The marine natural-derived inhibitors of

glycogen synthase kinase-3β phenylmethylene

hydantoins: In vitro and in vivo activities and
pharmacophore modeling.
Khanfar MA, Asal BA, Mudit M, Kaddoumi A, El Sayed KA
Bioorganic & Medicinal Chemistry 17 (2009) 6032-6039.

Presented by
Sarochin Santiwarangkool 4903052 PYPY/B
Faculty of Pharmacy, Mahidol University
September 7, 2010.
 Outline
Alzheimer’s disease (AD)
– Definition
– Pathogenesis
Glycogen synthase kinase-3β (GSK-3β)
Red sea sponge (Hemimycale arabica)
Studies for PMH analogues
– In silico screening
– In vitro & In vivo testing
– Pharmacophore model generation
Future design of GSK-3β inhibitors.
 Alzheimer’s disease
Definition
Alzheimer’s disease (AD) is a
progressive dementia affecting cognition,
behavior, and functional status with no
known cause or cure. Patients eventually
lose cognitive, analytical, and physical
functioning, and the disease is ultimately
fatal.
AD: Pathogenesis

Insoluble
Aβ
 GSK-3β
Glycogen synthase kinase-3β

ATP
Glycogen Activated
synthase GSK-3β Inactivated

ADP

Pi
 GSK-3β
Glycogen synthase kinase-3β
= “Tau phosphorylating kinase I”

ATP
Specific site:
Tau GSK-3β - Ser 199

ADP - Ser 396

Pi
http://www.jyi.org/articleimages/88/originals/img2.jpg
 GSK-3β
Glycogen synthase kinase-3β
= “Tau phosphorylating kinase I”

ATP Therapeutically
important for several
Tau GSK-3β neurodegenerative
diseases, including
AD.
ADP
Pi
Red sea sponge (Hemimycale arabica)
1: R = 4-OH  (Z)-5-(4-hydroxybenzylidene)-
hydantoin (PMH)

2: R = 4-SCH2CH3  (Z)-5-
(4(ethylthio)benzylidene)-hydantoin
- Potent in vitro anti-growth and anti-
invasive properties
- A potent and selective GSK-
3βinhibitor
high binding scores at the GSK-
3β ATP binding site
Studies for PMH analogues
In silico screening
– Molecular docking
In vitro & In vivo testing
– In vitro GSK-3β
inhibitory assay: a tau
phosphoELISATM [pS396]
– In vivo determination of
hepatic glycogen
contents
Pharmacophore model
generation
 NH at position 3 ↔

Molecular docking Carbonyl O of Asp

133. (2.11 Å)

Carbonyl O at
position 2 ↔
H-bonding
Backbone N of
Val135. (1.67 Å)
The highest docking score

Extensive interaction Rotating

with nucleotide- out of the
binding loop. plane
 Molecular docking
PMH 3: high selectivity for GSK-3β vs other kinases

Hydrophobic pocket: Ile 62, I-5

Glu63, Val70.

I-5
H-bonding: aniline
N at position 3
guanidine moiety
of Arg141. (3.13 Å)

PMH3

Arg141: selectivity residue for GSK-3β  important to improve the activity in the
process of designing new derivatives for GSK-3β inhibitors.
In vitro GSK-3β inhibitory assay

The most
potent
inhibition

Tau [pS396] phosphoELISATM kit

– Detection of tau phosphorylation at Ser396.
– Measuring IC50
Glycogenesis

GSK-3β
 GSK-3β
Glycogen synthase kinase-3β

Activated
ATP Inactivated
Glycogen
synthase GSK-3β

• ↓ glucose output
ADP
• ↑ glycogen synthesis

Pi
 Determination of hepatic glycogen
contents
A significant increase in rat’s liver
glycogen content in a dose-dependent
manner. (p<0.05)

In vivo Sprague Dawley rat model.

Measuring glycogen content from liver homogenate with
UV spectrophotometer.
Amount (mg) = (DU/DS) x (Volume of Extract
(mL)/Weight of Liver tissue (g)) x 0.09
Pharmacophore model generation

DISCOtechTM
– Active compound as GSK-3b inhibitor: IC50 ≤ 20 µM
– DISCO runs: varying tolerance and range of required features.
Pharmacophore model generation
Mod-2(b)-1
Model optimization
 Mod-2(b)-1

Mod-7
Mod-7

PMH:
PMH3: R = 4-N(CH2CH3)
 NH at position 3
↔
Carbonyl O of
Asp 133.

Carbonyl O at
position 2 ↔
H-bonding
Backbone N of
Val135.

Mod-7
Aniline N at
position 3 ↔
Guanidine moiety
of Arg141.

PMH3: R = 4-N(CH2CH3)
Future design: Potent & selective
GSK-3β inhibitors.
H-bonding with the hinge region: Asp133 &
Val135.
Filling the hydrophobic pocket: Val70 & Lys85
For example:
1.) Keeping hydantoin ring
2.) Placing COO- or negatively charged
moiety at C-9 or C-10.
3.) Placing benzyl or phenylethyl at C-12.
Thank you for
your attention.