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Novos Fármacos Moduladores da Proteína CFTR Novos Fármacos Moduladores da Proteína CFTR

Margarida D AmaralLlsboa, 26 Novembro 2016

Margarida D AmaralLlsboa, 26 Novembro 2016

Semana Europeia da Fibrose Quística – Jornada de Divulgação em Fibrose QuísticaSemana Europeia da Fibrose Quística – Jornada de Divulgação em Fibrose Quística

The CF Pathogenesis CascadeThe CF Pathogenesis Cascade

Amaral & Kunzelmann (2007) Trends Pharmacol Sci 28: 334‐341Amaral & Kunzelmann (2007) Trends Pharmacol Sci 28: 334‐341

2 Defective CF Genes2 Defective CF Genes

Deficient CFTR ProteinDeficient CFTR Protein

Abnormal Cl‐ PermeabilityAltered Ionic Transport

CFTR

Chloride

Sodium

ENaCCFTR

Chloride

Sodium

ENaCCFTR

Chloride

Sodium

ENaC

Abnormal Cl‐ PermeabilityAltered Ionic Transport

CFTR

Chloride

Sodium

ENaCCFTR

Chloride

Sodium

ENaCCFTR

Chloride

Sodium

ENaC

Decreased Water in ASLThick Mucus

Decreased Water in ASLThick Mucus

End stage lung diseaseEnd stage lung disease

Act here to rescue the basic defect and block the 

CF cascade!

Act here to rescue the basic defect and block the 

CF cascade!

Most current therapies in CF!Most current 

therapies in CF!Mucus Obstruction  & 

BronchiectasisMucus Obstruction  & 

Bronchiectasis

Bacterial InfectionBacterial Infection

InflammationInflammation

ScarringScarring

Cycle of DestructionCycle of 

Destruction

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CFTR: from Gene to ProteinCFTR: from Gene to Protein

CFTR Gene  ‐ 190 kbCFTR Gene  ‐ 190 kb

1     2       3       4      5      6a    6b      7      8       9    10    11     12     13   14a   14b   15    16    17a   17b  18   19    20     21    22    23     24             1     2       3       4      5      6a    6b      7      8       9    10    11     12     13   14a   14b   15    16    17a   17b  18   19    20     21    22    23     24             1     2       3       4      5      6a    6b      7      8       9    10    11     12     13   14a   14b   15    16    17a   17b  18   19    20     21    22    23     24             

Protein ‐ 1480 aa

out

in

F508del C

N R

TM1 TM2

NBD1NBD2

Translation + Glycosylation

Protein ‐ 1480 aaProtein ‐ 1480 aa

outout

inin

F508delF508del C

NN RR

TM1TM1 TM2TM2

NBD1NBD1NBD2NBD2

Translation + GlycosylationTranslation + Glycosylation

Folding + Traffic

Epithelial cells

Cl‐

Folding + TrafficFolding + Traffic

Epithelial cellsEpithelial cells

Cl‐Cl‐

In CF: ~2,000 mutations!In CF: ~2,000 mutations!In CF: ~2,000 mutations!

Transcription + SplicingTranscription + SplicingmRNA ‐ 6.5 kbmRNA ‐ 6.5 kb

ExonsExons1    2     3       4     5     6a     6b   7    8      9      10     11    12     13   14a   14b   15    16    17a   17b  18    19    20     21    22     23    24                 1    2     3       4     5     6a     6b   7    8      9      10     11    12     13   14a   14b   15    16    17a   17b  18    19    20     21    22     23    24                 

Functional Classes of CFTR Mutations/ TheratypesFunctional Classes of CFTR Mutations/ Theratypes

De Boeck & Amaral MD (2016) Lancet Respir Med 4: 662‐74De Boeck & Amaral MD (2016) Lancet Respir Med 4: 662‐74

No mRNA

No mRNANo mRNA

VIIVII

dele2,3(21kb)dele2,3(21kb)

1717‐1G>A1717‐1G>A

394delTT

Reduced stabilityReduced stability

VIVI

A455EA455E

c.120del23c.120del23

rF508delrF508del

Less protein

VV

3272‐26A>G3272‐26A>G

A455EA455E

Reduced synthesisReduced synthesis

3849+10kbC>T3849+10kbC>T

Less protein

Less function

IVIV

R117HR117H

Defective conductanceDefective 

conductance

L206WL206W

R334WR334W

IIIIII

G551DG551D

Defective gating

Defective gating

S549RS549R

G1349DG1349D

No function

IIII

F508delF508del

N1303KN1303K

Defective processingDefective processing

A561EA561E

No traffic  

W1282XW1282X

II

G542XG542X

R1162XR1162X

DefectivesynthesisDefectivesynthesis

No protein

wt‐CFTRwt‐CFTR

CFTRCFTR

Mutation‐specific therapies!Mutation‐specific therapies!

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Mutações de Classe I Ausência de ProteínaMutações de Classe I Ausência de Proteína

1717‐1G>A (b)1717‐1G>A (b)

G542X (a)G542X (a)

394delTT (a)394delTT (a)

Class I MutationsClass I Mutations

Aim: Suppress Premature Stop Codons (PTCs) with read‐through compounds

Aim: Suppress Premature Stop Codons (PTCs) with read‐through compounds

Translation of mRNA into proteinTranslation of mRNA into protein

*In clinical trial*In clinical trial

Ex: GentamycinPTC‐124 (Ataluren)*

Ex: GentamycinPTC‐124 (Ataluren)*

mRNAmRNA

mRNA Degradation:  Nonsense‐Mediated 

Decay (NMD)

mRNA Degradation:  Nonsense‐Mediated 

Decay (NMD)

PTCPTC

StopStop

RibosomesRibosomes

mRNAmRNA

Full‐length ProteinFull‐length Protein

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Supression of Class I Mutations: G542X, W1282XSupression of Class I Mutations: G542X, W1282X

Kerem et al (2008) Lancet 372: 719‐27Kerem et al (2008) Lancet 372: 719‐27

PTC124 (Ataluren) is now in Clinical Phase III for patients who are not under aminoglycoside antibiotics

Results from Clinical Phase III did not reach significance. However, a positive effect was observed for patients who were not under aminoglycoside antibiotics

Kerem et al (2014) Lancet Respir Med 2: 539‐47

Mutações de Classe IIAusência de TráfegoMutações de Classe IIAusência de Tráfego

R1066CR1066C

F508delF508del

A561EA561E

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F508del: The Most Frequent Disease-Causing MutationF508del: The Most Frequent Disease-Causing Mutation

NC

TM1 TM2

NBD2NBD1

R Domain

NNCC

TM1TM1 TM2TM2

NBD2NBD2NBD1NBD1

R DomainR Domain

outout

inin

Complex Structure: Difficult to fold into native conformation!Complex Structure: Difficult to fold into native conformation!

F508del‐CFTR: even more difficult to fold!F508del‐CFTR: even more difficult to fold!

Phe508Phe508Phe508

Semi‐folded…Semi‐folded…Unfolded…Unfolded… Folded!Folded!

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GolgiGolgi

Cell MembraneCell Membrane

The ER Quality Control Retains Misfolded ProteinsThe ER Quality Control Retains Misfolded Proteins

Endoplasmic Reticulum

Endoplasmic Reticulum

Way OutWay Out

ProteasomeProteasome

The Endoplasmic Reticulum "Quality Control"

The Endoplasmic Reticulum "Quality Control"

The ER Quality ControlThe ER Quality ControlER-GolgiTraffic

Proteasomal degradationProteasomal degradation

ER lumen ER lumen

2nd Checkpoint: Calnexin cycle2nd Checkpoint: Calnexin cycle

Vesicle formationSec23/24-mediated cargo

selection

1st Checkpoint:prolonged association

with Hsc70

1st Checkpoint:prolonged association

with Hsc70

3rd CheckpointAFT-mediated

4th CheckpointDi-acidic exit code

UbHsc70

ChipChip

Phe508

Calnexin

1st and 2nd CheckpointsFolding/association with

chaperones

Farinha & Amaral, Mol & Cell Biol 2005Roxo‐Rosa et al, PNAS 2006Farinha et al, Chem Biol 2013

Farinha & Amaral, Mol & Cell Biol 2005Roxo‐Rosa et al, PNAS 2006Farinha et al, Chem Biol 2013

ERADERAD GERADGERAD

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Low Temperature Rescues F508del-CFTRLow Temperature Rescues F508del-CFTR

Band CBand C

Band BBand B

From: GM Denning (1992) Nature 358, 761‐764.From: GM Denning (1992) Nature 358, 761‐764.

We need to find alternative ways to rescue the mutant protein!

We need to find alternative ways to rescue the mutant protein!

Why Study Rescuing Mechanisms of F508del-CFTR?Why Study Rescuing Mechanisms of F508del-CFTR?

If we understand how mutant CFTR can be rescued to the cell surface, we may be able to mimic such effects with small molecules

If we find more than one way of doing this, we can use different molecules for enhanced results

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Rescue of F508del-CFTR with CorrectorsRescue of F508del-CFTR with Correctors

The outcome of the Phase III clinical trial with VX‐809/VX‐770 combination therapy were significant but modest!

The outcome of the Phase III clinical trial with VX‐809/VX‐770 combination therapy were significant but modest!

Mutações de Classe III / IVAusência / Menos FunçãoMutações de Classe III / IVAusência / Menos Função

G551DG551DS549RS549R

G1349DG1349D

R117HR117H

R334WR334W

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Class III/IV Mutations Class III/IV Mutations Rescuing Class III/IV MutationsRescuing Class III/IV Mutations

MSD2MSD1

NBD1 NBD2

ADP + PiADP + Pi

ATPATP

Class III‐ Regulation Defect:The channel does not respond!Class III‐ Regulation Defect:

The channel does not respond!

Cl‐Cl‐

Aim: To activate the channelwith Potentiators! (e.g.: VX‐770)Aim: To activate the channel

with Potentiators! (e.g.: VX‐770)

Cl‐Cl‐

MSD2MSD1

Class IV‐ Conductance Defect:Less ions flow through the channel

Class IV‐ Conductance Defect:Less ions flow through the channel

PP PP

ATPATP

PKAPKA PP2APP2CPP2APP2C

R Domain

Cl‐Cl‐

TM2TM1

VX-770 Effectively Improves Lung Function and Sweat Cl- in CF Patients with G551DVX-770 Effectively Improves Lung Function and Sweat Cl- in CF Patients with G551D

Sweat chloride  Sweat chloride  

Ramsey  (2011) NEJMRamsey  (2011) NEJM

Day 15 Week 8 Week 16 Week 24 Week 32 Week 40 Week 48-60

-55

-50

-45

-40

-35

-30

-25

-20

-15

-10

-5

0

5

PlaceboVX-770

Ch

ang

e in

sw

eat

chlo

rid

e co

nce

ntr

atio

nm

mo

l/L (

mea

n, 9

5%

CI)

Treatment effect through Week 24– 47.9 mmol/LP < 0.0001

Treatment effect through Week 48

– 48.1 mmol/LP < 0.0001

FEV1 % PredictedFEV1 % Predicted

Slide kindly provided by Kris De BoeckSlide kindly provided by Kris De Boeck

Ivacaftor FDA‐approved for: G178R, S549N, S549R, G551S, G970R, G1244E, S1251N, S1255P, G1349D + R117H

Ivacaftor FDA‐approved for: G178R, S549N, S549R, G551S, G970R, G1244E, S1251N, S1255P, G1349D + R117H

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Mutações de Classe VMenos ProteínaMutações de Classe VMenos Proteína

3272‐26A>G3272‐26A>G

A455EA455E

3849 + 10 kb C >T3849 + 10 kb C >T

Class V: Reduced SynthesisClass V: Reduced Synthesis

Less Normal Protein. Ex: 2789+5G>A (c.2657+5G>A)Less Normal Protein. Ex: 2789+5G>A (c.2657+5G>A)

ex13 ex14b ex15+5

intr 14bCFTR Gene =precursor mRNACFTR Gene =

precursor mRNASplicingSplicingSplicing

++Normal 

TranscriptNormal 

Transcript1514b14a13

Alternative transcript

1514a13Alternative transcriptAlternative transcript

1514a13

Rescuing Class V MutantsRescuing Class V Mutants

DrugDrugDrug AON*AON*

oror

StopStop

ex14a

*AON‐Antisense oligonucleotide*AON‐Antisense oligonucleotide

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Real‐time PCRReal‐time PCR

AON1 almost completely restored exon inclusion 

AON1 almost completely restored exon inclusion 

% exon 14b inclusion 62 95 78 49 % exon 14b inclusion 62 95 78 49

Wt Mut AON1 AON2 CtrlWt Mut AON1 AON2 Ctrl14a 14b 1513

13 14a 15

RT‐PCRRT‐PCR 14a 14b 1513 Western blot Western blot 

CNXCNX

CFTRCFTRBand CBand CBand BBand B

Wt Mut AON1 CtrlWt Mut AON1 Ctrl

AON1 increases CFTR protein levels AON1 increases CFTR protein levels 

0

50

100

150

200

250

300

Mut AON1 Ctrl

CF

TR

(%

co

ntr

ol r

elat

ive

to M

ut)

*

N=4N=4

***

0

10

20

30

40

50

60

70

80

90

Mut AON1 AON2 Ctrl

Exo

n I

ncl

usi

on

(%

)

*** N=4N=4

**

Susana IgrejaSusana Igreja

Correction of Aberrant Splicing by AONs*Correction of Aberrant Splicing by AONs*

*AON‐antisense oligonucleotide*AON‐antisense oligonucleotideIgreja et al (2016) Hum Mutat 37: 209‐15Igreja et al (2016) Hum Mutat 37: 209‐15

Class V Mutations : Novel TherapiesClass V Mutations : Novel Therapies

1. Increase the Number of Channels at Membrane

1. Increase the Number of Channels at Membrane

2. Stimulate Channels already at the cell membrane

2. Stimulate Channels already at the cell membrane

Also potentiators of CFTR function (e.g., VX‐770)?? Also potentiators of CFTR function (e.g., VX‐770)??

Correct alternative splicing

Also with correctors of traffic (e.g., VX‐809)??

Correct alternative splicing

Also with correctors of traffic (e.g., VX‐809)??

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Mutações de Classe VIMenor EstabilidadeMutações de Classe VIMenor Estabilidade

rF508delrF508del

c.120del23c.120del23

Rescued F508del-CFTR is Also Class VIRescued F508del-CFTR is Also Class VI

CL‐

Rescued F508del‐CFTR has much lower surface stability 

than wt‐CFTR

Rescued F508del‐CFTR has much lower surface stability 

than wt‐CFTR

Moniz et al (2013) ACS Chem Biol 8: 432‐42Moniz et al (2013) ACS Chem Biol 8: 432‐42

HGF alone rescues ~10% of F508del‐CFTR  function and with VX‐809 it 

rescues ~25%

HGF alone rescues ~10% of F508del‐CFTR  function and with VX‐809 it 

rescues ~25%

Paulo MatosPaulo Matos

How to stabilize F508del‐CFTR at the cell surface?How to stabilize F508del‐CFTR at the cell surface?

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Mutações de Classe VIICFTR "Não-Resgatável"Mutações de Classe VIICFTR "Não-Resgatável"

CFTRdele2,3(21kb)CFTRdele2,3(21kb)

Gene TherapyGene Therapy

Non‐toxic Low efficiency Non‐toxic Low efficiency

High efficiency Imunogenic High efficiency Imunogenic

LiposomesLiposomes Viral vectorsViral vectors

CFTR cDNACFTR cDNA

Alton et al (2015) Lancet Resp Med. Epub:3JulAlton et al (2015) Lancet Resp Med. Epub:3Jul

The transgene is usually silenced!

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CF: Increased Sodium TransportCF: Increased Sodium Transport

ENaC is hyperactive in CF  There is excessive sodium absorption

ENaC is hyperactive in CF  There is excessive sodium absorption

CFTR

Chloride

Sodium

ENaCCFTRCFTR

ChlorideChloride

SodiumSodium

ENaCENaC

CFTR

Normal chloride transport 

ENaC

normal sodium transport 

CFTR

Reduced chloride transport

ENaC

Increased sodium transport

CFTRCFTR

ChlorideChloride

SodiumSodium

ENaCENaC

Cystic FibrosisCystic Fibrosis

Inhibition of DGKι Normalizes ENaCInhibition of DGKι Normalizes ENaC

FMP‐fluorescence in A549 cellsFMP‐fluorescence in A549 cells Human primary CF lung cells in Ussing chamber

Human primary CF lung cells in Ussing chamber

Inhibition of DGKι delays fluid absorptionInhibition of DGKι delays fluid absorption

Diana FariaDiana Faria

Almaça, Faria et al (2013) Cell 154: 1390‐1400Almaça, Faria et al (2013) Cell 154: 1390‐1400

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Anoctamin/TMEM16 FamilyAnoctamin/TMEM16 Family

Family of 10 members –some are Cl‐ channels

Family of 10 members –some are Cl‐ channels

Ano1 channel is expressed in airway epithelial cellsAno1 channel is expressed in airway epithelial cells

Credits: Park et al. (2011)

? Ano1: An alternative 

Cl‐ channel for epithelial 

Cl‐ secretion ?

? Ano1: An alternative 

Cl‐ channel for epithelial 

Cl‐ secretion ?

Bypass the lack of functional CFTR in CFBypass the lack of 

functional CFTR in CF

Já Conseguimos Tratar Todosos Pacientes com FQ?Já Conseguimos Tratar Todosos Pacientes com FQ?

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Novas Abordagens Personalizadas para o Tratamento da FQ

Novas Abordagens Personalizadas para o Tratamento da FQ

Our Approach: ex vivo TestingOur Approach: ex vivo Testing

Intestinal OrganoidsIntestinal Organoids

Culture/expansionCulture/expansion

RNARNA

Q‐PCR (relative expression of allele‐specific transcripts

Q‐PCR (relative expression of allele‐specific transcripts

Transplanted lungs

Primary HBE cell cultureMolecular Biology/Biochemistry& Ussing Chamber measurements

Transplanted lungs

Primary HBE cell cultureMolecular Biology/Biochemistry& Ussing Chamber measurements

PatientPatient

Parameters of expression / function of CFTR vs Clinical PhenotypeParameters of expression / function of CFTR vs Clinical Phenotype

Ussing Chamber measurementsUssing Chamber measurements

Nasal epithelial cellsNasal epithelial cells

Rectal BiopsyRectal Biopsy

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Testing VX-809 Efficacy in Primary HBE Cells from CF PatientsTesting VX-809 Efficacy in Primary HBE Cells from CF Patients

Nikhil AwatadeNikhil 

AwatadeF508del/F508delF508del/F508del

DMSO

DMSO

VX‐809

VX‐809

A561E/A561EA561E/A561E N1303K/G542XN1303K/G542X

Awatade et al (2014) EBioMedicine 2: 147–153Awatade et al (2014) EBioMedicine 2: 147–153

VX‐809 rescues A561E (and possibly also Y1092X) in human primary airway cells but not N1303K→theratype classification of mutationsVX‐809 rescues A561E (and possibly also Y1092X) in human primary airway cells but not N1303K→theratype classification of mutations

Summary of VX-809 Efficacy in Primary HBEsSummary of VX-809 Efficacy in Primary HBEs

Nikhil AwatadeNikhil Awatade

Awatade et al (2014) EBioMedicine 2: 147–153Awatade et al (2014) EBioMedicine 2: 147–153

Poster #P49Poster #P49

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Cada Pessoa com Fibrose Quística é ÚnicaCada Pessoa com Fibrose Quística é Única

"From Rare to Care… ""From Rare to Care… "

Intestinal OrganoidsIntestinal Organoids

Adapted from Sato T and Clevers H (2013) Science 340: 1190  Adapted from Sato T and Clevers H (2013) Science 340: 1190  

Human colon biopsy

Human colon biopsy

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The future: personalized ex vivo biomarkers? The future: personalized ex vivo biomarkers?

Test drug effect ex vivo on patients’ own tissue Test drug effect ex vivo on patients’ own tissue Dekkers 2013, Nature Med

Intestinal organoids

Human Nasal Epithelial CellsHuman Nasal Epithelial Cells

Penque et al (2000) Lab Invest 80: 857‐68 Penque et al (2000) Lab Invest 80: 857‐68 

10 μm10 μm

Luka ClarkeLuka Clarke

Artur KmitArtur Kmit

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ConclusõesConclusões

Já existem (pré‐)fármacos moduladores da CFTR: Potenciador (VX770/Ivacaftor)  G551D + 8 mutações 

Corretor/ Potenciador (VX809 /Lumacaftor + Ivacaftor) F508del/F508del;

Classe I  PTC Supressores de PTC (em ensaio)

Classe II  novos compostos estão a sair da "pipeline": diferentes corretores parecem atuar em sinergia

Classe V  AONs para correção do splicing

Classe VII  Screens de siRNAs identificaram genes reguladores da ENaC (e ativadores de canais de Cl‐

alternativos  em curso).

Os fármacos já existentes podem ser testados ex vivo para outras mutações terapias personalizadas.

Financiamento CFF (USA) CF Trust (UK) European Union FCT, Portugal Gilead Génese

Financiamento CFF (USA) CF Trust (UK) European Union FCT, Portugal Gilead Génese

Colaboradores Celeste Barreto et cols

HSM, Lisboa, Portugal Jeff Beekman et cols

UMC Utrecht, The Netherlands Cal Cotton

Case Western, USA José Fragata et cols

Santa Marta, Lisboa, Portugal  Karl Kunzelmann

Univ Regensburg, Germany Amparo Solé et cols

Hospital La Fé, Valencia, Spain Rainer Pepperkok

EMBL‐Heidelberg, Germany Dirceu, Fernando Ribeiro et cols

University of Campinas, Brazil Sygnature Discovery

Nottingham, UK

Colaboradores Celeste Barreto et cols

HSM, Lisboa, Portugal Jeff Beekman et cols

UMC Utrecht, The Netherlands Cal Cotton

Case Western, USA José Fragata et cols

Santa Marta, Lisboa, Portugal  Karl Kunzelmann

Univ Regensburg, Germany Amparo Solé et cols

Hospital La Fé, Valencia, Spain Rainer Pepperkok

EMBL‐Heidelberg, Germany Dirceu, Fernando Ribeiro et cols

University of Campinas, Brazil Sygnature Discovery

Nottingham, UK

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Biosystems & Integrative Sciences Institute:Functional Genomics and Proteostasis GroupBiosystems & Integrative Sciences Institute:

Functional Genomics and Proteostasis Group