, Tmax,
AUC, t1/2, hERG inhibition, CYP inhibition, CaCo2
permeability, Ames test
• GSK-3β IC50: 21 nM
• Tested against 50 common
neuroreceptors & enzymes
• Tested against 320 kinases
• Excellent selectivity index (SI)
Irina Gaisina
X-ray Co-crystal Structure with 9ING12
Characteristic hydrogen bonding of the
maleimide core to the carbonyl group of
Asp133 and amino group of Val135.
A water molecule acts as a bridge between
the carbonyl group of maleimide to Asp200
while another water molecule connects the
oxygen atom of the benzofuran ring to
Lys85.
The hydroxypropyl substituent on the
indole ring forms a hydrogen bond with
Arg141 but is largely solvent exposed.
9-ING-12 makes a number of hydrophobic
contacts with the side chains of Ile62,
Phe67, Val70, and Leu188.
ChemMedChem 2011, 1593-1602.
More Water Soluble Analogs
Attenuation of hyperactivity
in CLOCK mutant mice
GSK-3β IC50 between 5 and 600 nM
ChemMedChem 2011, 1593-1602.
Pyrazolones
ChemMedChem 2011, 1587-1592.
Diazepinoindoles
O
R1
R1
N
e,f,g
N
H
N
Boc
H
R1
R1
=H
43a:
1
43b: R = F
=H
39:
1
42: R = F
O
H
N
h or i
N
O
N
O
HN
24: R1 = H
25: R1 = F
O
R1
N
O
R1
a-d
O
H
N
O
27: R1 = H, R4 = 2-pyrazinyl
28: R1 = H, R4 = 4-pyridylmethyl
29: R1 = F, R4 = 1-piperidinyl
30: R1 = F, R4 = 4-piperidinopiperidin1-yl
31: R1 = F, R4 = 4-morpholinyl
R4
a
Reagents and conditions: (a) (i) ethanolamine, 10% Pd/C, MeOH, rt, 1 h; (ii) H2, 1 atm, rt, 3
h; (b) (Boc)2O, THF, aq. K2CO3, 0 ºC to rt, 5 h; (c) MsCl, N,N-diisopropylethylamine, THF, 0 ºC to
rt, 16 h; (d) NaH, DMF, 0 ºC, 1h; (e) ethyl chlorooxoacetate, Et2O, 0 ºC to rt, 16 h; (f)
benzofuran-3-yl-acetamide, t-BuOK, THF or DMF, 0 ºC to rt, 16 h; (g) trifluoroacetic acid,
CHCl3, rt, 2 h; (h) R4CO2H, EDC.HCl, HOBt, DMF, N-methylmorpholine, rt, 16 h; (i) R4COCl, N,Ndiisopropylethylamine, MeOH, rt, 3 h.
J.Med.Chem 2013, 5115-5129.
GSK3 & Translocator Protein
PDSP primary binding assay
Confidential data
Bryan Roth
UNC
The default concentration for primary binding experiments performed at the
Psychoactive Drug Screening Program (UNC, Chapel-Hill) is 10 μM. In the primary binding
assay, mean percent inhibition (n=4) was determined for each compound at the specific
receptor subtype. Only those compounds that showed >50% mean percent inhibition in
the primary binding assay were tested for subsequent secondary binding assays
GSK3 & TSPO
PDSP secondary binding assay
Confidential data
Data represent Ki (nM) values obtained from non-linear regression of
radioligand competition binding isotherms. Ki values are calculated from best fit
IC50 values using the Cheng-Prusoff equation.
Delineate the SAR profile of these maleimide-based GSK-3 inhibitors in TSPO binding
Steroidogenesis & TSPO
Steroidogenesis stimulation
of maleimide-based GSK-3β
inhibitors in MA-10 tumor
Leydig cells. MA-10 tumor
Leydig cells were incubated in
the presence or absence of
GSK-3β inhibitor, and at the
end of incubation, steroid
(progesterone) production
and cellular toxicity were
evaluated by RIA and MTT
assay, respectively.
J.Med.Chem 2013, 5115-5129.
GSK-3/TSPO/anxiety
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Hypothesis: GSK-3 inhibition could induce the production of neurosteroids
which in turn modulate the anxiety-like behavior in vivo
Lithium suppresses anxiety-like symptoms in mouse models of Fragile X
syndrome, Huntington’s disease, and cerebral ischemia
TSPO ligand emapunil (XBD-173) demonstrated fast-acting anxiolytic
effects without benzodiazepine-like side effects in human clinical trials
Recently the structure of translocator protein has been elucidated
emapunil
Science 2014, 1363-1366; 2009, 490-493.
GSK-3 / TSPO
Midzak & Papadopoulos
GSK-3 / TSPO
Midzak & Papadopoulos
SmartCube® Assay
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Clinically approved reference drugs grouped per indication
Drugs were injected 15 min prior to test
Digital videos of the mice processed with computer vision algorithms
to extract 2,000 dependent measures, e.g. grooming, rearing, etc.
Establish reference database of therapeutic class signatures
Front Neurosci 2011, 103
Chronic SmartCube® Assay
SUBCLASS DAY 1
CLASS DAY 1
Antidepressant
(medium dose)
ANTA 5HT2A
MAOAB I
NE/DA RI
NE RI - Tricyclic
5HT/NE RI
5HT Re Enh
SS RI
Triple RI
(higher dose)
ANTA α 2/5HT2/3
ANTA 5HT2A
MAOAB I
NE/DA RI
NE RI/Tricyclic
Anxiolytic
HG4-22
ING-135
HG4-22
ING-135
SUBCLASS DAY 13
CLASS DAY 13
ANTA mGLUR5
A 5HT1A
Benzo
ANTA CRF1
Sedative/Hypnotic
ANTA H1
Benzo
GABAA
NON Benzo
HG4-22
ING-135
Analgesic
A Opiate
NSAID
Hallucinogen
ANTA NMDA
A 5HT2
Anxiogenic
ANTA α 2
ANTA GABAA
A 5HT1B/1C/2C
β Carboline
IA GABA
ANTA NMDA
ANTA mACh
Typical
ANTA nACh
Moodstabil/Anticonv
GABA RI
NA CH Block
Carbamaz
Unknown
Side Effects
ANTA α 2
ANTA GABAA
A Opiate
A 5HT1A
AChase I
GABA RI
PDE4 I
Not Classified
Class and subclass analyses of HG4-22 (50 mg/kg)
and ING-135 (50 mg/kg) on day 1 and 13 in SC®
Cognitive Enhancer
A nACh
ANTA H3
AChase I
PDE4 I
ANTA 5HT2A
Atypical
Atypical
Typical
ING-135
DA RI
NE RI
5HT/DA/NE Re Enh
5HT/DA/NE RI
Unknown
Xantine
Antipsychotic (low)
(medium)
HG4-22
Psychostim/ADHD
Vehicle
5HT Re Enh
Unknown
J.Med.Chem 2013, 5115-5129.
Spinal Muscular Atrophy
http://www.treat-nmd.eu
Thoughts…
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Ready access to cell-based GSK-3 assays & immunoblotting,
looking at biomarkers, both the direct (pGSK3) & indirect ones e.g.
pCREB, p-beta-catenin, BDNF, etc.
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Animal models of anxiety
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PK and ADME/Tox in house
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Circadian rhythm-related experiments
Acknowledgements
Alan P. Kozikowski
Irina Gaisina, Wenwen Chen, Bob Chen
APK Group past & present
Vassilios Papadopoulos
Andrew Midzak
Psychogenics Team
Barbara Caldarone. Dani Brunner, Taleen Hanania
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NIMH
Late Professor Gertrude Neumark
Thank you!

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