Molecular Basis of Personalized Therapies for CF: Can We Treat All Patients?

Transcription

1 CF Europe Meeting Molecular Basis of Personalized Therapies for CF: Can We Treat All Patients? Margarida D Amaral Brussels, 13 June 2015 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 Different Functional Classes of CFTR Mutations CFTR wt CFTR I II III IV V VI No protein No traffic No Function Less Function Less Protein Less Stable G542X (a) 394delTT (a) G>A (b) Amaral (2015) J Int Med 277: ] R1066C A561E F508del G551D S549R G1349D R117H R334W A455E c.120del A>G A455E kb C >T rf508del 2

3 CF: From Genotypes to Theratypes PTC Supressors Correctors Potentiators Stabilizers wt CFTR G542X (a) 394delTT (a) G>A (b) Rescue Protein Synthesis R1066C A561E F508del Rescue Traffic G551D S549R G1349D R117H R334W Restore Channel Activity A455E c.120del A>G A455E kb C >T rf508del Stabilize Protein Amaral MD (2015) J Intern Med 277: Class III / IV Mutations No / Less Function R117H R334W G551D S549R G1349D 3

4 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 Slide kindly provided by Kris De Boeck 4

5 Class II Mutations No Traffic R1066C A561E F508del 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! 5

6 Unfolded Semi folded Folded! The ER Quality Control Retains Misfolded Proteins Cell Membrane The Endoplasmic Reticulum "Quality Control" Golgi Way Out Endoplasmic Reticulum Proteasome 6

7 20/06/2015 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 Low Temperature Rescues F508del-CFTR Band C Band B We need to find alternative ways to rescue the mutant protein! From: GM Denning (1992) Nature 358,

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! VX661-VX770 Combination Phase 2 Study Inclusion criteria: Homozygous F508del; FEV 1 : 40 90%; > 18 years Results Modest but stat significant sweat chloride changes Modest and variable but significant lung function changes at d28 Donaldson S VX 661, an investigational CFTR corrector, in combination with ivacaftor, a CFTR potentiator, in patients with CF and homozygous for the F508Del CFTR mutation: an interim analysis abstract WS 7.3 8

9 Treating CF Patients: Are we there yet? Do We Still Need to Discover New Drugs for CF? Despite exciting, novel drugs approved/under approval have had limited success because: 1) ivacaftor only applies to patients with gating mutations, i.e. ~5% of patients; 2) lumacaftor/ivacaftor combination therapy is limited to F508del homozygous, i.e. ~40% of patients and only showed 3% improvement in lung function. There is an unmet need for novel drugs for the majority (55%) of CF patients, namely: for those who have two very rare ("orphan") mutations; more effective novel drugs to restore F508del CFTR. 9

10 Class I Mutations No Protein G542X (a) 394delTT (a) G>A (b) PTC Mutations ~24% of all CFTR mutations comprise pathogenic mutations that generate premature termination codons (PTCs): 8.35% nonsense mutations (PTC mutations) 15.66% frameshift, most also leading to PTCs These mutations lead to a significant reduction or total absence of normal CFTR mrna (and protein) expression and most are associated with particularly severe clinical phenotypes. Therapies to correct them have been neglected. 10

11 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) 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 11

12 Identifying Novel F508del-CFTR Rescuing Compounds Why Study Rescuing Mechanisms of F508del-CFTR? If we understand how mutant CFTR can be rescued to the cell surface, we perhaps mimic such effects with small molecules If we find more than one way of doing this, we can use different molecules for enhanced results 12

13 Traffic Assay for High-Throughput Screens Anti Flag Ab Flag C Inducible Expression of tagged CFTR in Human Epithelial Respiratory Cells Total CFTR Cell surface CFTR Shehrazade Dahimène N mcherry mcherry Flag F508del CFTR mcherry Flagwt CFTR Almaça et al (2011) Methods Mol Biol 742: High-Content Microscopy Screens to Identify Novel F508del-CFTR Correctors Hugo Botelho Coat 384 well plates with sirnas or drugs Cells seeded on top (reverse transfection) Automated Imaging Analysis 13

14 Class V Mutations Less Protein 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 PTC Drug or AON* Alternative transcript Normal Transcript 13 14a b 14b 15 *AON Antisense oligonucleotide 14

15 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% Class VII Mutations "Unrescuable" CFTR CFTRdele2,3(21kb) 15

16 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 an excessive sodium absorption sirnas Screens (6,396 genes) by Automated Microscopy Joana Almaça 739 ENaC activators Data Analysis &e Bioinformatics Validation and Classification of Top Hits 7 ENaC activators were out of the known ENaC regulator pathways 16

17 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: Identifying Novel Regulators of TMEM16A/ Anoctamin 1 17

18 New Personalized Approaches to CF Treatment The future: personalized ex vivo biomarkers 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: Poster #P49 18

19 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: Human Nasal Epithelial Cells Luka Clarke 10 μm 200 μm Penque et al (2000) Lab Invest 80: Doucet et al (2003) J Histochem Cytochem 51:

20 Intestinal Organoids Human colon biopsy Adapted from Sato T and Clevers H (2013) Science 340: 1190 The future: personalized ex vivo biomarkers? Intestinal organoids Test drug effect ex vivo on patients own tissue Dekkers 2013, Nature Med 20

21 Personalized Medicine for Cystic Fibrosis Functional biomarkers for CF diagnosis, prognosis and drug response Patient s cells/ tissues Functional tests: Diagnosis Prognosis Drug response Functional Analyses Point ofcare analysis Comfortable for patient Easy to perform Reduces medical visits Cuts diagnosis time Lowers costs Assesses response to drugs Summary We already have (pro )drugs modulating CFTR: Potentiator (VX770/Ivacaftor) G551D + 8 mutations Corrector/ Potentitor (VX809 /Lumacaftor + Ivacaftor) F508del/F508del; Class I PTC Supressors Class II new compounds coming out of the "pipeline": different correctors seem to act synergistically Class V AONs to correct splicing Class VII High content sirna microscopy screens can help to identify regulators of ENaC and activators od alternative Cl channels. Existing drugs can be tested ex vivo for their effects on other mutations towards personalized therapies. 21

22 Funding CFF (USA) European Union FCT, Portugal Gilead Génese Collaborators Celeste Barreto et cols HSM, Lisboa, Portugal Jeff Beekman et cols UMC Utrecht, The Netherlands 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 Biosystems & Integrative Sciences Institute 22