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A network pharmacology approach to novel therapeutic strategies (Seminaris Tecnològics 2014)

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(1)

A network pharmacology approach to

novel therapeutic strategies!

Patrick Aloy!

http://sbnb.irbbarcelona.org!

(2)

Human biology is a complex system!

For every complex problem there is a solution that is simple,

neat and wrong (HL Mencken).!

The problem!

Jaeger et al. (in preparation)!

Duran-Frigola & Aloy, Genome Medicine (2012)!

High attrition rates in drug discovery.! !

Complex diseases are difficult to address.! !

Challenging drug side effect anticipation.! !

(3)

Targeting"

networks!

(4)

Promising target combinations!

Knight et al, Nat Rev Cancer (2010)!

Systems pharmacology in BC!

Pathway B!

[[PPII33KK--AAkktt ssiiggnnaalllliinngg]]!!

Pathway A!

[[EErrBBBB ssiiggnnaalllliinngg]]!! Drug A!

Cell growth!

Resistance to drug A! Drug B! Crosstalk! Extended! crosstalk!

(5)

in silico and experimental strategy validation!

MCF7 (ER+ / PR+)! SKBR3 (HER2+)!

MDA-MB-231 (Triple negative)! BT-4T4 (Triple positive)! U2OS (Osteosarcoma)!

MCF10A (Normal mammary epithelial)! !"##$%&'(&#&)*$

in silico and experimental strategy validation!

(6)

Is there a synergistic effect ?!

(7)

Drug"

reprofiling!

Magic bullets ???!

Drug-drug network!

Target-target network!

!

!

(8)

Phenotypic screens in human!

Duran-Frigola & Aloy. Genome Medicine 2012!

(9)

Alzheimer´s disease (AD)!

PPrreecclliinniiccaall AADD!!

!!

>20 years before diagnosis!

Short-term memory loss!

M

Miilldd aanndd m

mooddeerraattee AADD!!

!

2 - 10 years!

Reading problems!

Poor object recognition!

Poor direction sense!

SSeevveerree AADD!!

!!

1 - 5 years!

Poor judgment!

Short attention!

Visual problems!

PPrree--ssyym

mppttoom

maattiicc ddiiaaggnnoossee!!

DDiisseeaassee m

mooddiiffyyiinngg ttrreeaattm

meennttss!!

EEffffeeccttiivvee bbiioom

pprrooggrreessss aanndd rreessppoonnssee ttoo ttrreeaattm

maarrkkeerrss ttoo m

moonniittoorr

meenntt!

AD is a complex and heterogenic disease!

M1! M2! M3! Day 1! Day 2! Day 3! Day 4! Day 5! Day 6! Day 7! Day 8!

Neuronal dysfunction!

Amyloid plaques!

Oxidative stress!

Neurofibrilary tangles!

Environmental!

factors !

We need disease-modifying therapeutic treatments that also alleviate the

clinical symptoms!

(10)

Network biology and systems pharmacology!

GST

pull-downs

and

Co-IPs

!

Co-localization

!

Interaction discovery applied to AD!

200 PPI involving 8 AD causative/susceptibility genes

and 66 candidates, 17 of which appear up- or

down-regulated in AD patients (p< 3*10

-4

)!

HC AD-related network!

(11)

Soler-López*, Zanzoni*, et al. 2011. Genome Res! Soler-López et al. 2012. BioEssays!

ECSIT might act as a molecular link between oxidative stress,

inflammation and mitochondrial dysfunction in AD!

Myc ECSIT-myc 0 50 100 150 200

A!!40

% r esp ect to scr am bl e Myc ECSIT-myc 0 50 100 150 200

A!!42

% r esp ect to scr am bl e

H4-APP overexpressing human cells!

In ECSIT-ovexpressing cells, Ab40 & Ab42 levels decrease,

indicating that ECSIT does have an e!ect in APP processing"

Ab40 and Ab42 levels from treated cell supernatants measured by

ELISA immunoassays!

Overexpressed MycECSIT"

(12)

H4 human cells, whole lysates !

ECSIT-subcellular localization!

ECSIT



mitochondrial



isoform only appears in APP cells"

ECSIT FL

ECSIT mt

B-actin

Building networks is (ahem) easy, but how

about therapeutics ?!

ECSIT NDUFA4 Cmplx.I Cmplx. VI Cmplx. II Cmplx.III OXPHOS system GCDH LONP1 intramitochondrial proteolysis energy generation PSEN2 PSEN1 APP Aβ APOE APP APP translocation translocation Aβ Aβ secretion TRAF6 IFIT5 PRDX2 redox signaling inflammatory response redox signaling TLR pathway ER i inflflammattory LOXL4 CYP2 C18 MAPK pathway BLMH cytosol ECSIT NOS3 reported interaction novel interaction AD reported gene Electron trasnport chain

ETC

AD candidate gene ECSIT literature interactor

MEKK1

TGF-β/BMP pathway

transcription activation

BMP-target genes cellular differentiationapoptosis

nucleus plasma membrane Nf-kB Nf-kB TOM TO M APP

BACE1 PSEN1 PSEN2

mtROS production ABAD Aβ channel SMADs ECSIT TLR4 TLR pathway BMPr1a BMP pathway receptor/transporter cROS production CD14 SMAD7 Ub ECSIT Ub ECSIT SMAD4 RSMAD endocytosis ubiquitination AP-1 oxidative stress APOE Aβ oligomerization ETC Cmplx. V ACAD9 endosome P Aβ Ca+2 Ca+2 ATP pump Calcium signaling Ca+2 PSEN Ca+2 Ca+2 channel NMDAr C membrane excitability PTP Ca+2 Ca+2 MCU Ca+2 eration ∆ψ 2 +22 ∆ψ

Soler-López*, Zanzoni*, et al. 2011. Genome Res! Soler-López et al. 2012. BioEssays!

(13)

How can we USE the information hidden in

the complex maps?!

Newton equation!

Empirical potentials!

Structural bioinformatics!

ODEs!

KDD!

Systems biology!

A top-down approach to systems biology!

Physiological!

effects!

Known drugs!

Drug targets!

(14)

!

!

Activation or inhibition

(nodes) cause the AE!

of effector proteins

One same AE can be

caused by several

Motifs (mechanisms)!

AEs are coded

according to standard

terminology!

Comprehensive, hand-curated database (more than 15 000 articles reviewed)!

> 3 000 Effectors!

(individual proteins)!

> 800 Motifs!

(subnetworks)!

> 350 AEs!

(MedDRA)!

~50,000

relationships

!

Our own Rosetta stone!

MOTIVE

ID! MOTIVE NAME! MOTIVE DESCRIPTION! Organ or System affected! affected! Cell type affected!Tissue Proteins!Seed

1! pathology !Amyloid Amyloid plaques_Abnormal clusters of protein fragments build up

between nerve cells.! CNS! Brain!

Neurons ,! Endothelial cells,!

VSMCs,! Microglia!

26!

2! Tau pathology! Neurofibrillary tangles_insoluble twister fibers inside neurons ! CNS! Brain! Neurons!

14!

3! Oxidative Stress! Oxidative stress plays a role in the etiology and also in the

pathophysiology of the disease . ! CNS! Brain!

Microglia,

Astroglia !

77!

4! dysfunction Neuronal

and death !

Neuronal loss and brain conductance disturbances associated to memory

loss ! CNS! Brain! Neurons !

13!

#! #! #!

(15)

MOTIVE

Level! MOTIVE ID! Effector Protein! Name! Uniprot! Causative Effect! Relevance! Reference! Evidence !

M ! 1! Amyloid-precursor protein! APP! P05067! 1! 100! 20232515!Aß comes from the proteolitic processing of amyloid precursor

protein! M ! 1! receptor-related protein Low-density lipoprotein

1 85 kDa subunit ! LRP1 ! Q86SWO ! -1! 90! 20854368!Protein involved on Aß clearqance from the brain !

M ! 2! Microtubule-associated protein tau! MAPT, TAU! P10636! 1! 90! 20510151!

Tau is the component of Neurofibrillary tangles, the insoluble twister fibers inside neurons !

M ! 2! glycogen synthase kinase ! GSK3ß! P49841! 1! 90! 20510151!The main kinase that phosphorilates TAU protein!

M#! 3! Interleukin-1 beta! IL1ß! P01584! 1! 80! 20149872!

Neuroinflammatory changes (activated glial cells and elevated levels of proinflammatory IL-1$ and TNF-%) are present in AD brains!

M ! 3! Superoxide dismutase [Cu-Zn] ! SOD1! P00441! -1! 90! 10880853!proinflammatory IL-1$ and TNF-%!

M ! 4! Acetylcholinesterase! AChE! P22303! -1! 90! 20525543!Patients with AD has a decline of cholinergic pathways!

How does the actual data look like ?!

~50,000 relationships!

(16)

Soft (molecular) data, soft models!

Erectile

dysfunction!

Diarrhoea!

Headache!

Dyspepsia!

PDEs!

(17)

Tau pathology!

Amyloid pathology!

Oxidative stress!

16 distances per node/motif!

Artificial neural network!

Building predictive disease models!

Pujol et al (2010) US Patent Application 12/912.535!

The system is able to predict ~80% of all described

therapeutic indications and AEs, with undetermined FDR

(map-specific leave-one-target-out experiment)

.!

Case example: Diazepam!

!""#$%&'(# !""#$%&'(#

(18)

AD!

Amyloid plaques!

AX_ALZ_003! AX_ALZ_004! AX_ALZ_006!

Neurofibrilary tangles!

AX_ALZ_002!

Oxidative stress!

AX_ALZ_003! AX_ALZ_004! AX_ALZ_006! AX_ALZ_007!

Cognition!

AX_ALZ_003! AX_ALZ_004! AX_ALZ_005! AX_ALZ_006!

We excluded all compounds with any

prior art on AD and/or!

therpeutic indications in CNS !

The real test: reprofiling experiment in AD!

Neuronal dysfunction!

AX_ALZ_011+013!

Amyloid!

plaques!

Neurofibrilary !

tangles!

Oxidative!

stress!

Cognition!

AX_ALZ_002!

AX_ALZ_003!

AX_ALZ_004!

AX_ALZ_005!

AX_ALZ_006!

AX_ALZ_007!

(19)

Amyloid!

plaques!

Neurofibrilary !

tangles!

Oxidative!

stress!

Cognition!

AX_ALZ_002!

50%  pTAU/TAU!

AX_ALZ_003!

30% inh AChE!

AX_ALZ_004!

 Ab42 (x3)!

AX_ALZ_005!

AX_ALZ_006!

95% inh AChE! ~40% prot H

2

O

2!  Ab40 / Ab42!

AX_ALZ_007!

~40% prot H

2

O

2!

Cell-based assays and animal models!

Proton-pump inhibitors increase A

!

production by

modulating

!

- and

"

-secretase activities!

(20)

Proton-pump inhibitors increase A

!

production by

modulating

!

- and

"

-secretase activities!

Badiola et al. PLoS ONE 2013!

Experiment:!

4 mo!

6’5 mo!

10 weeks treatment !

daily dose by gavage !

Morris water-maze test

to check!

Memory impairment at

early-stage!

Low ! Mild ! Pathology level!

AD mice ( 3xTg AD)!

APPSwe, PS1M146V, tauP301L !

!

Healthy mice!

Group 1: Vehicle!

Group 2: Treatment!

Group 3: Vehicle!

Mice Groups:!

(21)

Morris water-maze test!

Learning process!

NE! SE! SW! NW! Day 1! Day 2! Day 3! Day 4! Day 5! Day 6! Day 7! Day 8!

Days!

Pathlength

(cm)

!

AD mice - vehicle!

AD mice - treatment!

Healthy mice!

10-14 animals / group!

Memory test!

Spatial memory measurements!

!

•! Ratio of time spent in the target quadrant vs opposite quadrant!

•! Area of influence around the platform!

! Platform crossings

$

Vehicle!

Vehicle!

Alzheimer Disease Mice!

(3xTg-AD)!

(Non-transgenic mice)!

Healthy Mice!

)#

*#

n = 14-24 animals/group!

Spatial memory test on 7m old 3xTg-AD !

Coma et al. US Patent Application 61/510,121 (2011) & 13/660,205 (2012) !

+",&-#"$

./011$

./012$

./0113./012$ 4"5'676"$

+",&-#"$

.8"'$9:$&6;<"6-"$'89<6=$

),"$>#'?985$

@&5"$A>"6)$&6$"'-,$

(22)

Is it disease-modifying ?!

0 50 100 150 VhAx 011 25uM Ax 013 10uM Ax 011+ 013 A!!!!40 % re sp ec t t o Veh ic le 0 50 100 150 Vh Ax 011 25uM Ax 013 10uM Ax 011+ 013 A!!!!42 % res pe ct to V eh ic le

PS70 cells

Mitterreiter S et al. J. Neurosci. 2010!

AAC

ChhEE iinnhhiibbiittiioonn!!

((iinn ccooll w

w JJaavviieerr SSaaeezz VVaalleerroo –– UUM

MH

H))!!

!

22% in blood and 0% in the brain!

Potential mechanism of action!

Ax_ALZ_011+013 reduce the synaptic noise in the

AD background and restore the synaptic plasticity!

(23)

!

•! Have set up a network biology approach to generate global

blockades in breast cancer!

•! Have set up a top-down systems biology approach that uses clinical

data to suggest therapeutically relevant biology in protein networks!

•! Have applied our strategy to AD (+ ALS and glaucoma), and found a

few compounds that show very interesting activities in cell and animal

models (positive and negative). Need to investigate them further!

•! Our approach is based on protein networks, and thus won´t work on

complex diseases that mainly involve other type of macromolecules!

•! Just our 5 cents towards network medicine approaches that should

take us beyond “cherry-picking” examples, and fully exploit the

information contained in the cell networks that we´re charting!

Concluding remarks!

SSBB&&N

NBB::!!

!

Montse soler!

Roberto Mosca!

Albert Pujol

!

Samira Jaeger!

Miquel Duran!

Laura Isús!

Teresa Juan!

Jofre Tenorio!

Rodrigo Arroyo!

Eva Capdevila!

Victor Alcalde !

Your Name Here !!

!

Acknowledgements!

C

Coollllaabboorraattoorrss::!!

FFuunnddiinngg aaggeenncciieess::!!

Gerhard Multhaup!

Alberto Lleó!

José M Mas, Mireia Coma!

& Teresa Sardón!

Javier Saez Valero!

(24)

Interactome3d and 3did websites

and databases updated!

Mosca et al. Nucleic Acids Res 2014! Mosca et al. Nat Methods 2013!

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