Cysc ﬁbrosis: a simple monogene$c loss of func$on disease ... · Hemoptysis, Cor pulmonale Liver Impaired Cl - secretion; obstructive biliary tract disease with liver cirrhosis

Cys$cfibrosis:asimplemonogene$clossoffunc$ondisease?

Thecys$cfibrosistransmembraneregulator

anditsregula$on

Gray balloons on the M7-M8 loop = N-linked glycosylation sites. R, R-domain NBD-1, NBD-2: Nucleotide binding domains

Transmembrane topology of CFTR.

Human ABC gene subfamilies

Subfamily Aliases Number Number Name of genes of pseudogenes ABCA ABC1 12 5 ABCB MDR 11 4 ABCC MRP 13 2 ABCD ALD 4 4 ABCE OABP 1 2 ABCF GGN20 3 2 ABCG White 5 2 Total 49 21

CFTR= ABCC7 on long arm of chromo- some 7

The dendrogram of 30 human ABC proteins represented by complete sequences in the sequence databases

Vitamin B12 transporter-complex BtuCD-BtuF (front view and rotated by 90°)

The periplasmatic binding protein BtuF (red) has been docked onto the transporter in its open substrate-free conformation. The membrane portion of BtuCD (blue and yellow) are connected to the nucleotide-binding domains (gree and cyan). The translocation pathway is closed on both sides.

Gray balloons on the M7-M8 loop = N-linked glycosylation sites. R, R-domain NBD-1, NBD-2: Nucleotide binding domains


Organ affected Transport defect and symptoms

Skin Defective reabsorption of NaCl with enhanced salt concentration in the sweat (Cl-> 60 mmol/L)

Lung Enhanced absorption of Na+ and decreased Cl- secretion; reduced airway surface liquid, increased mucus viscosity, reduced mucociliary clearance

Chronic airway inflammation

Recurrent bronchitis and pneumonia, bronchiectasis

Lung colonization with Staphylococcus aureus and Pseudomonas aeruginosa

Hemoptysis, Cor pulmonale

Liver Impaired Cl- secretion; obstructive biliary tract disease with liver cirrhosis

Pancreas Defective HCO3- secretion and impaired Cl- conductance; cystic degeneration of pancreatic tissue

Exocrine pancreatic insufficiency deficiency of digestive enzymes

Endocrine pancreatic insufficiency insulin-dependent diabetes mellitus

Small and large intestine

Hyperabsorption of NaCl, defective Cl- and HCO3 - secretion; meconium ileus, distal intestinal obstructive syndrome, meconium ileus equivalent in adults, anal prolapse

Kidney Eventually enhanced Na+ absorption in collecting duct; disturbed urinary concentration?

Reproductive tract Defective Cl- and HCO3 - secretion, hyperabsorption of NaCl; congenital bilateral absence of vas deference; male infertility, dysfunction of the endometrium

Organ Symptoms Standard treatment

Lung and respiratory tract

Nasal polyps and chronic sinusitis Surgery

Mucus impactions Physiotherapy, inhalative therapy, mucolytic compounds, DNAse

Recurrent bacterial infection and chronic colonization

Antibiotic treatment (orally, intravenously, inhalative)

Obstructive ventilatory disease and allergic pulmonary aspergillosis

2-Adrenoceptor agonists, glucocorticoids

Respiratory failure O2 therapy, lung transplantation

Gastrointestinal tract Meconium ileus Surgery

Exocrine pancreatic insufficiency Pancreatic enzyme replacement (lipase)

Malabsorption Substitution of fat-soluble vitamins

Failure to thrive Hypercaloric nutrition, feeding tube

Obstructive biliary disease Ursodesoxycholic acid

Endocrine pancreatic insufficiency Treatment of diabetes mellitus

Standard medication and therapy of cystic fibrosis

Proportion of Patients Free of Exacerbations of Respiratory Symptoms Requiring Parenteral Antibiotic Therapy

“An exacerbation of respiratory symptoms, prospectively defined in the study, was said to have occurred when a patient was treated with parenteral antibiotics for any 4 of the following 12 signs or symptoms: change in sputum; new or increased hemoptysis; increased cough; increased dyspnea; malaise, fatigue, or lethargy; temperature above 38°C; anorexia or weight loss; sinus pain or tenderness; change in sinus discharge; change in physical examination of the chest; decrease in pulmonary function by 10 percent or more from a previously recorded value; or radiographic changes indicative of pulmonary infection.“

Endpoint - Pulmozyme

Fuchs HJ et al; Effect of aerosolized recombinant human DNase on exacerbations of respiratory symptoms and on pulmonary function in patients with cystic fibrosis. The Pulmozyme Study Group; N Engl J Med. 1994 Sep 8;331(10):672-3.

Mean Percent Change in FEV1 from Base Line Throughout the 24-Week Study Period



Moss RB; Long Term Benefits of Inhaled Tobramycin in Adolescent Patients with Cystic

Fibrosis (Chest 2002; 121:55-63)

Endpoint - Tobramycin

The mean relative change in FEV1 percent predicted for the intent-to-treat adolescent population is shown over time. Baseline is the week that TSI therapy was initiated (week 0 for patients originally randomized to TSI treatment; week 24 for those originally randomized to placebo treatment). Shaded bars represent 28-day on-drug periods. Means at week 96 were derived from 27 TSI-arm and 37 placebo-arm patients who completed the trial. Ramsey et al12 reported a treatment effect of 23.03% at week 20 (p 0.001). † End of on-drug period measurement not recorded.

Endpoint - Tobramycin

Moss RB; Long Term Benefits of Inhaled Tobramycin in Adolescent Patients with Cystic Fibrosis (Chest 2002; 121:55-63)

The mean change in BMI from the beginning to the last visit of each 24-week study period is shown. BMI was calculated as weight in kilograms/height in meters squared. * = Patients in the original placebo group received TSI after week 24.

phenotype: autosomal recessive; carrier frequency 1:25⇒ heterozygote advantage ? (V. cholerae; S. Typhi)

190 kb; 27 exons

6.5 kb

1480 aa; 168 kDa

7q31.2

Figure 2 Schematic overview of the folding pathway of CFTR.

Crofelemer,anAn$secretoryAn$diarrhealProanthocyanidinOligomerExtractedfromCrotonlechleri,TargetsTwoDis$nct

Intes$nalChlorideChannels

Fig. 1. Cellular mechanisms of intestinal fluid secretion by enterocytes, showing chloride secretion through apical membrane chloride channels.

Crofelemer (Fulyzaq®) approved by the FDA in 2012 for 2012 by the FDA for "symptomatic relief of non-infectious diarrhoea in patients with HIV/AIDS on anti-retroviral therapy”.

Gray balloons on the M7-M8 loop = N-linked glycosylation sites. R, R-domain Channel lining = residues from M1, M3, M6, and M12: gate (that blocks ion conduction through the channel in the closed state):? linear current voltage relation anion selectivity (Cl-/Na+ permeability ratio=10-300) permeability sequence: I (1.8) > Br (1.2) > Cl (1.0) > F (0.2), R352 (at cytoplasmic end of M6) = major determinant of charge selectivity


Schematic illustrating the structure of CFTR and the relationships of its domains and associated proteins to the channel. Membrane-spanning domains, red; the water-filled transmembrane channel, blue; NBD-1, yellow; NBD-2, orange; bound ATP, white ovals; R-domain, green; the N- and C termini are indicated by black lines; the associated proteins syntaxin 1A (Syn 1A) and Na+/H+ exchanger regulatory factor/ezrin-binding protein 50 (NHERF/EBP-50), purple. Note the position of Arg-352, a major determinant of charge selectivity, at the narrow point in the channel. Cl ions probably do not interact with the NBDs and R-domain as they pass through the channel. The cytoplasmic vestibule has a binding site for large anions. The interactions with PP2C and other channels and transporters are not shown.

Regulation of CFTR Channel Gating

•  Phosphorylation is a prerequisite for channel activation PKA, PKC, PKG: PKC → PKA ⇑ 5 sites out of 10 on the R-domain

•  Channel gating requires ATP hydrolysis:

Km for ATP = 0.3 mM Turnover rate = ~1 molecule/s (≈rate of channel gating)

(a) Putative transmembrane arrangement of the cystic fibrosis transmembrane con-ductance regulator (CFTR), showing 2 transmembrane do-mains, 2 nucleotide binding folds and a large cytosolic regulatory domain (R-domain). (b) Cystic fibrosis transmembrane con-ductance regulator whole-cell Cl currents measured in a patch-clamp experiment with CFTR-expressing CHO cells. Cells were voltage clamped from -40 to +60 mV in steps of 20 mV. Currents are shown before (-IBMX) and after (+IBMX) stimu-lation with 100 µmol/L IBMX. (c) Recording of a CFTR single Cl channel current obtained from a cultured airway epithelial cell in a cell-attached patch-clamp experiment. The arrow indicates the closed state of the channel. (Vc = 100 mV; pipette filled with 145 mmol/L NaCl).

CFTR is a Transmembrane Conductance Regulator

because CFTR also regulates the conductance of other channels: •  ENaC: CFTR inhibits ENaC

mechanism = direct interaction? •  AQP3: CFTR increases H2O-permeability

mechanism? •  Kv LQT1 & ROMK1: CFTR increases K+-permeation

Cellular models of electrolyte secretion (a) and electrolyte absorption (b) in the airways and intestinal epithelium. (a) In secretory cells, Cl- is taken up from the basolateral (blood) side by the Na+/K+/2 Cl cotransporter. K+ recycles via basolateral K+ channels and Na+ is pumped out of the cell by the Na+/K+-ATPase. Cl - leaves the cell via luminal (apical) cystic fibrosis transmembrane conductance regulator (CFTR) Cl channels and Na+ is secreted via the paracellular shunt following the electrical driving force generated by the lumen negative transepithelial voltage. K+ is also secreted to the luminal side via luminal K+ channels. Depending on the tissue, intracellular cAMP is increased and secretion is activated by adenosine or prostaglandin E2 (PGE2).

Cellular models of electrolyte secretion in the airways and intestinal epithelium. (b) In absorptive epithelial cells, Na+ is taken up by luminal epithelial Na+ channels (ENaC). Cl- is transported via the paracellular shunt and probably via CFTR Cl channels. Na+ is pumped out of the cell by the basolateral Na+/K+-ATPase, whereas Cl- and K+ leave the cell via Cl- and K+ channels, respectively. In absorptive epithelial cells coexpressing CFTR and ENaC, stimulation of CFTR leads to inhibition of ENaC. Thus, cAMP-mediated stimulation may shift the epithelium from absorption towards secretion of NaCl. PKA, protein kinase A.

Model of the airway epithelium consisting of an

absorptive surface epithelium (expressing epithelial Na+ channels (ENaC) and cystic fibrosis transmembrane conductance regulator (CFTR))

and secretory submucosal glands (predominantly expressing CFTR)..

Under baseline conditions, non-cystic fibrosis (CF) airway epithelia show net absorption. In CF airways, hyperabsorption occurs due to an increase in epithelial Na+ conductance (surface epithelium) and lack of CFTR-mediated Cl secretion in submucosal glands.

Under baseline conditions, non-cystic fibrosis (CF) airway epithelia show net absorption. In CF airways, hyperabsorption occurs due to an increase in epithelial Na+ conductance (surface epithelium) and lack of CFTR-mediated Cl secretion in submucosal glands. Following cAMP-depen-dent stimulation, the non-CF, but not the CF, epi-thelium is able to switch from net absorption to net secretion by activation of secretion in submucosal glands and inhibition of absorption in the surface epithelium.

In CF, impairment of CFTR function causes reduced fluid production. Enhanced sodium absorption through epithelial Na+ channels (ENaC) and basolateral Na/K ATPase pumps results in increased fluid absorption leading to drier airways and impaired ciliary clearance.

Normal ciliary clearance of bacteria and foreign particles. Cilia are fine hair like structures which cover the lining of the airways like a carpet. They beat continuously and act like a conveyor belt to remove bacteria and particles stuck to the mucus layer, driving them from the lungs to the mouth.

The chest X-ray often underestimates the presence and severity of mild and moderate lung disease. The above diagram shows some of chest X-ray and CT scan changes which oocur in CF (e.g. 1 bronchiectasis/dilated airways, 2 mucus plugging). Treatment aims to prevent/slow down the development of lung damage which can occur insidiously.

Organ Symptoms Standard treatment

Lung and respiratory tract

Nasal polyps and chronic sinusitis Surgery

Mucus impactions Physiotherapy, inhalative therapy, mucolytic compounds, DNAse

Recurrent bacterial infection and chronic colonization

Antibiotic treatment (orally, intravenously, inhalative)

Obstructive ventilatory disease and allergic pulmonary aspergillosis

2-Adrenoceptor agonists, glucocorticoids

Respiratory failure O2 therapy, lung transplantation

Gastrointestinal tract Meconium ileus Surgery

Exocrine pancreatic insufficiency Pancreatic enzyme replacement (lipase)

Malabsorption Substitution of fat-soluble vitamins

Failure to thrive Hypercaloric nutrition, feeding tube

Obstructive biliary disease Ursodesoxycholic acid

Endocrine pancreatic insufficiency Treatment of diabetes mellitus

Standard medication and therapy of cystic fibrosis

VX-770; approved in 2012 by FDA and EMA as Kalydeco®

for mutations: G178R, S549N, S549R, G551S, G1244E, S1251N, S1255P, G1349D and R117H.

VX-770 (ivacaftor) acted as a potentiator, not an activator, of G551D- and F508del CFTR in recombinant cells.

Van Goor F et al. PNAS 2009;106:18825-18830

©2009 by National Academy of Sciences

Atarulen

VX-770 potentiated the gating activity of CFTR. Representative patch-clamp recording of the single channel current resulting from activation of G551D CFTR in FRT cells (A) and F508del

CFTR in NIH 3T3 cells (B) by 1 mM ATP and 75 nM PKA before and during VX-...



VRT-768 and VX-809 (lumacaftor) increased F508del-CFTR maturation and chloride transport in FRT cells.



VX-809 = Lumacaftor

Biochemical and functional data suggest that VX-809 (lumacaftor) acted at the level of the ER to allow a fraction of the F508del-CFTR pool to adopt a properly folded form.



Mean Percent Change in FEV1 from Base Line Throughout the 24-Week Study Period



Proportion of Patients Free of Exacerbations of Respiratory Symptoms Requiring Parenteral Antibiotic Therapy


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Cysc ﬁbrosis: a simple monogene$c loss of func$on disease ... · Hemoptysis, Cor pulmonale Liver Impaired Cl - secretion; obstructive biliary tract disease with liver cirrhosis