Margarida D Amaral Llsboa, 26 Novembro 2016

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1 Semana Europeia da Fibrose Quística Jornada de Divulgação em Fibrose Quística Novos Fármacos Moduladores da Proteína CFTR Margarida D Amaral Llsboa, 26 Novembro 2016 The CF Pathogenesis Cascade 2 Defective CF Genes Deficient CFTR Protein Abnormal Cl Permeability Altered Ionic Transport Chloride CFTR Sodium ENaC Act here to rescue the basic defect and block the CF cascade! Decreased Water in ASL Thick Mucus Mucus Obstruction & Bronchiectasis Cycle of Destruction Scarring Most current therapies in CF! Bacterial Infection Inflammation End stage lung disease Amaral & Kunzelmann (2007) Trends Pharmacol Sci 28:

2 CFTR: from Gene to Protein CFTR Gene 190 kb In CF: ~2,000 mutations! a 6b a 14b a 17b mrna 6.5 kb Transcription + Splicing a 6b a 14b a 17b Exons Cl Translation + Glycosylation Epithelial cells Folding + Traffic N TM1 F508del NBD1 TM2 R Protein 1480 aa out in NBD2 C Functional Classes of CFTR Mutations/ Theratypes CFTR No protein No traffic No function Less function Less protein Less protein No mrna wt CFTR I II III IV V VI VII Defective synthesis Defective processing Defective Defective Reduced gating conductance synthesis Reduced stability No mrna G542X R1162X W1282X F508del A561E N1303K G551D S549R G1349D De Boeck & Amaral MD (2016) Lancet Respir Med 4: R117H L206W R334W A455E c.120del A>G rf508del kbC>T A455E dele2,3(21kb) G>A 394delTT Mutation specific therapies! 2

3 Mutações de Classe I Ausência de Proteína G542X (a) 394delTT (a) G>A (b) Class I Mutations Aim: Suppress Premature Stop Codons (PTCs) with read through compounds Translation of mrna into protein Ribosomes mrna Ex: Gentamycin PTC 124 (Ataluren)* Full length Protein PTC *In clinical trial mrna Stop mrna Degradation: Nonsense Mediated Decay (NMD) 3

4 Supression of Class I Mutations: G542X, W1282X 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: Kerem et al (2008) Lancet 372: PTC124 (Ataluren) is now in Clinical Phase III for patients who are not under aminoglycoside antibiotics Mutações de Classe II Ausência de Tráfego R1066C A561E F508del 4

5 F508del: The Most Frequent Disease-Causing Mutation TM1 N Phe 508 NBD1 R Domain TM2 C out NBD2 in Complex Structure: Difficult to fold into native conformation! F508del CFTR: even more difficult to fold! Unfolded Semi folded Folded! 5

6 26/11/2016 The ER Quality Control Retains Misfolded Proteins Cell Membrane The Endoplasmic Reticulum "Quality Control" Golgi Way Out Endoplasmic Reticulum Proteasome ER-Golgi Traffic The ER Quality Control 4th Checkpoint Di-acidic exit code 3rd Checkpoint AFT-mediated Vesicle formation Sec23/24-mediated cargo selection 1st and 2nd Checkpoints Folding/association with chaperones Phe508 1st Checkpoint: prolonged association with Hsc70 ER lumen 2nd Checkpoint: Calnexin cycle Calnexin Ub Hsc70 Chip ERAD Farinha & Amaral, Mol & Cell Biol 2005 Roxo Rosa et al, PNAS 2006 Farinha et al, Chem Biol 2013 GERAD Proteasomal degradation 6

7 Low Temperature Rescues F508del-CFTR Band C Band B From: GM Denning (1992) Nature 358, We need to find alternative ways to rescue the mutant protein! 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 7

8 Rescue of F508del-CFTR with Correctors The outcome of the Phase III clinical trial with VX 809/VX 770 combination therapy were significant but modest! Mutações de Classe III / IV Ausência / Menos Função R117H R334W G551D S549R G1349D 8

9 Class Rescuing III/IV Class Mutations III/IV Mutations Class III Regulation Defect: The channel does not respond! Class IV Conductance Defect: Less ions flow through the channel Cl Cl MSD1 TM1 MSD2 TM2 ATP ADP + P i Aim: To activate the channel with Potentiators! (e.g.: VX 770) NBD1 R Domain P PKA ATP P PP2A PP2C NBD2 VX-770 Effectively Improves Lung Function and Sweat Cl - in CF Patients with G551D FEV 1 % Predicted Sweat chloride 5 0 Change in sweat chloride concentration mm ol/l (m ean, 95% CI) Placebo VX-770 Treatment effect through Week mmol/l P < Treatment effect through Week mmol/l P < Day 15 Week 8 Week 16 Week 24 Week 32 Week 40 Week 48 Ramsey (2011) NEJM Ivacaftor FDA approved for: G178R, S549N, S549R, G551S, G970R, G1244E, S1251N, S1255P, G1349D + R117H Slide kindly provided by Kris De Boeck 9

10 Mutações de Classe V Menos Proteína A455E A>G kb C >T Class Rescuing V: Reduced Class V Synthesis Mutants Less Normal Protein. Ex: G>A (c g>a) ex13 ex14a ex14b +5 ex15 CFTR Gene = intr 14b precursor mrna Splicing Stop Drug or AON* Alternative transcript Normal Transcript 13 14a a 14b 15 *AON Antisense oligonucleotide 10

11 Correction of Aberrant Splicing by AONs* RT PCR 13 14a 14b 15 Western blot Susana Igreja Wt Mut AON1 AON2 Ctrl % exon 14b inclusion Exon Inclusion (%) Real time PCR *** N=4 *** Mut AON1 AON2 Ctrl AON1 almost completely restored exon inclusion Igreja et al (2016) Hum Mutat 37: a 14b a 15 CFTR Band C Band B CFTR (% control relative to Mut) CNX Wt Mut AON1 Ctrl Mut AON1 Ctrl AON1 increases CFTR protein levels * N=4 *AON antisense oligonucleotide Class V Mutations : Novel Therapies 1. Increase the Number of Channels at Membrane Correct alternative splicing Also with correctors of traffic (e.g., VX 809)?? 2. Stimulate Channels already at the cell membrane Also potentiators of CFTR function (e.g., VX 770)?? 11

12 Mutações de Classe VI Menor Estabilidade c.120del23 rf508del Rescued F508del-CFTR is Also Class VI CL Paulo Matos Rescued F508del CFTR has much lower surface stability than wt CFTR How to stabilize F508del CFTR at the cell surface? Moniz et al (2013) ACS Chem Biol 8: HGF alone rescues ~10% of F508del CFTR function and with VX 809 it rescues ~25% 12

13 Mutações de Classe VII CFTR "Não-Resgatável" CFTRdele2,3(21kb) Gene Therapy CFTR cdna Liposomes Viral vectors Non toxic Low efficiency Alton et al (2015) Lancet Resp Med. Epub:3Jul The transgene is usually silenced! High efficiency Imunogenic 13

14 CF: Increased Sodium Transport Chloride Sodium CFTR ENaC CFTR ENaC Normal chloride transport normal sodium transport Chloride CFTR Sodium ENaC Cystic Fibrosis CFTR Reduced chloride transport ENaC Increased sodium transport ENaC is hyperactive in CF There is excessive sodium absorption Inhibition of DGKι Normalizes ENaC FMP fluorescence in A549 cells Diana Faria Human primary CF lung cells in Ussing chamber Inhibition of DGKι delays fluid absorption Almaça, Faria et al (2013) Cell 154:

15 Anoctamin/TMEM16 Family Family of 10 members some are Cl channels Ano1 channel is expressed in airway epithelial cells Credits: Park et al. (2011) Bypass the lack of functional CFTR in CF? Ano1: An alternative Cl channel for epithelial Cl secretion? Já Conseguimos Tratar Todos os Pacientes com FQ? 15

16 Novas Abordagens Personalizadas para o Tratamento da FQ Our Approach: ex vivo Testing Parameters of expression / function of CFTR vs Clinical Phenotype Patient Rectal Biopsy Intestinal Organoids Ussing Chamber measurements Nasal epithelial cells Transplanted lungs Primary HBE cell culture Molecular Biology/Biochemistry & Ussing Chamber measurements Culture/expansion RNA Q PCR (relative expression of allelespecific transcripts 16

17 Testing VX-809 Efficacy in Primary HBE Cells from CF Patients F508del/F508del A561E/A561E N1303K/G542X Nikhil Awatade VX 809 DMSO Awatade et al (2014) EBioMedicine 2: Summary of VX-809 Efficacy in Primary HBEs Poster #P49 Nikhil Awatade VX 809 rescues A561E (and possibly also Y1092X) in human primary airway cells but not N1303K theratype classification of mutations Awatade et al (2014) EBioMedicine 2:

18 Cada Pessoa com Fibrose Quística é Única "From Rare to Care " Intestinal Organoids Human colon biopsy Adapted from Sato T and Clevers H (2013) Science 340:

19 The future: personalized ex vivo biomarkers? Intestinal organoids Test drug effect ex vivo on patients own tissue Dekkers 2013, Nature Med Human Nasal Epithelial Cells Luka Clarke 10 μm Penque et al (2000) Lab Invest 80: Artur Kmit 19

20 Conclusões 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 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 20

21 Biosystems & Integrative Sciences Institute: Functional Genomics and Proteostasis Group 21