CtiFCystic F Fib iFibrosis: From Gene DFrom Gene ... · From Gene DFrom Gene D Molecular ... Francis Collins Jack Riordan Lap-Chee Tsui ber 1989. ... e G551D mutation of cystic fibrosis

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

[email protected]

From:

Welsh, M.J. & Smith, A,E.

Sci. Am. 273, 36-43, 1995

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.

Carrier Father

R r

R R R rNormal (25%) Carrier (

Carrier Mother

R r

R r r r(50%) Affected (25%)

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.

Cystic FibroA lAccumula

Normal Subject

osis Airways t Mate Mucus

CF Patient

Cystic FibroA lAccumula

Wine JJ. Sci Transl Med. 2010;2:29ps20.

osis Airways t Mate Mucus


Ducts and Tubes Become Blo

Investigate the Bioch

Identify the De



Drug T

way to Developing r Genetic Disordersr Genetic Disordersocked by Thick Sticky Mucus

hemical Abnormality

efective Gene



herapy

apical

Lumen

apical

ABSORPTION

basolateralbasolateral

SECRETION

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-

Norrmal Airway Epithelia

ClearanceC ea a ce

Na+

Cl-

CF Airway Epithelia

Na+

Cl-



Defective Chloride Tran

Identify the De



Drug T


nsport Across Epithelia

efective Gene



herapy

8th Septem

Francis Collins

Jack Riordan

Lap-Chee Tsui

mber 1989




Identification and Clon



Drug T



ning of the CFTR Gene



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





A Chloride Channel wit


Drug T




th Complex Regulation


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

Wild-type CFTR

F508del-CFTR

1 pA

0 15

Cl-Cl-

0 2015Ti ( )Time (s)

Zhiwei Cai

Out

In

Cl-Cl

PP

RR

Closed

Open

Cl-

MS

D2

M

ATP

ADPPi

ATP

Cl-

PP

RR

PP

Cl-

MS

D2

MATP

ADPPi

ATP

Cl-

PP

RR

Wild-type CFTR

PP

yp

Cl-

ATPATP

Cl-

F508del-CFTR

Cl-

Apical

MS

D2

Cl

ATP

M

ADPPi

GolgiER

P

ER

NN

RR

R mRNA

PP

R mRNA

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




D f t i th Bi thDefects in the Biosynthes

Drug T

way to Developing or Cystic Fibrosisor Cystic Fibrosisocked by Thick Sticky Mucus




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.

Apical

GolgiERER

XXDefectiveProcessing

NF508del

N

XR mRNAR mRNA

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


Decreased Chloride SecretionDecreased Chloride Secretion





TherapyTherapy

Airway ClearanceAirway ClearanceBronchodilators, MucolyticsBronchodilators, Mucolytics

AntibioticsAntibioticsAntibioticsAntibiotics

AntiAnti--inflammatory inflammatory agentsagents

Lung transplantLung transplant

Pathogenesis ThePathogenesisDefective CF GeneDefective CF Gene

The


Decreased Chloride SecretionDecreased Chloride Secretion





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




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




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.

Documents

CtiFCystic F Fib iFibrosis: From Gene DFrom Gene ... · From Gene DFrom Gene D Molecular ... Francis Collins Jack Riordan Lap-Chee Tsui ber 1989. ... e G551D mutation of cystic fibrosis