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C ti FCystic FFrom Gene DFrom Gene D
MolecularDavid N. She
School of PhysiologyUniversity of Br
E-mail: D.N.Shepp
Fib iFibrosis: Discovery toDiscovery to r Medicineeppard, Ph.D.pp ,
y and Pharmacology,istol, Bristol, UK
Steps on the PathwDrug Therapies forDrug Therapies for
Describe the Cli
Investigate the Bioch
Identify the De
Characterise the Normal Fu
U d t d H M t ti Di tUnderstand How Mutations Disrupt
Drug T
way to Developing r Genetic Disordersr Genetic Disorders
nical Phenotype
hemical Abnormality
efective Gene
unction of the Gene Product
t th F ti f th G P d tt the Function of the Gene Product
herapy
An Early Reference
“Woe to that ckissed on thekissed on thesalty. He is bewmust die”Folklore from Medie al NortFolklore from Medieval Nort
e to Cystic Fibrosis
child which whenforehead tastesforehead tastes
witched and soon
thern E ropethern Europe
(Cystic Fibro
Dorothy Andersen
A complex genetic diseaorgans systems includirespiratory tract, the gastrliver, sweat glands and the
CYSTIC FIBROSIS
osis of the Pancreas or M i id i )Mucoviscidosis)
ase affecting a number ofng the lung and upperrointestinal tract, pancreas,e genitourinary tract.
Organs Affected b
Respiratory Airways
P
Liver
Small
Pancreas
Reproductive Tract
Intestine
Tract
by Cystic Fibrosis
The genetic defect thatunderlies cystic fibrosiscauses ducts and tubesto become blocked bythick sticky mucus.thick sticky mucus.
Steps on the PathwDrug Therapies forDrug Therapies for
Ducts and Tubes Become Blo
Investigate the Bioch
Identify the De
Characterise the Normal Fu
U d t d H M t ti Di tUnderstand How Mutations Disrupt
Drug T
way to Developing r Genetic Disordersr Genetic Disordersocked by Thick Sticky Mucus
hemical Abnormality
efective Gene
unction of the Gene Product
t th F ti f th G P d tt the Function of the Gene Product
herapy
Chloride MovemeW t SWater Se
apical
PKA
CC
PKA: Protein Kinase A
nts Drive Salt and tiecretion
basolateral
Cl- Cl-Cl Cl
Chloride MovemeW t SWater Se
apical
PKA
CC
PKA: Protein Kinase A
nts Drive Salt and tiecretion
basolateralH OH2O
Cl- Cl-Cl Cl
Na+
apical NORMA
ClPKA
Cl-
Paul Quinton
CYSTIC FIB
Paul Quinton
PKA
CYSTIC FIB
Cl-
basolateralAL
ClCl-
BROSISBROSIS
Cl-
Steps on the PathwDrug Therapies forDrug Therapies for
Ducts and Tubes Become Blo
Defective Chloride Tran
Identify the De
Characterise the Normal Fu
U d t d H M t ti Di tUnderstand How Mutations Disrupt
Drug T
way to Developing r Genetic Disordersr Genetic Disordersocked by Thick Sticky Mucus
nsport Across Epithelia
efective Gene
unction of the Gene Product
t th F ti f th G P d tt the Function of the Gene Product
herapy
Steps on the PathwDrug Therapies forDrug Therapies for
Ducts and Tubes Become Blo
Defective Chloride Tran
Identification and Clon
Characterise the Normal Fu
U d t d H M t ti Di tUnderstand How Mutations Disrupt
Drug T
way to Developing r Genetic Disordersr Genetic Disordersocked by Thick Sticky Mucus
nsport Across Epithelia
ning of the CFTR Gene
unction of the Gene Product
t th F ti f th G P d tt the Function of the Gene Product
herapy
The Structure of CFTR
0
A D
198
300
500 700
600
I
400
CFTR, cystic fibrosis transmembrane cond
900
IA
A
S
L
1000
1300
I 1200
1400
800
1100
ductance regulator
C
Sit f CSite of CommonF508del Mutation
Out
Pore
ln
ATP
ln
CNucleotide
Binding Domain 1
Cytoplasm RegulatoryDomain
Carbohydrate
Cell Membrane
Mike Welsh
ATP
Cl-Nucleotide
Binding Domain 2
From:Welsh, M.J. & Smith, A.E.Sci. Am. 273, 36-43, 1995
Phosphate
Steps on the PathwDrug Therapies forDrug Therapies for
Ducts and Tubes Become Blo
Defective Chloride Tran
Identification and Clon
A Chloride Channel wit
U d t d H M t ti Di tUnderstand How Mutations Disrupt
Drug T
way to Developing r Genetic Disordersr Genetic Disordersocked by Thick Sticky Mucus
nsport Across Epithelia
ning of the CFTR Gene
th Complex Regulation
t th F ti f th G P d tt the Function of the Gene Product
herapy
Location of Some CF-AssociatMissense Mutations
0
A D
198
300
500 700F508del
600
I
400
900
ted
IA
A
S
L
1000
1300
I 1200
1400
800
1100
How Do Mutationa Loss of Cl- Chaa Loss of Cl Cha
The mutant channel might• The mutant channel might apical membrane.
• The mutant channel might
ns in CFTR Cause annel Function?annel Function?
not be delivered to thenot be delivered to the
have altered properties.
F508del-CFTR
Biochemical AnalysBiochemical Analys
wwttCFTRCFTR
Maturation signs!Maturation signs!
Band CBand CBand BBand B
From: Prof. Margarida D. Amara
R Fails to Mature
sis of F508delsis of F508del--CFTRCFTR
F508del F508del CFTRCFTR
No maturation signs!No maturation signs!
Band BBand B
al, University of Lisboa, Portugal
Wiapical
Golgi
ClCl-
F5F5Golgi
Cl-
ld-Type CFTR basolateral
NER
mRNA
508del-CFTR508del CFTR
NER
mRNAmRNA
Cl-
Apical
MS
D2
Cl
ATP
M
DefectiveRegulation XADPX
Pi
GolgiER
P
ER
XXDefectiveProcessing
NF508del
N
XRR
R mRNA
PP
R mRNA
Steps on the PathwDrug Therapies foDrug Therapies foDucts and Tubes Become Blo
Defective Chloride Tran
Identification and Clon
A Chloride Channel wit
D f t i th Bi thDefects in the Biosynthes
Drug T
way to Developing or Cystic Fibrosisor Cystic Fibrosisocked by Thick Sticky Mucus
nsport Across Epithelia
ning of the CFTR Gene
th Complex Regulation
i d F ti f CFTRsis and Function of CFTR
herapy
TemperatureProcessing of
From: Denning, G.M. et al
e-Sensitive F508del-CFTR
l. Nature 358, 761-764, 1992
F508deGolgi
Cl-Cl-
F508deF508deGolgi
Cl-
el-CFTR @37°C
NER
mRNA
el-CFTR @25°Cel CFTR @25 C
NER
mRNAmRNA
Drug Strategies to F ldi D f tFolding Defect o
Golgi
Cl-
CFCF
• CFTR correctors rescue the proCFTR allowing the mutant proteCFTR allowing the mutant prote
Rescue the Protein f F508d l CFTRof F508del-CFTR
NER
mRNA
TR Correctors(+)
TR Correctors
otein folding defect of F508del-ein to traffick to the cell surface.ein to traffick to the cell surface.
Cl-
Apical
MS
D2
Cl
ATP
M
DefectiveRegulation XADPPi
X
GolgiER
P
ER
CFTRCorrectors
NF508del
N
XRR
R mRNA
PP
R mRNA
Control
Phloxine B (5 μM)
1 s1 pA
P
0.10 *MB
2
i (pA)
-1.0
* Po
0.05
MB(m
1
i (pA)
-0.5*
0.000.0
[PB] (μM)0 5
0.00
[PB] (μM)0 5
0.0
G551DG551D
IBI
8
BD
40 * IBI(s)
4
BDms)
20Cl-
0
*0
[PB] (μM)0 5
0
[PB] (μM)0 5
0
Zhiwei Cai
Control
Phloxine B (5 μM)
1 s1 pA
P
0.10 *MB
2
i (pA)
-1.0
* Po
0.05
MB(m
1
i (pA)
-0.5*
0.000.0
[PB] (μM)0 5
0.00
[PB] (μM)0 5
0.0
G551DG551D
IBI
8
BD
40 * IBI(s)
4
BDms)
20
0
*0
[PB] (μM)0 5
0
[PB] (μM)0 5
0
Zhiwei Cai
Cl-
CFTR Pote
ATPATP
Cl-
F508del-CFTR
Cl-
entiator
MS
D2
M
ATP
ADPPi
ATP
Cl-
PP
RR
“Rescued”
PP
F508del-CFTR
Cl-
Apical
MS
D2
Cl
ATP
M
CFTR Potentiator
ADPPi
GolgiER
P
ER
CFTRCorrectors
NF508del
N
RR XR mRNA
PP
R mRNA
Pathogenesis of CFPathogenesis of CF Defective CF GeneDefective CF Gene
Defective/Deficient CFTRDefective/Deficient CFTR
Decreased Chloride SecretionDecreased Chloride SecretionCFTRCFTRCFTRCFTR
Bronchial ObstructionBronchial Obstruction
InfectionInfectionInfectionInfection
InflammationInflammation
BronchiectasisBronchiectasis
Lung DiseaseLung Disease
ChlorideChlorideChlorideChloride
PathogenesisPathogenesisDefective CF GeneDefective CF Gene
Defective/Deficient CFTRDefective/Deficient CFTR
Decreased Chloride SecretionDecreased Chloride Secretion
Bronchial ObstructionBronchial Obstruction
InfectionInfectionInfectionInfection
InflammationInflammation
BronchiectasisBronchiectasis
TherapyTherapy
Airway ClearanceAirway ClearanceBronchodilators, MucolyticsBronchodilators, Mucolytics
AntibioticsAntibioticsAntibioticsAntibiotics
AntiAnti--inflammatory inflammatory agentsagents
Lung transplantLung transplant
Pathogenesis ThePathogenesisDefective CF GeneDefective CF Gene
The
Defective/Deficient CFTRDefective/Deficient CFTR
Decreased Chloride SecretionDecreased Chloride Secretion
Bronchial ObstructionBronchial Obstruction
InfectionInfectionInfectionInfection
InflammationInflammation
BronchiectasisBronchiectasis
erapeutic Strategieserapeutic StrategiesGene TherapyGene Therapy
CFTR C tCFTR C tCFTR CorrectorsCFTR CorrectorsCFTR PotentiatorsCFTR Potentiators
CFTR Bypass TherapyCFTR Bypass Therapy
The CFF Drug Dev01/06/09 snapshot, see: http://www.cff.
To Patients
01/06/09 snapshot, see: http://www.cff.
Phase 3
Phase 2Phase 2
Phase 1
Pre-Clinical
ResearchResearch
velopment Pipelineorg/research/DrugDevelopmentPipeline/org/research/DrugDevelopmentPipeline/
The CFF Drug Dev01/06/09 snapshot, see: http://www.cff.
To Patients
01/06/09 snapshot, see: http://www.cff.
VX-770, CFT
Phase 3
Phase 2
VX-809, CFT
Phase 2
Phase 1
Pre-Clinical
ResearchResearch
velopment Pipelineorg/research/DrugDevelopmentPipeline/org/research/DrugDevelopmentPipeline/
TR Potentiator
TR Corrector
The CFF Drug Dev
To Patients VX-770, CFT
Phase 3
Phase 2
VX-809, CFTVX-770, CFT
Phase 2
Phase 1
Pre-Clinical
ResearchResearch
velopment Pipeline
TR Potentiator
TR Corrector +TR Potentiator
FDA Approves Kalydeco (VX-770Underlying Cause of Cystic Fibroy g yJanuary 31, 2012
The Cystic Fibrosis Foundation today applapproval of Kalydeco™ (ivacaftor; previousthe search for a cure for cystic fibrosis.
The drug was developed by Vertex PharmThe drug was developed by Vertex Pharmsignificant funding support from the Cystic
The FDA approved Kalydeco (kuh-LYE-depopulation those ages 6 and older with thepopulation, those ages 6 and older with thedrug is taken in pill form.
“Today marks an important milestone infibrosis,” said Robert J. Beall, Ph.D., presFoundation. “Kalydeco addresses the unbehind the drug has opened exciting neth t t ll l d t dditi l ththat may eventually lead to additional thliving with CF.”
) — First Drug That Targets the osis
auds the Food and Drug Administration’s sly known as VX-770), a major advance in
aceuticals Inc with scientific clinical andaceuticals Inc., with scientific, clinical and Fibrosis Foundation.
h-koh) for a segment of the CF e G551D mutation of cystic fibrosis Thee G551D mutation of cystic fibrosis. The
n our journey to find a cure for cystic sident and CEO of the Cystic Fibrosis nderlying cause of CF, and the science ew doors to research and development h i th t ill b fit lherapies that will benefit more people
The CFF Drug Dev
To Patients VX-770, CFT
Phase 3
Phase 2
VX-809, CFTVX-770, CFT
Phase 2
Phase 1
Pre-Clinical
ResearchResearch
velopment Pipeline
TR Potentiator
TR Corrector +TR Potentiator
Steps on the PathwDrug Therapies foDrug Therapies foDucts and Tubes Become Blo
Defective Chloride Tran
Identification and Clon
A Chloride Channel wit
D f t i th Bi thDefects in the Biosynthes
Rational TherapRational Therap
Drug T
way to Developing or Cystic Fibrosisor Cystic Fibrosisocked by Thick Sticky Mucus
nsport Across Epithelia
ning of the CFTR Gene
th Complex Regulation
i d F ti f CFTRsis and Function of CFTR
peutic Strategiespeutic Strategies
herapy
Prolonged Surviva
http://www.cff.org/ECommitment/2006_fall/people/one_o
al is Now the Norm
on_one-danny_bessette.html
Further RCommentaries:Human genetics: One gene, twentJul 9; 460(7252):164-9Jul 9; 460(7252):164-9.Genetics. The promise of a cure: 2Frankel J. Science. 2009 Jun 19; Personalized medicine. New cystiprice. Kaiser J. Science. 2012 Feb
Reviews:Cystic fibrosis Welsh MJ Smith ACystic fibrosis. Welsh MJ, Smith A
Reading:
ty years. Pearson H. Nature. 2009
20 years and counting. Couzin-324(5934):1504-7.c fibrosis drug offers hope, at a b 10;335(6069):645.
AE Sci Am 1995 Dec; 273(6):52-9AE. Sci Am. 1995 Dec; 273(6):52-9.