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26/11/2016
1
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
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