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The marine naturalThe marine natural--derived inhibitors ofderived inhibitors ofglycogen synthase kinaseglycogen synthase kinase--33 phenylmethylenephenylmethylene

hydantoins:hydantoins: In vitroIn vitro andand in vivoin vivo activities andactivities and

pharmacophore modeling.pharmacophore modeling.Khanfar MA, Asal BA, Mudit M, Kaddoumi A, El Sayed KAKhanfar MA, Asal BA, Mudit M, Kaddoumi A, El Sayed KA

Bioorganic & Medicinal ChemistryBioorganic & Medicinal Chemistry 1717 ((20092009)) 60326032--60396039..

Presented by

Sarochin Santiwarangkool 4903052 PYPY/B

Faculty of Pharmacy, Mahidol University

September 7, 2010.

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OutlineOutline

Alzheimers disease (AD)Alzheimers disease (AD) DefinitionDefinition

PathogenesisPathogenesis

Glycogen synthase kinaseGlycogen synthase kinase--33 ( (GSKGSK--33))Red sea sponge (Red sea sponge (HemimycalearabicaHemimycalearabica))

Studies for PMH analoguesStudies for PMH analogues In silico screening

In vitro & In vivo testing Pharmacophore model generation

Future design of GSKGSK--33 inhibitors. inhibitors.

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Alzheimers diseaseAlzheimers disease

DefinitionDefinition

Alzheimers disease (AD) is a Alzheimers disease (AD) is aprogressive dementia affecting cognition,progressive dementia affecting cognition,behavior, and functional status with nobehavior, and functional status with noknown cause or cure. Patients eventuallyknown cause or cure. Patients eventuallylose cognitive, analytical, and physicallose cognitive, analytical, and physical

functioning, and the disease is ultimatelyfunctioning, and the disease is ultimatelyfatal.fatal.

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AD: PathogenesisAD: Pathogenesis

InsolubleA

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GSKGSK--33

Glycogen synthase kinaseGlycogen synthase kinase--33

Glycogensynthase

ATP

ADP

GSK-3

Pi

Inactivated

Activated

Pi

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GSKGSK--33

Glycogen synthase kinaseGlycogen synthase kinase--33

= Tau phosphorylating kinase I= Tau phosphorylating kinase I

Tau

ATP

ADP

GSK-3

Pi

Specific site:

- Ser 199

- Ser 396

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http://www.jyi.org/articleimages/88/originals/img2.jpg

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GSKGSK--33

Glycogen synthase kinaseGlycogen synthase kinase--33

= Tau phosphorylating kinase I= Tau phosphorylating kinase I

Tau

ATP

ADP

GSK-3

Pi

Therapeutically

important for several

neurodegenerative

diseases, includingAD.

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Red sea sponge (Red sea sponge (HemimycalearabicaHemimycalearabica))

1: R = 4-OH ((ZZ))--55--((44--hydroxybenzylidene)hydroxybenzylidene)--

hydantoin (PMH)hydantoin (PMH)

2: R = 4-SCH2CH3 ((ZZ))--55--((44(ethylthio)benzylidene)(ethylthio)benzylidene)--hydantoinhydantoin

-- PotentPotent in vitroin vitro antianti--growth and antigrowth and anti--

invasive propertiesinvasive properties

-- A potent and selective GSKA potent and selective GSK--

33inhibitorinhibitorhigh binding scores at thehigh binding scores at the GSKGSK--33 ATP binding siteATP binding site

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Studies for PMH analoguesStudies for PMH analogues

In silicoIn silico screeningscreening

Molecular dockingMolecular docking

In vitroIn vitro &&In vivoIn vivo testingtesting

In vitroIn vitro GSKGSK--33inhibitory assay: a tauinhibitory assay: a tauphosphoELISAphosphoELISATMTM

[pS[pS396396]]

In vivoIn vivo

determinationdeterminationof hepatic glycogenof hepatic glycogencontentscontents

PharmacophorePharmacophore modelmodelgenerationgeneration

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Molecular dockingMolecular docking

Rotatingout of the

plane

H-bonding

Carbonyl O atposition 2

Backbone N ofVal135.(1.67 )

NH at position 3

Carbonyl O of Asp133.(2.11 )

Extensive interactionwith nucleotide-binding loop.

The highest docking score

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Molecular dockingMolecular docking

H-bonding: anilineN at position 3

guanidine moietyof Arg141.(3.13 )

Hydrophobic pocket: Ile 62,Glu63, Val70.

I-5

I-5

PMH3

Arg141: selectivity residue forGSK-3 important to improve the activity in the

process of designing new derivatives for GSK-3 inhibitors.

PMH 3: high selectivity forGSK-3 vs other kinases

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In vitro GSKIn vitro GSK--33 inhibitory assay inhibitory assay

Tau [pSTau [pS396396] phosphoELISA] phosphoELISATMTM kitkit

Detection of tau phosphorylation at SerDetection of tau phosphorylation at Ser396396..

Measuring ICMeasuring IC5050

The mostpotent

inhibition

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GSK-3

Glycogenesis

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Determination of hepatic glycogenDetermination of hepatic glycogen

contentscontents

In vivo Sprague Dawley rat model.In vivo Sprague Dawley rat model.

Measuring glycogen content from liver homogenate withMeasuring glycogen content from liver homogenate withUV spectrophotometer.UV spectrophotometer.

Amount (mg) = (DU/DS) x (Volume of ExtractAmount (mg) = (DU/DS) x (Volume of Extract(mL)/Weight of Liver tissue (g)) x(mL)/Weight of Liver tissue (g)) x 00..0909

A significant increase in rats liverglycogen content in a dose-dependentmanner. (p

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Pharmacophore model generationPharmacophore model generation

DISCOtechDISCOtechTMTM Active compound as GSKActive compound as GSK--33b inhibitor: ICb inhibitor: IC5050 2020 MM

DISCO runs: varying tolerance and range of required features.DISCO runs: varying tolerance and range of required features.

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Pharmacophore model generationPharmacophore model generation

ModMod--22(b)(b)--11Model optimization

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Mod-2(b)-1

Mod-7

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Mod-7

PMH:

PMH3: R = 4-N(CH2CH3)

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H-bonding

Carbonyl O atposition 2

Backbone N ofVal135.

NH at position 3

Carbonyl O ofAsp 133.

Aniline N at position3 Guanidine

moiety of Arg141.

Mod-7

PMH3: R = 4-N(CH2CH3)

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Future design: Potent & selectiveFuture design: Potent & selective

GSKGSK--33 inhibitors. inhibitors.HH--bonding with the hinge region: Aspbonding with the hinge region: Asp133133 &&

ValVal135135..

Filling the hydrophobic pocket: ValFilling the hydrophobic pocket: Val7070 & Lys& Lys8585

For example:For example:

11.) Keeping hydantoin ring.) Keeping hydantoin ring

22.) Placing COO.) Placing COO-- or negatively chargedor negatively charged

moiety at Cmoiety at C--99 or Cor C--1010..33.) Placing benzyl or phenylethyl at C.) Placing benzyl or phenylethyl at C--1212..

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Thank you for

your attention.