Rescue of ∆F508-CFTR by Kinase Inhibitors - TSpace

Rescue of ∆F508-CFTR by Kinase Inhibitors

by

Duy (Leo) Nguyen

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Graduate Department of Biochemistry

University of Toronto

© Copyright by Duy (Leo) Nguyen (2013)

ii

Rescue of ∆F508-CFTR by Kinase Inhibitors

Duy (Leo) Nguyen

Master of Science

Department of Biochemistry

University of Toronto

2013

Abstract

ΔF508-CFTR is a trafficking mutant that is retained in the ER, unable to reach the plasma

membrane. To identify corrector of this mutant, we screened a kinase inhibitor library enriched

for compounds clinically available or in clinical trials for the treatment of other diseases, using

our recently developed high-content functional screen. Several inhibitors of receptor tyrosine

kinases exhibited strong rescue of ∆F508-CFTR. Moreover, prominent rescue was also observed

with inhibitors of four major pathways: Ras/Raf/MEK/ERK, TAK1/p38, Wnt/GSK-3β, and

PI3K/Akt/mTOR. A complimentary siRNA screen was also performed to identify pathways

involved in the rescue. FGFR1 and several proteins downstream of FGFRs were identified

suggesting a possible role of these receptors in regulating ΔF508- CFTR trafficking. Moreover,

the use of compounds clinically available or in clinical trials for other diseases can expedite

delivery of treatment for CF patients.

iii

Acknowledgements

First, I would like to thank my supervisor, Dr. Daniela Rotin, not only for giving me an

opportunity to work on this very interesting project, but also for teaching me self-discipline, and

providing me guidance and encouragement through the whole Master program. I also want to

thank my committee members, Dr. Christine Bear and Dr. Walid Houry, for their support and

suggestions during the committee meetings.

Secondly, I want to thank Agata Trzcinska-Daneluti, who is a mentor, a colleague, and a

friend. She not only performed the Cellomics and flow cytometry studies, but also has been

giving me helpful suggestions throughout the project. I want to thank Dr. Chong Jiang for

teaching me all the techniques, especially the Ussing chambers, since I started in the lab, and

Ruth Milkereit for helping me extract the macrophages for my phosphoprotein experiment. I also

want to thank all of lab members, Avi, Chen, Ryan, Phillip, Wioletta, and Yunan, who made my

journey much more fun and pleasant.

Lastly, I want to thank my wife, An, and my family for believing and supporting me.

Without them, I would not be able to finish this program.

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Table of Contents

Abstract iii

Acknowledgements iv

Table of Contents v

List of Tables viii

List of Figures ix

Abbreviations xi

Chapter 1: Introduction

I. Cystic Fibrosis

1. Overview and pulmonary pathogenesis in CF patients 2

2. Current clinical management of CF 3

II. CFTR and CF-causing mutations

1. CFTR structure 6

2. Channel gating by ATP binding and hydrolysis 9

3. Regulation of CFTR by phosphorylation 12

4. Overview of CF-causing mutations 13

5. ∆F508-CFTR mutation and its defects 15

III. Chaperone systems involved in the processing of CFTR

1. ER-associated and cytosolic chaperone systems 22

a. Hsp70 and its cochaperones 22

b. ER membrane-bound and luminal chaperones 26

c. Hsp90 in the processing of CFTR 27

2. Peripheral chaperone systems 28

IV. Screens for correctors of the ∆F508-CFTR defects

1. High-throughput screens for correctors of ∆F508-CFTR 30

2. Discovery of VX-809 and VX-770 and their clinical trials 32

v

3. Our screens using high-content Cellomics assays 33

V. Project rationale and goals 37

Chapter 2: Materials and Methods

1. Media and reagents 39

2. Small molecules kinase inhibitor library 39

3. Cells 40

4. Cellomics YFP halide exchange screen 41

5. Data analysis 42

6. Immunoblotting 42

7. Flow cytometry 43

8. Short-circuit (Isc) measurements in Ussing chambers 43

9. Isolation of bone marrow-derived macrophages (BDMM) 44

10. Phosphoprotein analysis 44

11. shRNA knockdown and qPCR quantification of knockdown 47

Chapter 3: Results

I. Screen for kinase inhibitors that correct ∆F508-CFTR function using high-content

functional Cellomics assays

II. Validation of the hits

1. Maturation of ∆F508-CFTR

2. Functional analysis of correction of ∆F508-CFTR by the kinase

inhibitors

3. Effect of kinase inhibitors on ∆F508-CFTR chloride channel activity in

primary Human Bronchial Epithelial (HBE) cells harvested from CF

patients

III. Dose response curves of rescue of ∆F508-CFTR in MDCK cells treated with

select kinase inhibitors

IV. Analyses of E6201, a derivative of (5Z)-7-Oxozeaenol

1. E6201 did not rescue ∆F508-CFTR maturation and function

2. Comparison of the effect of E6201 and (5Z)-7-Oxozeaenol on different

signaling pathways

V. Validations of hits of esi-RNA screen

48

57

62

54

66

69

74

80

vi

Chapter 4: Discussion

I. Kinase inhibitor screen 89

II. E6201 and (5Z)-7-Oxozeaenol 94

III. esiRNA screen 95

Future directions

I. Testing the effect of knocking down top hit genes on rescuing ∆F508-CFTR

function using Ussing chamber

II. Elucidate the mechanism of rescuing ∆F508-CFTR by Oxozeaenol 99

III. Elucidate the pathways through which FGFR1 regulates the rescue of ∆F508-CFTR 100

Summary 100

Conclusion 102

References 103

99

vii

List of Tables

Table I. Hit compounds and their validations. 50

Table II. Hit genes of the siRNA screen. 81

viii

List of Figures

Figure 1: Pulmonary pathogenesis in Cystic Fibrosis 4

Figure 2: Schematic diagram and three-dimensional structural model of CFTR. 7

Figure 3: Gating of CFTR channel by ATP hydrolysis. 10

Figure 4: Classes of CFTR mutations. 16

Figure 5: ∆F508-CFTR trafficking and folding defects. 19

Figure 6: ER-associated and peripheral quality control systems involved in the

trafficking of CFTR.

Figure 7: Principles of the Cellomics assays to test rescue of mutant CFTR. 35

Figure 8: Ussing chamber schematic diagram and ∆Isc calculations. 45

Figure 9: Representative hits of the high-content screen. 52

Figure 10: Effect of select kinase inhibitors on ∆F508-CFTR maturation analyzed

by immunoblotting.

Figure 11: Effect of kinase inhibitors on cell surface expression of ∆F508-CFTR

analyzed by flow cytometry.

Figure 12: Effect of compounds’ treatment on ∆F508-CFTR channel activity in

MDCK cells stably expressing ∆F508-CFTR.

Figure 13: Effect of compounds’ treatment on ∆F508-CFTR activity in primary

Human Bronchial Epithelial (HBE) cells harvested from lungs of

∆F508/∆F508 homozygote patients undergoing lung transplant.

Figure 14: Dose response curves of select kinase inhibitors for rescue of ∆F508-

CFTR expressed in MDCK cells.

Figure 15: E6201 does not rescue the function of ∆F508-CFTR. 70

Figure 16: E6201 does not rescue the maturation and function of ∆F508-CFTR

by immunoblotting and Ussing chambers.

Figure 17: Signaling pathways inhibited by (5Z)-7-Oxozeaenol and E6201. 75

23

55

58

60

63

67

72

ix

Figure 18: Both (5Z)-7-Oxozeaenol and E6201 inhibit the phosphorylation of

similar downstream signaling targets.

Figure 19: Validation of hits of the esiRNA screen by Cellomics assays. 82

Figure 20: Validation of hits of the esiRNA screen by immunoblotting. 85

78

x

Abbreviations

∆F508-CFTR deletion of phenylalanine at position 508 in CFTR

∆Isc difference in maximal stimulated current

293MSR genetically engineered HEK 293 cell line expressing the human

macrophage scavenger receptor

4PBA sodium 4-phenylbutyrate

5% Blotto 5% dry milk made with PBST

ABC adenine nucleotide-binding cassette

AMP-PNP adenylyl imidodiphosphate

ASL airway surface liquid

ATP adenosine triphosphate

BHK baby hamster kidney cell

BMM macrophage extracted from mouse bone marrow

CAMK2B calcium/calmodulin-dependent protein kinase II beta

cAMP cyclic adenosine monophosphate

CF cystic fibrosis

CFTR cystic fibrosis transmembrane conductance regulator

CHIP carboxyl terminus of Hsc70 interacting protein

http://pubs.acs.org/doi/abs/10.1021/bi00789a009

xi

CK2 casein kinase 2

CL4 cytoplasmic loop 4

Corr-4a corrector 4a

CPDR Canadian CF patient registry report

DAG diacylglycerol

DMEM Dulbecco’s Modified Eagle’s Medium

DMSO dimethyl sulfoxide

DNA deoxyribonucleic acid

DNase deoxyribonuclease

DNDS 4,4-dinitrostilbene-2,2- disulfonic acid

dTDP thymidine diphosphate

dTMP thymidine monophosphate

dTTP deoxythymidine triphosphate

DTYMK deoxythymidylate kinase

EC50 half maximal effective concentration

ECL enhanced chemiluminescence

ENaC epithelial sodium channel

xii

ER endoplasmic reticulum

ERAD ER-associated degradation

ERK extracellular signal-regulated kinase

ESCRT endosomal sorting complex required for transport

esiRNA endonuclease-prepared siRNA

FACS fluorescence-activated cell sorting

FBS fetal bovine serum

FGFR fibroblast growth factor receptor

FIG mixture of forskolin, IBMX, Genistein

FRS2 factor receptor substrate

G551D-CFTR glycine to aspartic acid substitution mutation at position 551 in CFTR

GAB1 Grb2-associated binding protein 1

GFP green fluorescent protein

Gly GlyH-101

GRB2 growth factor receptor-bound protein 2

Gsk3β glycogen synthase kinase 3 beta

HA hemagglutinin

HBE human bronchial epithelial

xiii

HBSS Hank’s balanced salt solution

Hdj human DnaJ homologue

HEK human embryonic kidney cell

HRP horseradish peroxidase

Hsc heat shock cognate protein

HSF1 heat shock factor 1

Hsp heat shock protein

HTS high-throughput screen

IBMX 3-isobutyl-1-methylxanthine

ICD intracellular domain

IP3 inositol triphosphate

IP5 inositol pentakisphosphate

IPMK inositol polyphosphate multikinase

Isc short-circuit current

IκB inhibitor of kappa-B

JNK c-Jun N-terminal kinase

LPS lipopolysaccharide

MAP3K mitogen-activated protein kinase kinase kinase

xiv

MDCK Madin-Darby canine kidney epithelial cell

MEK MAPK/ERK kinase

MSD membrane-spanning domain

mTOR mammalian target or rapamycine

NDB nucleotide-binding domain

NFκB nuclear factor kappa-light-chain-enhancer of activated B cell

NOS2 nitric oxide synthase 2

PAL mixture of pepstatin, aprotinin, and leucine

PBS phosphate buffered saline

PBST phosphate buffered saline with Tween 20

PDGFR platelet derived growth factor receptor

PI3K phosphoinositide 3 kinase

PIAS1 Protein inhibitor of activated STAT1

PIP2 phosphatidylinositol 4, 5-bisphosphate

PKA protein kinase A

PKC protein kinase C

PLC phospholipase C

xv

PMSF phenylmethylsulfonyl fluoride

PRKAR2B cAMP-dependent protein kinase type II-beta regulatory subunit

PRKAR2B cAMP-dependent protein kinase type II-beta regulatory subunit

R domain regulatory domain

RE regulatory extension

RhoA Ras homologue gene family member A

RI regulatory insertion

RIPK receptor-interacting protein kinase

RNA ribonucleic acid

RNAi RNA interference

ROCK Rho-associated, coiled-coil containing protein kinase

RPS6KC1 ribosomal protein S6 kinase delta-1

RSK ribosomal S6 kinase

RTK receptor tyrosine kinase

RT-qPCR quantitative real time polymerase chain reaction

SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis

shRNA small/short hairpin RNA

siRNA small-interfering RNA

xvi

SOS protein son of sevenless

STAT1 signal transducer and activator of transcription 1

SUMO small ubiquitin-like modifier

TAK TGF-beta activated kinase

TGF transforming growth factor

TMPK thymidylate kinase

TPR tetratricopeptide

USP ubiquitin specific protease

VEGFR vascular endothelial growth factor receptor

WT-CFTR wild-type CFTR

YFP yellow fluorescent protein

1

CHAPTER 1

INTRODUCTION

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I. CYSTIC FIBROSIS

1. Overview and pulmonary pathogenesis in CF patients:

Cystic Fibrosis (CF) is an autosomal recessive disorder, most common among Caucasian

populations, and affects 1 in 2500 live births (Ratjen and Doring, 2003). It is caused by

mutations in the gene encoding the protein Cystic Fibrosis Transmembrane Conductance

Regulator(CFTR), which is a chloride channel expressed in most secretory and absorptive

epithelial cells. Besides functioning as a chloride channel, CFTR is also known to be permeable

to HCO3- (Devor et al., 1999; Tang et al., 2009) and to regulate other membrane proteins,

especially the sodium channel ENaC. For example, in the sweat glands, CFTR activity is

required for ENaC activation (Reddy et al., 1999), and both channels’ activities are needed to

reabsorb ions back into the sweat ducts. In airways, ENaC activity is elevated in CF, the exact

opposite of what is seen in sweat glands and ducts (Reddy, 2003; Boucher, 2004).

Even though CF is a pleiotropic disease affecting various organs such as the intestine,

liver, pancreas, and vas deferens, the major morbidity and mortality are due to chronic lung

inflammation and disease. CF pulmonary pathogenesis is considered as a failure of the innate

defence mechanisms of the lungs against pathogens. In normal airways, anion secretion is

mediated by CFTR and other alternative chloride channels, coupled with sodium absorption by

ENaC to maintain the fluid homeostasis of the airway surface liquid (ASL). Proper volume

homeostasis of the ASL maintains the viscosity of the mucus layer to promote appropriate cilia

movement and efficient mucocilliary clearance of bacteria. However in the airways of CF

patients, reduced Cl- secretion, due to the lack of functional CFTR on the apical surface, and

hyper-absorption of Na+, due to elevated activity of ENaC, which is negatively regulated by

CFTR in airway cells (Stutts et al., 1995; Stutts et al., 1997; Rubenstein et al., 2010; Gentzsch et

3

al., 2010), lead to dehydration of the ASL. This increases the viscosity of the mucus layer, and

deposition of the thickened mucus collapses the cilia and impairs the mucocilliary clearance

mechanism. Moreover, the deposited layer of mucus creates an environment that promotes

bacterial colonization, commonly by Pseudomonas aeruginosa, and eventually leads to chronic

infection of the lungs (Boucher RC, 2004)(Figure 1). Combined with excessive inflammatory

response due to both infection and dysregulation of inflammatory response in airway cells, CF

patients eventually suffer from irreversible airway damage and respiratory failure (Davies et al.,

2007; Bodas and Vij, 2011).

2. Current clinical management of CF

CF is considered to be a deadly disease of young people (Gadsby DC, 2006). Even

though at birth the airways are uninfected, lung infection and inflammation occur soon after

(Gibson et al., 2003). In 1938, when CF was first recognized as a separate disease, 70% of babies

with CF died within their first year of life due to the inability to absorb nutrients in the intestine

(Garattini et al., 2011). With immense improvements in the treatments of CF, the median

predicted survival age of Canadians with CF in 2011 was estimated to be around 48 years

(Canadian CF patient registry report (CPDR), 2013). However, an ultimate curative treatment for

CF is currently unavailable.

To date, the main goals of the treatments of cystic fibrosis are to relieve the symptoms of

the disease and to enhance the quality of life (Antunovic et al., 2013). The treatments target

nutrition, relief of airway obstruction, and suppression of airway infection and inflammation

(Davis PB, 2006; Garattini et al., 2011). First, long-term nutritional management is very

important for CF patients as about 90% of patients suffer from pancreatic insufficiency, which is

4

Figure 1: Pulmonary pathogenesis in Cystic Fibrosis. In normal airways, the balance between

Na+

absorption (mediated by ENaC in the apical membrane) and anion secretion (mediated by

apical CFTR and alternative anion channels) determines the volume of the fluid on airway

surfaces. This maintains the viscosity of the ASL and allows the proper cilia movement to clear

out bacteria and deposited mucus. In CF airways, lack of functional CFTR leads to reduced

chloride secretion and augments sodium absorption; this, in turn, dehydrates the ASL leading to

the deposition of thicken mucus on the airway surface and impeding the mucocilliary clearance

mechanism. Failure of the clearance mechanism results in bacterial colonization, hyper

inflammation, and chronic infections of the lungs (Modified from Frizzell & Pilewski, 2004).

6

caused by the obstruction and damage of the pancreatic duct and results in malabsorption and

deficiency of (especially fat-soluble) nutrients including vitamins (Ooi and Durie, 2012).

Moreover, CF patients also require extra energy to overcome increased work of breathing and

constant battles against infections (http://www.cysticfibrosis.ca), and might experience episodes

of hypochloremia or hyponatremia due to the excessive loss of salt through sweat (Priou-

Guesdon et al., 2010). Therefore, the dietetic management includes very high-caloric intake

(120-150% of the normal recommended daily allowance), daily supplementation of pancreatic

enzymes, fat soluble vitamins (A, D, E, K), and sodium chloride (Antunovic et al., 2013;

Garattini et al., 2012; http://www.cysticfibrosis.ca).

The second main target of CF treatments is to clear out obstruction in the airway. These

therapies include daily airway clearance physiotherapies such as postural drainage, chest

percussion, positive expiratory pressure, and breathing exercises. Complementary treatments to

enhance mucociliary clearance of repiratory secretions may include mucolytics, such as DNase

or hypertonic saline, in combination with bronchodilators to clear out mucus and enlarge the

luminal diameter of the airway. The last main area of treatments is to suppress infection and

inflammation using different antibiotics and anti-inflammatories, including inhaled, oral, or

intravenous medications depending on the type of drug and the severity of the infections

(Antunovis et al., 2013; http://www.cysticfibrosis.ca). In addition to these main areas, CF

patients have to endure additional treatments during an exacerbation or onsets of other

complications such as CF-related diabetes, bone disease that might result in long period of

hospitalization.

Even though these therapies, which have to be carried out across the lifespan of CF

patients, have significantly improved the survivorship and quality of life of the patients, they are

http://www.cysticfibrosis.ca/



7

still complex and time-consuming processes which greatly affect and become burdens to the

lives of not only the patients but also their family. Eventually, however, lung failure is inevitable,

and lung transplantation is required. Therefore, a drug that could target the basic molecular cause

of the disease is much needed.

II. CFTR AND CF-CAUSING MUTATIONS:

1. CFTR Structure

CFTR was first identified as a member of the adenine nucleotide-binding cassette (ABC)

family of transporters (Riordan et al., 1989). Although it has the core structural architecture of an

ABC transporter, CFTR is the only member of this family functioning as Cl- channel (Kartner et

al., 1991). Like other members of the family, CFTR consists of two symmetrical halves, each

consisting of one nucleotide-binding domain (NDB), which possesses a binding site for ATP,

and one membrane-spanning domain (MSD), which is comprised of six transmembrane

segments (Figure 2). These two halves are linked together by a unique, highly unstructured

regulatory (R) region/domain. While the two MSDs form the anion-selective pore of the channel,

the two NBDs form a head-to-tail dimer with the two ATP binding sites at the interface (Riordan

JR, 2008). To date, no high-resolution structure of the full-length CFTR channel has been solved.

However, models of the structure of the full-length CFTR have been built based on the structure

of the bacterial transporter Savv1866 (Mornon et al., 2008, 2009;

8

Figure 2: Schematic diagram and three-dimensional structural model of CFTR. A)

Schematic diagram of CFTR with two halves, each consists of one MSD (membrane-spanning

domain) and one NBD (nucleotide binding domain), linked together by the unstructured R region

(domain). B) CFTR three-dimensional built based on the experimental structural model of the

bacterialtransporter Savv1866. The intracellular loops of MSDs (ICL1, ICL2 of MSD1 and

ICL3, ICL4 of MSD2) provide the contacts with NBDs to create the MSD/NBD interfaces

(Lyczak et al., 2002 ; Mornon et al., 2008).

9

(A)

(B)

10

Serohijos et al., 2008).Even though there are some limitations due to the use of Savv1866

structure as a template, this three-dimensional model still permits better insights into the

interactions between the domains of CFTR and the molecular mechanism underlying the activity

and gating of the channel.

2. Channel gating by ATP binding and hydrolysis

The gating mechanism of the channel has been extensively studied. Unlike other

members of the ABC family that use energy from ATP hydrolysis to transport substances against

a concentration gradient, the binding and hydrolysis of ATP in the NBDs of CFTR regulate the

opening and closing of the channel via conformational change caused by the formation and

disruption of the NBD1-NBD2 dimer complex. Like several other members of the ABC family,

the hydrolysis rate of ATP is different between the two domains. The ATP in the binding site of

NBD1 is negligibly slowly hydrolyzed while ATP in the site of NBD2 readily undergoes

hydrolysis (Aleksandrov et al., 2008). Upon binding of ATP to the NBD2 binding site, the two

domains come together to form a dimer complex and open the channel. When hydrolysis of the

ATP at this site occurs, the complex falls apart and the channel closes until the next ATP binding

event (Gadsby et al., 2006).The gating sequence of events are depicted in Figure 3.

The hydrolysis of ATP in the ATP-binding site 2 in NBD2 facilitates the rapid opening

and closing of the channel. However, the hydrolysis of ATP itself is not responsible for closing

and opening of the channel. AMP-PNP, a non-hydrolysable analogue of ATP, can lock the

channel in the open state (Hwang et al., 1994). This suggests that the presence of ATP in the

second binding site is responsible for opening of the channel. This locked open state can also be

observed when the protein is mutated at residue K1250 that abolishes ATP hydrolysis, and leads

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Figure 3: Gating of CFTR channel by ATP hydrolysis. The ATP-binding site in NBD1 has

high affinity and low hydrolysis rate. Thus, the rapid opening of the channel is caused by the

ATP hydrolysis at the second ATP-binding site in NBD2. The opening and closing of the

channel follows 4 steps. Step 1: ATP binds to the NBD2 binding site to initiate the process. Step

2: The conformational change caused by the formation of the NBD dimer causes the channel to

open. Step 3: The dimer is disrupted due to hydrolysis of ATP. Step 4: Pi and ADP release

restores channel to its basal conformation (Gadsby et al., 2006).

13

to prolonged binding of ATP (Gunderson and Kopito, 1995). The NBD dimer crystal structure of

other ABC transport reveals that the γ-phosphate of ATP forms a hydrogen bond with the

conserved serine residue (S548 in NBD1, and S1346 in NBD2) and the main chains of the

glycine residues (G550, G551 in NBD1, and G1349 in NBD2) (Hwang TC and Sheppard DN,

2009). In particular, theG551D mutation causes a severe channel gating defect (Bompadreet al.,

2007). The hydrolysis of ATP at the second nucleotide binding site might disrupt these hydrogen

bonds and the dissociation of ADP then closes the channel.

3. Regulation of CFTR by phosphorylation

Another difference between CFTR and other ABC family members is the unique R

region/domain that contains a cluster of dibasic (R-R/K-X-S/T) or monobasic (R-X-S) consensus

sites for phosphorylation, mainly by cAMP-dependent protein kinase A (PKA) (Seibert et al.,

1999). The R domain, until phosphorylated, restrains channel activity. Partial deletion of this

domain produces a constitutively active channel (Ostedgaard et al., 2002). Phosphorylation of

the consensus sites on the R domain provides another level of regulation of CFTR channel

gating. PKA phosphorylation is a pre-requisite for channel opening (Ostedgaard et al., 2001) and

has been shown to increase channel activity by at least 100-fold (Csanady et al., 2005).

Originally, one model for this gating of the channel was that when unphosphorylated, the R

domain blocked the pore of the channel, and thus inhibited any anion transport; upon

phosphorylation by PKA, the accumulation of negative charges created an electrostatic push to

repel the R domain out of the pore and relieved this inhibition (Cheng et al., 1991).This model,

however, was proved to be too simplistic. Later studies have shown that phosphorylation of the

R domain is a very dynamic and complex process, which may cause both stimulatory and

inhibitory effects (Gatsby and Nairn, 1999).

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Results from site-directed mutation studies (Seibert et al., 1995; Seibert et al., 1999),

combined with evidence of structural rearrangement of the R domain upon phosphorylation

(Dulhanty and Riordan, 1994; Dulhanty et al., 1995), support another theory of the gating

mechanism of the channel. In this model, conformational change of the R domain, rather than the

accumulation of negative charges, is responsible for its regulatory function. Even though it still

remains unstructured and disordered independently of phosphorylation, the R domain has been

shown recently to contain segments of helical structure that most likely interact with other

domains of CFTR.NMR studies performed byte Forman-Kay lab have shown that

phosphorylation reduced not only the helicity of these helical segments but also their interactions

with NBD1. These interactions might play an important role in conferring the regulatory effect

of the R domain on CFTR (Baker et al., 2007).

CFTR is also phosphorylated by other kinases. However, aside from the phosphorylation

of CFTR by PKA and PKC, phosphorylation by other kinases has not been extensively studied.

PKC phosphorylation causes a modest activation of the channel (Dulhanty and Riordan, 1994)

and potentiates the PKA-mediated activation of CFTR, probably by facilitating subsequent PKA

phosphorylation by exposing sites that are otherwise inaccessible (Chang et al., 1993; Jia et al.,

1997). However, the exact mechanism of how PKC directly regulates the channel is unknown.

The R domain does not undergo conformational change when phosphorylated by PKC (Dulhanty

et al., 1994). Yet, PKC does slowly phosphorylate PKA sites (Jia et al., 1997). Recently, another

kinase, Casein kinase2 (CK2), was also shown to regulate CFTR through direct phosphorylation.

Mutating the two CK2 phosphorylation sites diminished both channel conductance and

trafficking of the protein to the plasma membrane (Luz et al., 2011).

15

The R domain is not the only region of CFTR that contains phosphorylation sites.

Mutation of Ser-422, close to the N-terminal of NBD1, in CFTR that already contains nine

mutations in the R domain further reduces channel anion flux and cAMP response (Chang et al.

1993). This phosphorylation site lies in the recently defined regulatory insertion (RI) region of

NBD1 (residues 403-437) (Lewis et al., 2004, 2005). NMR studies have shown that

phosphorylations of the RI region and another region called C-terminus regulatory extension

(RE) at the end of the NBD1 disrupt their binding with NBD1 and expose the binding site for the

first coupling helix of the N-terminal intracellular domain (ICD) of NBD1 (Kanelis et al., 2010).

The helices of ICD are thought to be involved in transmitting the change in conformation during

the formation of NBD1/2 dimer to the MSDs in the regulation of channel opening (Ward et al.

2007). In short, the regulation of CFTR through phosphorylation has been shown to be a

dynamic and complex process which still requires further investigation, and various kinases may

play a role in regulating both channel activity and trafficking.

4. Overview of CF-causing mutations

To date, more than 1800 mutations in the CFTR gene have been identified (CPDR,

2011). However, most of these mutations are rare; and there are only 24 mutations that have been

identified with a frequency of 0.1% or higher (Sick Kids CF mutations database,

www.genet.sickkids.on.ca). These mutations can be classified into six classes depending on their

consequences. Class I mutations produce a stop codon leading to premature transcription

termination signals. These mutations result in truncated or no protein expression. Class II

mutations are usually missense mutations causing the protein to misfold, leading to premature

degradation and failure to reach the apical membrane. The most common CF-causing mutation,

∆F508, belongs to this class. CFTR bearing class III mutations still properly folds and results in

16

Figure 4: Classes of CFTR mutations. Class I mutations produce premature transcription

termination signals resulting in truncated or no protein expression. Class II mutations are

missense mutations causing the protein to misfold, leading to premature degradation. Class III

mutations cause defective channel regulation, resulting in decreased channel activity. Class IV

mutations result in reduced channel conductance due to lower chloride permeability and opening

probability. Class V mutations cause partial defect in producing or processing the protein,

resulting in reduced number of functional channels. Class VI mutations lead to higher rate of

degradation of the channel after biosynthesis (Anderson, 2010).

18

normal trafficking to the cell surface; however, it suffers a defect in its regulation, resulting in

severely decreased channel activity. A representative from this class is the G551D substitution.

This mutation in the ATP binding site on NBD1 is the third most common CFTR mutation that

results in defects in binding and hydrolysis of ATP (Li et al., 1996). Class IV mutations result in

reduced channel conductance due to lower chloride permeability and opening probability. Class

V mutations cause partly defective production or processing of the protein, resulting in a

reduction in the number of functional channels. Finally, Class VI mutations have only been

characterized recently. Such mutations reduce the channel stability causing an abnormally high

degradation after biosynthesis, and affect CFTR’s regulation of other proteins (Figure 4)(Ratjen

F, 2003; Anderson P, 2010; Okiyoneda and Lukacs 2012).

5. ∆F508-CFTR mutation and its defects

The most common mutation, identified in approximately 90% of CF patients, is the

deletion of phenylalanine at position 508, or ∆F508, a class II mutation. This deletion mutation

lies on the interface between NBD1 and the cytosolic loop 4 (CL4) of the MSD2 domain

(Mornon et al., 2008). It destabilizes the NBD1 thermodynamically and kinetically, and affects

the stability of the NBD1-MSD2interface (Rabeh et al., 2012), and the folding of NBD2 domain

(Du et al., 2005). This consequently disrupts the domain-domain interaction and their assembly

to form the complete channel, and thus causes the protein to be misfolded, kinetically trapped in

the endoplasmic reticulum (ER) and eventually targeted for degradation via ubiquitination by the

ER-associated degradation (ERAD) pathway and the proteasome (Riordan, 2008) (Figure 5).

Normally, the biosynthesis of CFTR starts by being synthesized and core glycosylated in

the ER. After exiting the ER, it is processed through the Golgi and presented on the cell surface

19

as a fully glycosylated mature CFTR (Cheng et al., 1990).CFTR is a large protein, 1480 amino

acid long, which exhibits a very inefficient folding and processing- up to 80% of the wild-type

(WT) CFTR gets degraded during the biosynthesis stage at the ER (Lukacs et al., 1994).This

results from a combination of slow domain assembly and fast degradation by ERAD (Lukacs and

Verkman, 2012). With the ∆F508 mutant, the efficiency is even lower with99% of the mutant

protein targeted for degradation before it reaches the plasma membrane (Ward and Kopito,

1994).This leads to the absence or very low density of the mutant channel on the plasma

membrane, which gives rise to the CF phenotype. This difference can be observed as the very

distinct migration patterns on SDS-PAGE. The WT protein appears as two bands: a prominent

band C represents the fully glycosylated mature form of CFTR (~180kDa), and a less intense

band B represents the core glycosylated immature form (~150kDa). On the other hand, ∆F508-

CFTR shows up predominantly as band B with little or no band C.

In addition, ∆F508 is a temperature-sensitive mutation; some of the mutant channels can

be rescued and reach the plasma membrane by incubation at lower temperature (26-30oC)

(Denning et al., 1992). However, even when rescued to the plasma membrane, ∆F508-CFTRis

quickly internalized by the membrane-associated chaperone system. While WT-CFTR has a half-

life of about 16h on the plasma membrane and is efficiently recycled back to the cell surface

after internalization, the rescued ∆F508 is quickly removed from the plasma membrane with the

half-life of about 2h; misfolding prevents this mutant protein from recycling back to the surface

and promotes its ubiquitination and degradation (Sharma et al., 2004; Swiatecka-Urban et al.,

2005).

Besides reducing the availability of the protein on the apical surface, ∆F508 mutation

also reduces the Cl- permeability of the channel. On the cell surface, ∆F508-CFTR only

20

Figure 5: ∆F508-CFTR trafficking and folding defects. A) ∆F508 belongs to class II CF-

causing mutation which facilitates the misfolding of the protein leading to its early degradation at

the ER. The WT CFTR can properly fold, escapes the ER and is further processed in the Golgi

and presented on the plasma membrane as the mature, fully glycosylated channel. Images of

normal and ∆F508 primary airway cells demonstrate the distribution of WT and mutant CFTR,

shown as green fluorescence. WT-CFTR is primarily present on the apical surface while ∆F508-

CFTR mostly resides in the cytosol (Riordan JR, 2008) C) CFTR homology structure shows the

location of ∆F508 at the interface between NBD1and CL4 of MSD2 (upper panel).This mutation

destabilizes the interface and disrupts the assembly of the domains (lower panel) (Lukacs GL

and Verkman AS, 2012).

21

(A)

(B)

22

exhibits partial channel activity in response to PKA (Bear et al., 1992). Therefore, it is very

complicated to find a drug for the treatment of ∆F508. Drugs used to treat ∆F508 have to correct

two main problems in order to achieve near normal lung function in CF patients:

i)prevent premature degradation of the protein and promote its trafficking to the cell surface;

ii) reduce the internalization rate and improve the recycling efficiency at the plasma membrane.

To tackle these problems, a more complex approach is probably required. Recent studies have

shown that stabilizing both NBD1 folding and the NBD1-MSD2 interface are required to fully

reverse the defects of ∆F508-CFTR (Rabeh et al., 2012; Mendoza et al., 2012). Indeed,

suppressant mutations that correct one of these folding defects of ∆F508-CFTR only led to

partial rescue of the channel; while combining mutations that stabilizing both NBD1 and NBD1-

MSD2 interface produced a synergistic rescue (Rabeh et al., 2012).Since CFTR folding and

assembly are monitored by complex systems of chaperones, affecting the chaperones involved in

the processing of CFTR, via a combinational drug therapy, may be one possible approach to

achieve a dual correction effect.

III. CHAPERONE SYSTEMS INVOLVED IN THE PROCESSING OF CFTR

Since the ∆F508 mutant is improperly folded, it is prone to aggregation, and accumulates

in the intracellular compartments (Qu and Thomas, 1996). Therefore, degradation of misfolded

protein is necessary to prevent the formation of large aggregations, which are toxic to cells.

However, when degradation occurs too rapidly, the protein might not have sufficient time for

proper folding. This can be applied to the case of CFTR since the nonubiquinated ∆F508

23

intermediates exist in a folding competent conformation. Inactivating the chaperones, which

target these intermediates for proteasomal degradation via ubiquitination, or treating the cells at

lower temperature, allows the misfolded intermediates to fold properly and reach the cell surface

(Younger et al., 2004). However, in vivo, CFTR biosynthesis is scrutinized at multiple quality

control checkpoints by complex systems of chaperones from the ER-associated chaperones to the

peripheral quality control systems at the plasma membrane (Lukacs and Verkman, 2012). A

summary of the chaperone systems involved in CFTR trafficking and recycling is depicted in

Figure 6.

1. ER-associated and cytosolic chaperone systems

The synthesis of a multi-domain protein, such as CFTR, is a very complex, multi-stage

process controlled by various chaperone systems. It requires not only the proper folding of

individual domains, but also appropriate domain-domain interactions and arrangements. For

CFTR, the first step in the process is the folding of the nascent chain protein which is controlled

by the ER-associated chaperones, both membrane-bound and cytosolic (Chanoux and

Rubenstein, 2012). One of the reasons why CFTR biosynthesis is very inefficient is due to its

uneconomically rigorous early folding steps. The majority of CFTR is degraded in the pre-Golgi

compartments, and thus, never reach the cell surface (Ward and Kopito, 1994).

a. Hsp70 and its cochaperones

One of the first chaperones described to bind to the nascent CFTR chain and mediate the

folding cotranslationally is the heat shock cognate 70 (Hsc70) protein, which belongs to the heat

shock protein 70 (Hsp70) family and is localized to the cytosolic face of the ER. Initially,

Hsc/Hsp70 was thought to be able to distinguish between the mutant and WT protein and have a

prolonged association with the mutant ∆F508 compared to WT-CFTR, and thus retain the mutant

24

Figure 6: ER and peripheral quality control systems involved in the trafficking of CFTR.

A) The ER quality control system involves mainly the complexes of Hsp70 and Hsp90 and their

cochaperones. These complexes sense the folding state of the nascent CFTR chain and target

the misfolded protein for degradation via ubiquitination by E3 ligase such as CHIP. Hsp70 and

Hsp90 can facilitate both degradation and folding of the nascent chain depending on the

cochaperone associated with them. Hdj-2 promotes folding of nascent chain while CHIP

facilitates its degradation when in complex with Hsp70. Cochaperone Aha1 promotes the

degradation pathway of CFTR via Hsp90 (Wang et al., 2006). B) There are overlapping

components between the peripheral and ER quality control. Hsp70/90 and their cochaperones

are also involved in the peripheral machinery that promotes the endocytosis of CFTR from the

plasma membrane and ubiquitination of the protein. Ubiquitinated protein is then removed from

the recycling pool via the ESCRT complex and degraded in the lysosome. Deubiquitination by

USP10 facilitates the recycling of internalized channel back to the plasma membrane (Lukacs

GL and Verkman AS, 2012).

25

(A)

(B)

26

in the ER for later degradation (Yang et al., 1993).However, later studies have shown that

Hsc/Hsp70 can facilitate both the folding and degradation of CFTR nascent chains depending on

the association of other co-chaperones. Meacham et al. showed that Hdj-2 formed a complex

with Hsc/Hsp70, which bound to and promoted the folding of the ribosomal-bound intermediates

protein during the expression of NBD1 (Meacham et al., 1999). This effect dramatically

decreases after the subsequent expression of the R domain and MSDII. TheHdj-2/Hsc70

complex preferentially binds to ∆F508-CFTR and prevents the aggregation of NBD1.

Traditionally, molecular chaperones were thought of as proteins that aided in the folding

of other proteins by promoting their self-assembly (Ellis, 1987). However, in many cases, such

as with CFTR, chaperones can also target the partially folded peptide chain for degradation. The

C-terminus of the Hsc70-Interacting protein (CHIP) is another co-chaperone that can form a

complex with Hsc/Hsp70 (Meacham et al., 2001). However, unlike Hdj-2, CHIP, which is an

ubiquitin ligase, forms a complex with Hsc/Hsp70 to sense the folded state of the nascent chain

of CFTR and targets aberrant proteins for degradation (Meacham et al., 2001).In the case of

CHIP, it acts as an E3 ligase in cooperation with the E2 UbcH5a. These two proteins, when in a

complex with Hsc70, facilitate the degradation of nascent CFTR chains (Younger et al, 2004).

Other cochaperones of Hsc70 have been less well studied. Both HspBP1 and BAG-2

were shown to stimulate the maturation of CFTR by inhibiting the cochaperone CHIP (Alberti et

al., 2004; Arndt et al., 2005). Moreover, Saxena et al. also showed that Hsp105 worked both

independently and as a cochaperone of Hsc70 to stabilize CFTR at an early stage of synthesis,

and to promote the posttranslational folding. In addition, it was also proved to bind preferentially

to ∆F508-CFTR at both the ER and cell periphery and to enhance the expression of the mutant

protein on the cell surface (Saxena et al., 2012).

27

b. ER membrane-bound and luminal chaperones

While the CHIP/Hsc70 complex primarily recognizes the CFTR post-translationally,

another ER membrane-associated ubiquitin ligase complex, consisting of the E3 RMA1, the E2

Ubc6E, and Derlin-1, can recognize folding defects cotranslationally during the synthesis of

MSD1 and target the misfolded nascent CFTR for degradation (Younger et al., 2006).

Overexpression of RMA1, but not CHIP, promoted the degradation of G91R-CFTR, a mutation

that resides in MSD1and prevents proper folding of the protein (Xiong et al., 1997). Moreover,

Derlin-1 coimmunoprecipitated with MSD1. Therefore, in this complex, Younger et al. proposed

that Derlin-1, an ER membrane protein, sensed the folding status of MSD1/2, and formed a

complex with the protein that failed to assemble correctly. Subsequently, Derlin-1 recruited

RMA1 and Ubc6e to facilitate ubiquitination and degradation of CFTR (Younger et al., 2006).

The role of ER luminal chaperones such as calnexin is less well understood(Chanoux and

Rubenstein, 2012). Calnexin was initially thought to bind to immature CFTR and retain ∆F508-

CFTR in the ER due to the fact that it has a prolonged interaction with the mutant compared to

WT-CFTR (Pind et al., 1994). However, other studies have shown that calnexin has a positive

regulatory role in the synthesis of ∆F508-CFTR. Overexpression of calnexin created a pool of

∆F508-CFTR but reduced the degradation and aggregation of the mutant protein (Okiyoneda et

al., 2004). Moreover, knocking down calnexin did not seem to improve the trafficking of ∆F508-

CFTR (Okiyoneda et al. 2008). The role of calnexin is controversial but combined data from

various studies suggests that calnexin alone is not sufficient for the retention of ∆F508-CFTR in

the ER. Other ER luminal chaperones might be a better therapeutic target for CF. A recent study

by Suaud et al. suggested that ERp29 (ER luminal protein of 29 kDa), when overexpressed,

increased both the functional and surface expression of WT and ∆F508-CFTR (Suaud et al.,

28

2011). This ER luminal protein was shown to be upregulated by the compound sodium 4-

phenylbutyrate(4PBA) (Suaud et al., 2011), which was shown to correct ∆F508-CFTR by

altering the expression of chaperones, such as Hsc70 (Rubenstein et al., 1997; Rubenstein and

Zeitlin, 2000).

c. Hsp90 in the processing of CFTR

Another chaperone associated with CFTR maturation that has received considerable

attention is Hsp90, which was shown to stabilize the CFTR folding intermediates (Loo et al.,

1998). However, the activity of Hsp90 depends on the presence of its co-chaperones. Hsp90

cochaperone Aha1 was shown to down-regulate the rescue of misfolding CFTR to the cell

surface. A 50-70% knock down of Aha1led to a significant increase in both band B and band C

of∆F508-CFTR and an increase in halide conductance in CFBE41o- expressing ∆F508-

CFTR(Wang et al., 2006). Aha1 was proposed to increase the binding of Hsp90 to its client

through increasing the ATPase activity of Hsp90 (Wang et al., 2006). Other cochaperones of

Hsp90 were also found to affect Hsp90 in mediating the folding of CFTR. For example,

cochaperone p23was determined to be important in stabilizing and preventing degradation of

∆F508-CFTR (Wang et al., 2006). Recently, Hsp90 was also shown to have a negative impact on

FK506-binding protein 38 (FKBP38), which was localized to the ER membrane and promoted

the posttranslational processing and cell surface expression of CFTR. Mutation on the TPR motif

of this protein, which is required for the binding to Hsp90, uncoupled the two proteins, and

reduced CFTR synthesis but improved the maturation of the channel (Banasavadi-Siddegowda et

al., 2011).

29

2. Peripheral chaperone systems

One of the defects of rescued ∆F508-CFTR is its very rapid removal from the plasma

membrane due to its inability to recycle back to the surface after being internalized(Sharma et

al., 2004; Lukacs et al., 1993; Cholon et al., 2010). This process is the responsibility of the

peripheral chaperone systems. Recent studies done by the Lukacs group have identified

overlapping chaperone systems working as both peripheral and ER control machinery for CFTR

(Okiyoneda et al., 2010). Using siRNA to knock down 33 E3 ligases involved in the down-

regulation of plasma membrane proteins and CFTR ERAD, Okiyoneda et al. determined that

CHIP was the main E3 ligase responsible for the ubiquitination of complex glycosylated

(mature) ∆F508-CFTR. Knocking down CHIP reduced the internalization of mutant CFTR and

partially restored its recycling. Ablation of CHIP also delayed the delivery of internalized

proteins to lysosome for degradation via the endosomal sorting complex required for transport

(ESCRT 0-III) components, which redirected the mutant protein away from the recycling

pathway (Okiyoneda et al., 2010).

Moreover, Hsp70, Hsp90 and a subset of their cochaperones such as Aha1, Hdj-2, and

BAG-1 were also identified to be part of the peripheral quality control machinery. Either

knocking down Hsp70 or Hsp90 or breaking apart their interactions with CHIP via mutating the

TPR domain prevented channel down regulation from the plasma membrane. Ablation of the

cochaperones also reduced the ubiquitination of the mutant channel and its endocytosis

(Okiyoneda et al., 2010). These chaperones and cochaperones are also involved in the quality

control of CFTR at the ER and post-translation (Chanoux and Rubenstein, 2012). Other proteins

have been identified to be a part of the peripheral quality control. C-Cbl functions as an adaptor

30

protein at the plasma membrane promoting the endocytosis of CFTR by a ubiquitin-independent

mechanism. At the early endosomes, however, c-Cbl ubiquitinates and targets CFTR for

degradation (Ye et al., 2010). Moreover, the Ubiquitin Specific Protease-10(USP10)

deubiquitinates CFTR and regulates the recycling back to the plasma membrane of the

internalized channel (Bomberger et al. 2009). Knocking down USP10 reduced both the presence

of CFTR on the apical surface and the channel activity.

The studies of these chaperones systems will shed light on therapeutic approaches to

correct the trafficking defects of ∆F508-CFTR. Targeting the chaperones involved in the

processing of CFTR, rather than CFTR itself, might be a promising approach. However, since

CFTR quality control is such an intricate and complex system with redundant roles, to overcome

the various check points of this network will not be a simple task.

IV. SCREENS FOR CORRECTORS OF THE ∆F508-CFTR DEFECTS

The ∆F508 mutation causes several defects in the processing and function of CFTR. The

protein not only suffers from impaired trafficking from the ER to the plasma membrane, but also

rapid removal from the cell surface due to an impaired recycling mechanism. However, the

∆F508-CFTR trafficking defect is correctable, since treating cells expressing the mutant CFTR at

low temperature or with chemical chaperones such as glycerol can restore the surface expression

of the mutant (Denning et al., 1992; Sato et al., 1996).The fact that ∆F508-CFTR, when it

reaches the plasma membrane, still shows partial channel function (Bear et al., 1993) has

encouraged many groups to screen for small molecules that can rescue ∆F508-CFTR. Moreover,

it was suggested that a rescue of about 10-15% of the ∆F508-CFTR retained in the ER might

31

have therapeutic benefits to the patients (Johnson et al., 1992; Farmen et al. 2005; Zhang et al.,

2009).

1. High-throughput screens for correctors of ∆F508-CFTR

Without any indication of specific drug targets for the rescue of ∆F508-CFTR, high-

throughput screens (HTSs) of large libraries of compounds using functional or biochemical cell-

based assays have become the most practical approach. In these assays, the rescue of ∆F508-

CFTR can be indicated as an increase in the anion transport or through detecting the appearance

of the mutant protein on the cell surface (Pedemonte and Galietta, 2012). With the application of

HTSs, the last decade has witnessed the identifications of a number of compounds that could be

used to correct CF defects. Several of these compounds have shown promising potential. In

general, CF drugs can be divided into two different types: correctors, which correct the

trafficking defect of ΔF508-CFTR, and potentiators, which increase channel activity, e.g. in the

case of G551D-CFTR.

The first HTS was performed by the Verkman group, including an initial screen of a

library of 150,000 chemically diverse compounds and a second screen of 1,500 analogs of active

compounds (Pedemonte et al., 2005). In this screen, they identified the bithiazole corr-4a that

could rescue ∆F508 function in primary human airway epithelial cells obtained from ∆F508

homozygous CF patients (a rescue of about 8% of chloride conductance in non-CF samples, and

at about the same level of conductance as in samples treated at 27oC). Later studies also yielded

bithiazole analogs with improved potency with EC50 as low as 300nM (Yu et al., 2008; Ye et al.,

2010).

32

Since the first screen, several other groups have utilized HTS to identify other correctors

of ∆F508-CFTR. By screening a library of 42,000 compounds using BHK cells stably expressing

∆F508-CFTR bearing three tandem extracellular hemagglutinin (HA) epitote tags and

monitoring the appearance of ∆F508-CFTR on the cell surface as an indicator of the protein

rescue, Robert et al. identified a particular compound, the approved drug sildenafil, that showed

rescue of ∆F508 (Robert et al., 2008). Later on, sildenafil was shown to have a dual effect on

the mutant protein as it worked both as a corrector and potentiator of ∆F508-CFTR (Leier et al.,

2012). However, the author also concluded that the high doses of the drug required for the rescue

of CFTR might limit its application for therapeutic utilization.

The mechanisms of how these correctors work remain mostly unknown. However,

several groups have utilized a method aiming at specific targets based on our current knowledge

of CFTR structure and folding mechanism. The mutation ∆F508 lies on the interface domain

between NBD1 and CL4 of MSD2, and destabilizes the NBD1 and mostly the interface between

NBD1 and MSD1/MSD2 leading to protein misfolding (Mormon et al., 2008; Serohijos et al.,

2008; Lukacs and Verkman, 2012). Based on this conformational information, Sampson et al.,

utilizing a technique called differential scanning fluorimetry which detects ligands that bind and

stabilize purified protein, identified the phenylhydrazone RDR1, which is able to bind and

thermally stabilize purified NBD1 (Niesen et al., 2007; Sampson et al., 2011). Correctors that

bind directly to the protein and stabilize its folding are called pharmaceutical chaperones (PC).

Other correctors that also function as a PC are corr-4a, which was demonstrated to specifically

correct the folding at the ER of ∆F508 but not other mutant CFTR (Pedemonte et al., 2005; Loo

et al., 2008; Grove et al., 2009), and MBP, which was shown to directly bind to NBD1 to

increase CFTR trafficking (Becq et al., 1999; Stratford et al., 2003).

33

The approach to identify pharmaceutical chaperones seems promising since it has less

off-target effects due to the specificity of the binding of these compounds to CFTR and no

alteration of the cellular system (Sampson et al., 2011). Moreover, since it stabilizes the

conformation of the protein, it might correct the processing defects at both the ER and plasma

membrane. Using the homology structure of CFTR, researchers from Epix Pharmaceuticals have

performed structure-based screening of compounds that can increase the stability of the protein

by binding to the interfaces between domains of CFTR thus promoting folding and escape from

the ER (Kalid et al., 2010). Several of these compounds have shown dual activity, working both

as correctors and potentiators. The author suggested that this effect was due to the fact that they

used a model of CFTR in the conducting state, and thus stabilizing this stage not only promoted

the folding but also increased the open probability of the channel.

2. Discovery of VX-809 and VX-770 and their clinical trials

The most successful screen so far was performed by Vertex Pharmaceuticals, which

identified several compounds in the quinazolinone class acting primarily at the ER level to

facilitate folding of the protein (Van Goor et al., 2006). These compounds could rescue Cl-

transport in CF bronchial epithelial cells up to 20% of that in WT cells. This screen has led to

further discovery of some very promising drugs: the corrector VX-809 (Van Goor et al., 2011)

and the potentiator VX-770 (Van Goor et al., 2009). The latter of the two, commercially known

as Ivacaftor or Kalydeco, was recently approved by the FDA for the treatment of CF patients

bearing the mutation G551D, which affects channel gating activity (Ramsey et al., 2011).

Eckford et al., recently showed that VX-770 potentiated the activity of the purified reconstituted

channel in the absence of ATP and had an additive effect with ATP on channel regulation,

suggesting that it bound to a non-canonical site on CFTR (Eckford et al., 2012).

34

Unfortunately, VX-809 was not proven as successful as VX-770. The strong efficacy of

VX-809 in primary cells, exhibited a 25% rescue of ∆F508-CFTR, combined with its safety and

tolerability in vivo, has pushed the compound to a phase II clinical trials (Clancy et al., 2012).

However, except for a significant improvement in the sweat Cl-, the compound did not improve

lung function of ∆F508-CFTR patients. Due to its lower than expected efficacy in patients, VX-

809 is currently going through a phase II studies in combination with VX-770 (Boyle et al.,

2011; Pettit, 2012). Even though this study has shown some promising results, an ideal

combination of the two drugs has not been identified. These results also highlight the difficulty

in correcting the trafficking of ∆F508-CFTR.

3. Our screens using high-content Cellomics assays

So far, most of the HTS performed by various groups have focused on the ability of these

compounds to rescue the phenotype of mutant CFTR without understanding the pathways or

target proteins involved. Moreover, these drugs usually have to go through various clinical trials

stages before approval for clinical use, which usually take a substantial amount of time. Taking a

different approach, our lab had developed a high-content functional screen using Cellomics

KineticScan technology aiming at identifying proteins and small molecules (which are already in

the clinic or in clinical trials for other diseases) that correct the trafficking defect of ∆F508-

CFTR (Trzcinska-Daneluti et al., 2009). In this screen, we utilized human HEK283 MSR

GripTile cells that stably express ∆F508-CFTR and a mutant YFP, YFP(H148Q/I152L) whose

fluorescent signal can be quenched by halide exchange (I- for Cl

-) (Galietta et al., 2001) (Figure

7). Therefore, the quenching of fluorescent signal with time due to the iodide influx into the cells

in exchange for Cl- after exposing the cells to activators of CFTR (Forskolin/IBMX/Genistein)

and a high iodide media acts as an indicator of CFTR activity.

35

Using this approach, our lab identified several proteins that when overexpressed rescue

the function of ∆F508-CFTR. Among the hits were several chaperones, Golgi-associated,

trafficking, signalling proteins and transcription factors. One of the best hits identified was

STAT1 (Signal Transducer and Activator of Transcription 1). Knocking down of PIAS1, an

inhibitor of STAT1, also rescued ∆F508-CFTR, further supporting our findings. PIASI had been

shown to be elevated in CF epithelial cells leading to reduced activation of STAT1 and NOS2,

probably via the activation of the RhoA/ROCK pathway upstream of PIAS1(Kelley and Elmer.,

2000; Kreiselmerier et al., 2003). Recently, RhoA and ROCK were shown to be involved in the

complex with ezrin and actin that, via reorganizing the actin cytoskeleton, tethers ∆F508-CFTR

to the cytoskeleton and stabilizes it at the apical surface (Favia et al, 2010). Increasing the

activity of RhoA can rescue CFTR-dependent chloride efflux.

The above Cellomics is also suitable to identifying small molecules that can rescue

∆F508-CFTR function, or proteins that inhibit ∆F508-CFTR rescue via RNAi screen. Moreover,

one advantage of the screen, focusing on proteins, is that it can identify the pathways that are

involved in the rescue of ∆F508CFTR.

36

Figure 7: Principles of the Cellomics assays to test rescue of mutant CFTR. The mutant YFP

protein (H148Q/I152L), which is expressed in HEK293-GT cells, is halide sensitive, and its

fluorescence is quenched by iodide. The assay is performed with HEK293GT expressing the

mutant YFP and either WT-CFTR or ∆F508-CFTR. In cells expressing WT channel, when

exposed to high iodide/ low chloride media and stimulated with FIG

(Forskolin/IBMX/Genistein), the Cl-/I

- exchange via CFTR leads to quenching of fluorescent

signal. In cells expressing ∆F508-CFTR, the quenching is significantly less due to the absence of

functional channel on the cell surface. The quenching of fluorescent signal works as an indicator

of CFTR activity on the cell surface.

38

V) PROJECT RATIONALE AND GOALS:

To identify small molecule correctors of ∆F508-CFTR, our lab performed a Cellomics

screen of a library of 231 kinase inhibitors biased toward drugs that are already in the clinic or

are in clinical trials for the treatment of cancer or inflammation. Such drug repurposing has the

potential to expedite the development of treatment for CF if any of these small molecules can

indeed rescue ∆F508-CFTR. We also performed a complementary siRNA screen for kinases and

related proteins that suppress the rescue of ∆F508-CFTR to help us identify the pathways

involved.

In this thesis, I describe my work done to validate the top hits from the kinase inhibitors

screen and initial validation results of the hits of the siRNA screen. I also present the assessments

of the effect of E6201, a derivative of (5Z)-7-Oxozeaenol (Oxozeaenol), which was one of the

top hits of our kinase inhibitors screen, on the rescue of ∆F508-CFTR, and attempt to elucidate

the pathways involved in the rescue of ∆F508-CFTRby Oxozeaenol.

39

Chapter 2

Materials and Methods

40

1. Media and Reagents

Dulbecco's Modified Eagle's Medium (DMEM), F12 nutrient mixture, Dulbecco's

Phosphate Buffered Saline (D-PBS) with and without calcium or magnesium, fetal bovine serum

(FBS), trypsin, G418, Blasticidin, and Zeocin were obtained from Invitrogen (Carlsbad, CA).

SuperSignal West Femto Maximum Sensitivity kit was from Pierce (Rockford, IL), and

Affinipure goat anti-mouse antibody (Cat.#115005062) was from Jackson ImmunoResearch

(West Grove, PA). The small molecules kinome library was obtained from the Ontario Institute

for Cancer Research (OICR-see below). The mouse M3A7 anti-CFTR monoclonal antibody was

obtained from Millipore (Billerica, MA), and the anti-β-actin monoclonal antibody was from

Sigma (A5441). Mouse anti-HA.11 monoclonal antibody was from Covance (MMS-101R), and

Alexa Fluor 647-labeled goat anti-mouse antibody was from Invitrogen (A21236). The small

molecules kinase inhibitors used for validation of the compound kinome screen were from Tocris

(Bristol, UK), Selleck Chemicals and EMD Chemicals (San Diego, CA). The High Capacity cDNA

Reverse Transcription kit was obtained from Applied Biosystems, and the Platinum® SYBR® Green

qPCR SuperMix-UDG was from Invitrogen. The kinome esiRNA library was obtained from Dr. Laurence

Pelletier (The Samuel Lunenfeld Research Institute – see below). TRC (The RNAi Corsotium) shRNA

clones were a kind gift from Dr. Jason Moffat (University of Toronto), and pGIPZ shRNA clones were

provided by SIDNET (SPARCS), The Hospital for Sick Children.

2. Small Molecules Kinase Inhibitor Library

The OICR (Ontario Institute of Cancer Research) Kinase Inhibitor Cassette that was

screened contains 231 compounds that are reported to inhibit at least 68 kinases. These inhibitors

were purchased from a panel of more than 20 different vendors, or synthesized when not

commercially available. The library was designed to cover as many targets and drug-like

http://www.mcponline.org.myaccess.library.utoronto.ca/cgi/redirect-inline?ad=Invitrogen

http://www.mcponline.org.myaccess.library.utoronto.ca/cgi/redirect-inline?ad=Covance


41

compounds as possible. In cases where there are multiple compounds targeting the same primary

kinase, it was anticipated that having multiple chemotypes with different properties and

selectivity profiles would enrich the screening set. Approximately 25% of the library consists of

inhibitors that have made it into the clinic, an additional 25% being compounds in different

phases of discovery (lead generation or optimization), and the remaining 50% are tool

compounds that have not been advanced to the clinic but are known to be active inhibitors

against various kinase targets.

3. Cells

HEK293 MSR GripTite (293MSR-GT) cells stably expressing ΔF508-CFTR or wild type

CFTR (WT-CFTR) protein were stably transfected with eYFP(H148Q/I152L) cDNA in

pcDNA3.1/zeo vector using calcium phosphate (Trzcinska-Daneluti et al., 2009). At 24 h post-

transfection, the cells were seeded onto 5 × 10 cm dishes at various densities (in order to easily

pick individual clones) and selected under 100 μg/ml Zeocin. Individual clones were picked and

expanded. Expression of WT-CFTR or ΔF508-CFTR was validated by immunoblotting using

M3A7 anti-CFTR monoclonal antibodies. Expression of eYFP(H148Q/I152L) was validated by

fluorescent microscopy. 293MSR-GT cells stably co-expressing eYFP(H148Q/I152L) and

ΔF508-CFTR or WT-CFTR protein were cultured in DMEM medium supplemented with 10%

FBS, 1× nonessential amino acids, 0.6 mg/ml G418, 10 μg/ml Blasticidin, and 50 μg/ml Zeocin,

at 37 °C, 5% CO2 in humidified atmosphere. Baby Hamster Kidney (BHK) cells stably

expressing wild type (CFTR-3HA) or mutant (ΔF508-CFTR-3HA) protein with the triple

hemagglutinin (3HA) tag at the ectodomain were a kind gift from D. Y. Thomas (McGill

University, Montreal). The cells were propagated as monolayer cultures in DMEM-F12 medium

(1:1) supplemented with 5% FBS and 0.5 mM Methotrexate at 37 °C, 5% CO2. Madin Darby

42

Canine Kidney (MDCK) cells stably expressing ΔF508-CFTR protein were cultured in DMEM

supplemented with 10% FBS, 1×PenStrep and 5 μg/ml Blasticidin at 37 °C, 5% CO2. Before the

short-circuit current (Isc) studies, MDCK cells were grown on permeable millicell inserts (12

mm, Millipore) for 4 days and then treated with 10 μM kinase inhibitors for 48 h. Primary

human bronchial epithelial (HBE) cells homozygous for ΔF508-CFTR or WT-CFTR were kindly

provided by Dr. P. Karp at the University of Iowa Cell Culture Facility, and propagated on

collagen-coated permeable millicell inserts (12 or 6.5 mm, Millipore) (Zabner et al., 1996). Prior

to the Ussing chamber assay the ΔF508-CFTR inserts were treated with 10 μM kinase inhibitors

or 0.2% DMSO (vehicle control) for 48 h at 37 °C. Parental MDCK and RAW 264.7 cells were

cultured in DMEM supplemented with 5%FBS, 1×PenStrep at 37oC, 5%CO2.

4. Cellomics YFP Halide Exchange Screen

Cellomics halide exchange assay was performed as follow. Briefly, 50,000 293MSR-GT

cells (stably expressing eYFP(H148Q/I152L) and ΔF508-CFTR) per well were seeded in the 96-

well plates. The next day ΔF508-CFTR cells were treated (in triplicate) with 10 μM small

molecule kinome library (separate compound in each well), 0.2% DMSO (vehicle control), or

corr-4a (positive control) at 37 °C, or incubated at 27 °C (positive control). A 10 μM dose was

chosen based on a preliminary screen data (not shown) as a dose that covers a wide range of

inhibiting concentrations but is not toxic to ΔF508-CFTR cells. After 48 h of incubation the

medium was replaced with 152 μL of chloride solution (137 mM NaCl, 2.7 mM KCl, 0.7

mMCaCl2, 1.1 mM MgCl2, 1.5 mM KH2PO4, 8.1 mM Na2HPO4, pH 7.1), in the absence or

presence of FIG (25 μM Forskolin, 45 μM IBMX, 50 μM Genistein) at 37 °C. After 20 min

incubation, 92 μl of iodide buffer (137 mM NaI, 2.7 mM KCl, 0.7 mM CaCl2, 1.1 mM MgCl2,

1.5 mM KH2PO4, 8.1 mM Na2HPO4, pH 7.1) was added (final I−

concentration 52 mM). Using

43

the Cellomics VTI (Thermo Fisher), and a modified target activation algorithm, objects

(individual cells or sometimes clusters of cells) were defined by eYFP(H148Q/I152L)

fluorescence intensity, and the decrease in fluorescence intensity over 24-s time course, at 30 °C,

5% CO2 was recorded. The number of primary objects was used as an indicator of cell toxicity

(cell death). Valid wells contained between 70 and 300 objects per field. After collecting and

analyzing data, a second run of the screen was performed with compounds preselected based on

the first run (∼100 compounds, each in triplicate).

5. Data Analysis

Compounds with a difference in fluorescence intensity between unstimulated (−FIG) and

stimulated (+FIG) samples lower than 0.08 were rejected after the first run of the screen. The rest

of the compounds were subjected to the secondary screen. Only the compounds that exhibited a

difference in average fluorescence intensity between unstimulated and stimulated cells of at least

0.10 were further analyzed. Compounds that displayed a difference in average fluorescence

intensity of at least 0.17 were considered Tier I hits. Compounds that showed a difference in

average fluorescence intensity lower than 0.17 were considered Tier II hits. Representative

compounds of both groups were selected for further validation of the ΔF508-CFTR rescue.

6. Immunoblotting

Prior to immunoblotting ΔF508-CFTR cells were treated with 15 μM kinase inhibitors or

0.3% DMSO (vehicle control) for 48 h at 37 °C, or incubated for 48 h at 27 °C (positive control),

or transfected with shRNA construct as described above. Cells were then rinsed in cold PBS and

lysed in lysis buffer (50 mM Hepes pH7.5, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10%

glycerol (v/v), 1% Triton X-100 (v/v), 2 mM phenylmethylsulfonyl fluoride, 2× PAL inhibitors).

44

Proteins were resolved on SDS-PAGE, transferred to nitrocellulose membranes and

immunoblotted with anti-CFTR monoclonal antibodies (M3A7, 1:1000) or anti-β-actin

antibodies (1:10000). Membranes were washed with 5% Blotto, incubated with HRP-conjugated

goat anti-mouse antibody (1:5000), and washed with PBST. Signal was detected with

SuperSignal West Femto reagent.

7. Flow Cytometry

The rescue of ΔF508-CFTR was also validated by flow cytometry as described

previously. Briefly, at 48 h after adding 10 μM kinase inhibitor or 0.2% DMSO (vehicle control),

BHK cells were trypsinized, washed, and resuspended in ice-cold FACS buffer (PBS

supplemented with 2% FBS). To stain CFTR at the cell surface, cells were incubated with anti-

HA.11 monoclonal antibody (1:25) or AF647-labeled goat anti-mouse antibody (1:200) as a

control, for 1 h at 4 °C. Subsequently, the cells were washed with the cold FACS buffer and

incubated with AF647-conjugated goat anti-mouse antibody (1:200) at 4 °C for 1 h. They were

then washed as above and resuspended in FACS buffer with 1 μg/ml propidium iodide (PI). The

flow-cytometric analysis was performed using LSRII System (BD Biosciences). The data from

10,000 live (propidium iodide negative) cells were collected and analyzed with FlowJo v.7.6.4

software. Cell toxicity, as defined as >10% of cells staining positive for PI, was only observed

for Ki8751 treatment. Alsterpaullone treatment resulted in altered cellular morphology

(increased cell granularity and size) but not toxicity.

8. Short-circuit Current (Isc) Measurements in Ussing Chambers

Cell inserts or Snapwells, seeded with polarized MDCK or HBE cells (expressing ΔF508

or WT -CFTR), were mounted on an Ussing chamber apparatus (Physiological Instruments, San

45

Diego, CA) and studied under voltage clamp conditions. The buffer used in the assay composed

of 1x Hank’s Balanced Salt Solution (HBSS) supplemented with 21mM of NaHCO3, 1.2mM of

CaCl2, and 1.2mM of MgCl2.Prior to stimulation of CFTR, ENaC channels were inhibited with

10 μM amiloride (Sigma), and non-CFTR chloride channels were blocked with 250 μM DNDS

(4,4′-dinitrostilbene-2,2′-disulfonate, Sigma). CFTR currents were then stimulated using FIG,

and after the indicated time (min) inhibited using 15–50 μM GlyH-101 (Gly). Data were

recorded and analyzed using Analyzer 2.1.3. Dose-response analyses (EC50) for the top kinase

inhibitor hits were carried out with increasing inhibitor doses between 1 nM to 10 μM, applied to

MDCK cells stably expressing ΔF508-CFTR. A few of the tested compounds (PKC412,

GDC0941, PD184352, Go6976, Alsterpaullone, Kenpaullone) were toxic to MDCK cells,

resulting in loss of cell monolayer integrity and loss of resistance, detected in the Ussing

chambers. These were thus excluded from the data analysis. Schematic diagram of Ussing

chamber and one sample recorded currents are depicted in figure 8 to demonstrate the principle

of Ussing chamber and how the difference in short-circuit current (ΔIsc) was determined.

9. Isolation of Bone Marrow-derived Macrophages (BDMMs)

BMMs were prepared by culture of bone marrow isolated from femurs and tibias of

C57BL/6 mice (12-14 weeks of age). After red blood cell lysis, the cells were cultured in RPMI

1640 medium supplemented with 20% FBS, 100 units/ml penicillin, 100 µg/ml streptomycin,

and 40 ng/ml macrophage colony-stimulating factor (R & D Systems). LPS stimulations were

performed between 7 and 9 days of cell culture. Experimental and animal care were performed in

accordance with institutional guidelines.

10. Phosphoprotein analysis

46

Figure 8: Ussing chamber schematic diagram and ΔIsc calculations. (A) Schematic

diagram of Ussing chamber. The cell monolayer is mounted in the middle of a U-shaped

chamber to offset any pressure difference. The two halves of the chamber are filled with equal

amount of symmetrical buffer in order to remove any chemical, mechanical and electrical driving

forces. The ion transport across the epithelial monolayer produces a potential difference (voltage

difference). The voltage difference generated is measured using two voltage electrodes placed

near the tissue/epithelium. This voltage is cancelled out by introducing a current via two other

current electrodes. This current is called the Short-circuit current (Isc), and is equal to the net ion

transport across the epithelium (Clarke, 2009). (B) In Ussing chambers analysis, the effect of the

treatment is determined by the difference in stimulated short-circuit current (ΔIsc) between the

treated and control cells. One example of recorded currents is shown. The graph shows currents

recorded between cells treated with (5Z)-7-Oxozeaenol and DMSO control. After the insert is

mounted onto the chamber, amiloride (10 μM ) and DNDS (250 μM ) are added to inhibit the

activity of ENaC and other Cl- channels (not shown on the graph) prior to the stimulation with

FIG (25 μM Forskolin, 25 μM IBMX, and 50 μM Genistein). Then the current is inhibited with

GlyH101 (15µM). To calculate the ΔIsc, currents are normalized to 1 at the point of adding FIG.

ΔIsc is measured as the difference between the maximal current recorded between the two

samples.

http://en.wikipedia.org/wiki/Electrodes

47

Isc(

µA

mp

/ cm

2)

(A)

(A)

GlyH101

∆Is

c

FIG1

48

For analysis of phosphoproteins, RAW264.7 cells or BMMs were treated with indicated

concentrations of Oxozeaenol or E6201 for 1 hour, then stimulated with LPS (100ng/mL) for 5

minute, placed on ice, and washed with ice-cold PBS. The cells were lysed in lysis buffer

(150mMNaCl, 50 mM HEPES, 10% glycerol, 1% Triton X-100, 2 mM EDTA, 10 µg/ml

leupeptin, 10 µg/ml aprotinin, 1 µg/ml pepstatin A, 1 mM PMSF, and 1 mM Na3VO4) and

cleared by centrifugation at 14,000 rpm for 10 min. Equal amounts of proteins were resolved by

SDS-PAGE, transferred to nitrocellulose membrane, and analyzed by immunoblotting with the

indicated antibodies, followed by secondary antibodies and ECL detection (GE Healthcare)

11. shRNA Knockdown and qPCR quantification of knockdown

Prior to the Cellomics halide exchange assay ΔF508-CFTR cells (stably expressing eYFP

(H148Q/I152L) were transfected with target genes or luciferase (nonsilencing control) shRNA

constructs using Lipofectamine 2000, according to the manufacturer's instructions. Medium was

changed 6 h after transfection, and ΔF508-CFTR cells were placed at 37 °C, 5% CO2. 48 h after

transfection the cells were incubated with media containing Puromycin (5 μg/ml, 3 days).

Cellomics halide exchange assay was performed as described above.

Knockdowns were validated by two-step RT-qPCR. Total RNA was isolated using the

RNeasy 96 kit (Qiagen, Dorking, Surrey, UK), and cDNA was prepared using the High Capacity

cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). Real time PCR

reactions were performed using Platinum® SYBR® Green qPCR SuperMix-UDG (Invitrogen)

and CFX96 Real-Time System (BioRad). Primers were obtained from Integrated DNA

Technologies. For standard curves, real time PCR was performed on a fivefold dilution series

DNA.


49

Chapter 3

Results

I performed the majority of the work presented in this thesis, with the exception of the Cellomics

and flow cytometry experiments that were performed by Dr. Agata Trzcinska-Daneluti (Figure

9,11,15B, 19B). The validation results of the kinase inhibitors screen were published in the

journal Molecular & Cellular Proteomics under the title ‘Use of kinase inhibitors to correct

ΔF508-CFTR function’ (Trzcinska-Daneluti, A.M., Nguyen, L. et al., (2012) Mol Cell

Proteomics, 11(9): 745-757).

50

I. Screen for kinase inhibitors that correct ΔF508-CFTR function using high-content

functional Cellomics assays

Using a high-content functional screen our lab previously developed, a library of 231

kinase inhibitors, biased toward FDA-approved drugs or compounds currently in clinical trials

mainly for the treatment of cancer and inflammation, was screened to identify compounds that

could correct ΔF508-CFTR. Several other compounds were also included in the screen such as

(5Z)-7-oxozeaenol, SU5402, and Kenpaullone, which were small molecule kinase inhibitors that

mimicked the effect of knockdown of several of the kinases identified in an independently

performed esiRNA kinome screen. In the current small molecule screen, 293MSR-GT cells

stably co-expressing the Cl− sensitive eYFP (H148Q/I152L) mutant and ΔF508-CFTR (ΔF508-

CFTR cells) were treated with 10µM of each inhibitor from the library for 48h at 37oC. Cells

were then stimulated for 20 min using a mixture of Forskolin (25 μM)/IBMX (45 μM)/Genistein

(50 μM) (FIG) and exposed to low Cl-/high I

- solution. The quenching of fluorescence caused by

Cl-/I

- exchange, presumably by CFTR or its mutant, was recorded and quantified over time. The

list of hit compounds (Table I) consisted of those that exhibited at least 0.1 difference in average

fluorescence intensity between unstimulated (-FIG) and stimulated (+FIG) cells. The hit

compounds included several receptor tyrosine kinase inhibitors (RTKs), especially inhibitors of

FGFR1 (Fibroblast Growth Factor Receptor 1) and its downstream signaling targets (e.g.

SU5402, SU6668, Ki8751), as well as inhibitors of several important cellular signaling pathways

such as Ras/Raf/MEK/ERK (e.g. (5Z)-7-Oxozeaenol, RDEA-119), Wnt/GSK-3β (e.g. GSK-3β

Inhibitor II, Kenpaullone), TAK1/p38 (e.g. (5Z)-7-oxozeaenol, SKF86002), PI3K/Akt/mTOR

(e.g. PI-103, FPA124, 10-DEBC). Figure 9 (B-H) depicts examples of kinase inhibitors that

exhibited rescue of ΔF508-CFTR (e.g. (5-Z)-7-Oxozeaenol, SU5402, GSKInhII, Kenpaullone)

51

Table 1: Hit compounds and their validations. Hits were validated by immunoblotting in

293MSR-GT cells (WB), flow cytometry in BHK cells (Flow), and short circuit current (Isc)

analysis in Ussing chambers on epithelial MDCK cells (MDCK) or on primary Human Bronchial

Epithelial (HBE) cells harvested from lungs of ΔF508/ΔF508 homozygote patients undergoing

lung transplant. 293MSR-GT, BHK, and MDCK cells were stably expressing ΔF508-CFTR. For

MDCK, (+) indicates rescue, (−) no observed rescue and (*) indicates increased toxicity in

MDCK cells. For HBE, (+) indicates rescue observed in a sample from a patient, with (/)

separating between samples from different patients, (−) indicates no observed rescue. For the flow

experiments, (+) indicates >10% rescue, (±) 5–10% rescue, (−) indicates no rescue, (*) indicates

increased cell toxicity and (#) indicates morphological changes observed in the treated BHK cells.

For the immunoblotting experiments, (+) indicates strong rescue of ΔF508-CFTR (manifested as

increase in amount of band C in comparison to vehicle-alone control), (±) poor rescue, (−) no

rescue, and (*) indicates increased toxicity in 293MSR-GT cells. For dose response experiments

(EC50), MDCK cells were treated with increasing concentrations (1 nM to 10 μM range) of select

compounds prior to Isc analysis in Ussing chambers, (§) indicates compounds that rescue ΔF508-

CFTR function at 10 μM only.

52

Compound Pathway Target Cas Number PubChem

ID

Clinical

Trials

MDCK EC50

(MDCK)

[nM]

HBE Flow

(BHK)

WB

(293GT)

(5Z)-7-oxozeaenol Ras/Raf/MEK/ERK or Tak1/p38 ERK1/2, TAK1

MAP3K 66018-38-0 CID 9799061

Phase I, II

(E6201) + 60

+ +/+ +

+/− +/+/+ + +

SU6668 (Orantinib) Ras/Raf/MEK/ERK PDGFRβ,

VEGFR2, FGFR1,

EGFR

252916-29-3 CID 5329099 Phase I + 1047 + +/+ + *

SU5402 Ras/Raf/MEK/ERK VEGFR2, FGFR1,

PDGFRβ 215543-92-3 CID 5289418 + 12.9

+/+/+/− /+

+ + +

EKI-785, CL-387,785 Ras/Raf/MEK/ERK EGFR 194423-06-8 CID 2776 + 124.6 −/+ ± +

FPA 124 PI3K/Akt/mTOR Akt/PKB

902779-59-3

CID

16034833

+ § +/+ − −

Gsk-3β Inhibitor II Wnt/GSK-3β GSK-3β GSK-3β 478482-75-6 CID 6539732 + 127.8 + +/+ +

AZD0530, Saracatinib,

NSC-735464

Src-Bcr-Abl activates:

Ras/Raf/MEK/ERK (proliferation);

JAK/STAT (proliferation);

PI3K/Akt (mitochondrion)

pathways

Src/Abl

379231-04-6 CID

10302451 Phase II + 165.7 + + − ±*

7-Cyclopentyl-5-(4-

phenoxyphenyl)-7H-

pyrrolo[2,3-d]pyrimidin-

4-ylamine (Lopac-C-

8863)

T-cell receptor signaling;

phosphorylates PKC and PI3K;

activates Ras/Raf/MEK/ERK

pathway

Lck

213743-31-8 CID 6603792 + 147.4 +/+ + − ±*

PD173074 Ras/Raf/MEK/ERK FGFR1 219580-11-7 CID 1401 + § +/+ − −

PD0325901 Ras/Raf/MEK/ERK MEK1/2 391210-10-9 CID 9826528 Phase I + 6.1 + +/+ − + −

PI-103 PI3K/Akt/mTOR p110 PI3Ks,

mTORC1/2, DNA-

PK

371935-74-9 CID 9884685 + 16.4 −/+ + −

RDEA-119, AR-119,

BAY869766

Ras/Raf/MEK/ERK MEk1/2 923032-37-5

CID

44182295 Phase I, II + 41 +/− + −

SKF-86002 Tak1/p38 p38 MAP 72873-74-6 CID 5228 + 140.5 +/− ± −

GW5074 Ras/Raf/MEK/ERK Raf1 220904-83-6 CID 5924208 − − +/+ + −

Kenpaullone Wnt/GSK-3β GSK-3β, cdks, Lck 142273-20-9 CID 3820 −* −* + + + +/+ − +

Alsterpaullone Wnt/GSK-3β GSK-3β, cdks, Lck 237430-03-4 CID 5005498 −* −* −/+ +# *

Ki8751 VEGFR2 activates:

Ras/Raf/MEK/ERK pathway via

PKC, Akt/PKB pathway via PI3K

VEGFR2,

PDGFRα, FGFR2 228559-41-9 CID

11317348 − − − +/+/+ +*

# +

10-DEBC (Akt specific

inhibitor X)

PI3K/Akt/mTOR Akt/PKB 201788-90-1

CID

16760284 − − +/− − +

53

Figure 9: Representative hits of the high-content screen. Average normalized fluorescence

intensity values of ∆F508-CFTR cells (which co-express eYFP(H148Q/I152L) that were (A)

transfected with shRNA for FGFR1, or (B) treated with 10 µM SB431542 (non-corrector), (C)

(5Z)-7-Oxozeaenol, (D) SU5402, (E) GSK-3 Inhibitor II, (F), RDEA119, (G) Kenpaullone, (H),

Ki8751, and grown at 37 °C. After 48 h (5 days in case of shRNA knockdown of FGFR1) cells

were stimulated with FIG (25µM Forskolin, 45µM IBMX and 50µM Genistein), and quenching

of fluorescence during Cl-/I

- exchange of 70–300 cells was quantified simultaneously and re-

corded by the Cellomics. FGFR1 knockdown was 90% (as determined by RT-qPCR)

[Experiments for this figure were carried out by Dr. Agata Trzcinska-Daneluti]

55

and one kinase inhibitor that did not rescue the function of mutant CFTR (SB431542). Figure 9A

displays the rescue of ΔF508-CFTR after shRNA knockdown of FGFR1, a target of several

kinase inhibitors identified in the screen.

II. Validation of the Hits

1. Maturation of ΔF508-CFTR

Among the positive hits from the initial screen using Cellomics, 41 representative

compounds were chosen for further analysis and validations of ΔF508-CFTR rescue using

alternative methods. These compounds were selected based on their inhibition of kinases that

participate in four major signaling pathways identified by the screen (e.g. Ras/Raf/MEK/ERK,

Wnt/GSK-3, PI3K/Akt/mTOR and TAK1/p38). First, the effect of the compounds on the

maturation of ΔF508-CFTR was examined by immunoblotting. As previously mentioned, in

SDS-PAGE, WT-CFTR appears as two bands, a prominent band C around 180 kDa representing

the mature, fully glycosylated form of CFTR, and a minor band B around 150 kDa representing

the core glycosylated, immature form. ΔF508-CFTR, which exhibits impaired maturation,

migrates primarily as band B. For this assay, HEK293GT cells stably expressing ΔF508-CFTR

were treated with either DMSO (negative control) or 15µM of selected top hit compounds.

Treatment of the cells at 27oC was used as a positive control, which, as shown in figure 10, led to

a robust increase in band C. Moreover, treatment with some of the indicated compounds (e.g.

(5Z)-7-Oxozeaenol, Kenpaullone) also led to an increase in the maturation of ΔF508-CFTR

(appearance of band C) compared to vehicle control treatment, albeit a less robust rescue than

low temperature. Unfortunately, several compounds exhibited adverse effect on the cells and

thus could not be tested by immunoblotting. In figure 10, TCS2312 illustrates one such

compound. Treatment of cells with this compound abolished both forms of CFTR.

56

Figure 10: Effect of select kinase inhibitors on ∆F508-CFTR maturation analyzed by

immunoblotting.293MSR-GT cells stably expressing ∆F508-CFTR were treated with

15µMkinase inhibitors or 0.3% DMSO (vehicle control), as indicated, grown at37 °C for 48 h,

and the appearance of the mature protein, band C, monitored by immunoblotting with anti-CFTR

antibodies. Band B represents the immature protein. DMSO represents vehicle-alone control,

27oC represents temperature rescue of ∆F508-CFTR at27 °C, 37 °C represents untreated ∆F508-

CFTR control, and WT represents WT-CFTR. Top panels depict the anti-CFTR immunoblot and

bottom panels depict actin (loading) control.**represents cellular toxicity.

58

In addition, flow cytometry was performed to demonstrate the appearance of ΔF508-CFTR at the

plasma membrane. Toward this end, BHK cells stably expressing ΔF508-CFTR bearing a 3HA

tag on the ectodomain of the protein were treated with either 0.2% DMSO (negative control) or

10µM kinase inhibitors; cells grown at 27oC were used as a positive control. To quantify the

amount of ΔF508-CFTR reaching the cell surface, nonpermeabilized BHK cells were then

immunostained with antibodies directed against the 3HA epitote tag before being subjected to

flow cytometry. Figure 11 depicts results of treatment at 27oC (panel A) or with several

compounds (panels B-E), as well as cell surface expression of WT-CFTR (panel F). A summary

of the percentage of increase in cell surface expression of ΔF508-CFTR after treatment with the

kinase inhibitors is shown in panel G. Collectively, these results show that surface expression of

ΔF508-CFTR was rescued by as much as 40-50% after treatment with several inhibitors such as

Oxozeaenol, SU5402, RDEA119 and SU6668. This was about half of the rescue observed by

treatment at lower temperature (about 80% increase in cell surface expression).

2. Functional Analysis of Correction of ΔF508-CFTR by the Kinase Inhibitors

To further analyze the effect of the kinase inhibitors on ∆F508-CFTR, the rescue of

∆F508-CFTR activity was determined by short-circuit current (Isc) analysis in Ussing chambers

using epithelial MDCK cells that stably express ΔF508-CFTR. Cells were grown on inserts with

permeable membrane until they reached confluency, and then treated with 10 μM kinase

inhibitor or 0.2% DMSO (vehicle control) for 48h. Inserts were then mounted in Ussing

chambers for short-circuit current studies. CFTR channel was stimulated with FIG in the

presence of ENaC inhibitor Amiloride and DNDS, an inhibitor of Na+/HCO3

− cotransporters and

Cl−/HCO3

−exchangers; and currents across the layer of cells were recorded. Figure 12A-E

59

Figure 11: Effect of kinase inhibitors on cell surface expression of ∆F508-CFTR analyzed

by flow cytometry. BHK cells stably expressing ∆F508-CFTR-3HA were placed at (A) 27 °C

(positive control) for 48 h, or (B) treated with 10 μM (5Z)-7-oxozeaenol, (C) SU5402, (D)

SU6668, or (E) RDEA-119/AR-119/BAY869766, at 37 °C. (F) BHK cells stably expressing

WT-CFTR. Flow cytometry was then performed on non-permeabilized cells following

immunostaining for the HA epitope located at the ectodomain of ∆F508-CFTR or WT-CFTR, to

quantify the amount of cell-surface CFTR in the analyzed cells. (G) Summary of increase in cell

surface expression of ∆F508-CFTR (%change in fluorescence intensity) of the hits analyzed by

flow cytometry (two independent experiments, 10,000 live cells per treatment per experiment).

[Experiments for this figure were carried out by Dr. Agata Trzcinska-Daneluti]

61

Figure 12: Effect of compounds treatment on ∆F508-CFTR channel activity in MDCK cells

stably expressing ∆F508-CFTR. Representative short-circuit current (Isc) traces of MDCK

∆F508-CFTR monolayers treated with vehicle (DMSO) alone (black lines), or 10 µM of (A)

(5Z)-7-oxozeaenol, (B) SU5402, (C) SU6668, (D) GSK-3β Inhibitor II, (E) 7-Cyclopentyl-5-(4-

phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4- ylamine (C8863) (grey lines), for 48 h prior to

analysis in Ussing chambers. ENaC was inhibited with 10 µM amiloride; non-CFTR chloride

channels were blocked with 250 µM DNDS. CFTR currents were stimulated with FIG (25 µM

Forskolin, 25 µM IBMX and 50 µM Genistein) at time 0 and after the indicated times (arrows)

inhibited using 15 µM GlyH-101 (Gly). (F) Representative short-circuit currents mediated by

untransfected MDCK cells and MDCK cells that stably express WT-CFTR or ΔF508-CFTR. (G)

Summary of the increase in short-circuit currents (∆Isc) in MDCK cells stably expressing

∆F508-CFTR that were treated by the analyzed compounds (relative to DMSO vehicle control

alone). Data are mean ± S.E. (n): number of experiments (*: p<0.05; **: p<0.01, paired one-

tailed Student’s t-test).

62

**

**

**

**

**

** **

*

*

*

* *

63

displays the effects of treatments with representative compounds (e.g. Oxozeaenol, SU5402,

SU6668, GSK-3β Inhibitor II, 7-Cyclopentyl-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-

4-ylamine/C8863), and others on ΔF508-CFTR function (i.e. chloride channel activity).

Stimulated current in cells stably expressing WT-CFTR is also shown in comparison with that in

cells expressing ΔF508-CFTR and in parental MDCK cells (Figure 12F). Parental MDCK cells

showed no response to either FIG or the CFTR inhibitor, GlyH101. In addition, there was some

channel activity in the untreated ΔF508-CFTR MDCK cells, suggesting some mutant CFTR had

escaped from the ER to the surface in the overexpression system. DMSO control treatment

(0.2%) did not improve this activity. Summary of the difference in maximal stimulated currents

(∆Isc) between cells treated with inhibitors and vehicle control (Figure 12G) shows various

degrees of functional rescue of ΔF508-CFTR with several of these hit compounds. The ∆Isc

between cells expressing WT-CFTR and those expressing the mutant protein was about

8µAmp/cm2. On average, the top compounds (Oxozeaenol, SU5402, SU6668, RDEA-119, PI-

103) exhibited an increase of 20-30% (1.5 to 2.5µAmp/cm2) in stimulated current compared to

what was observed in cells expressing WT-CFTR.

3. Effect of Kinase Inhibitors on ΔF508-CFTR Chloride Channel Activity in

Primary Human Bronchial Epithelial (HBE) Cells Harvested from CF Patients

Even though some of these compounds were proven to be able to rescue the function of

ΔF508-CFTRin vivo in MDCK cells, it was necessary to determine if these inhibitors could

rescue function in bronchial epithelial cells from CF patients. To this end, we examined the

effect of treatment with selected compounds on primary HBE cells obtained from patients that

were homozygous for ΔF508-CFTR and had undergone lung transplant. Since the samples were

limited, only the compounds that rescued the function of ΔF508-CFTR in MDCK cells and/or in

64

Figure13: Effect of compounds treatment on ∆F508-CFTR activity in primary Human

Bronchial Epithelial (HBE) cells harvested from lungs of ∆F508/∆F508 homozygote

patients undergoing lung transplant. Representative short-circuit currents (Isc) mediated by

∆F508-CFTR human bronchial epithelial (HBE) monolayers treated with vehicle (DMSO) alone

(black lines), or 10 µM of (A) (5Z)-7-oxozeaenol, (B) SU5402, (C) SU6668,(D) GSK-3β

Inhibitor II, (E) 7-Cyclopentyl-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-ylamine

(C8863), (F) Kenpaullone (grey lines) for 48 h prior to analysis in Ussing chambers. ENaC

sodium channels were inhibited with 10 µM amiloride; non-CFTR chloride channels were

blocked with 250 µM DNDS. CFTR currents were stimulated with FIG (25 µM Forskolin, 25

µM IBMX and 50 µM Genistein) as indicated, and after the indicated times (black arrows)

inhibited using 15µM (panels A, B, E, F) or 50 µM (panels C, D) GlyH-101 (Gly). In panels C

and D, half of the Gly solution (25 µM) was added twice sequentially, as indicated. (G)

Representative short-circuit currents mediated by HBE cells from non-CF controls (WT-CFTR).

(H) Summary of increase in short-circuit currents (∆Isc) in HBE cells stably expressing ∆F508-

CFTR that were treated with the indicated compounds. Data from individual patients are shown.

Where several replica were tested from the same patient (see Table I), the average value is

shown. Bars represent median values. The baseline currents (before amiloride addition) ranged

between 6 –20 µAmp/cm2 for WT-HBE and 19 – 40 µAmp/cm

2for ∆F508-CFTR HBE. After

adding amiloride, the currents for both WT and ∆F508-CFTR HBE were ~0 –3 µAmp/cm2.

66

several other validation assays were chosen to be tested in HBE cells. Because there was

variation between cells obtained from different patients due to differences in the severity of the

disease and conditions of each patient, the effects of the treatments were assessed by comparing

samples from each patient with the control treatment (vehicle alone) from the same patient. This

eliminated patient-to-patient variability.

Like the assays in MDCK cells, primary HBE cells were grown on permeable inserts and

then treated with either 10µM of selected compounds or 0.2% DMSO control. Representative

traces with cells treated with Oxozeaenol, SU5402, SU6668, GSK-3β Inhibitor II, 7-

Cyclopentyl-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-ylamine (C8863), and

Kenpaullone are shown in Figure 13A-F. Cells obtained from different patients behaved

differently; and some still exhibited partial activity even with the control treatment. Non-CF

HBE obtained from normal lung was used for comparison (Figure 13G). In non-CF samples, the

stimulated current was about 2µAmp/cm2

compared to unstimulated sample. Most of the

compounds showed variable rescue of ΔF508-CFTR in samples from different patients (Figure

13H), as expected. However, on average, the compounds enhanced the current in HBE samples

to approximately 10% of WT activity (0.2µAmp/cm2). In some cases, the enhancement of the

current was up to 40- 50% of activity of WT HBE.

Combined together, these results suggest that several of these kinase inhibitors can

improve the function of ΔF508-CFTR in both tissue culture cells and in HBE obtained from

patients. This occurs likely by improving the maturation and surface expression of the mutant

channel. However, the possibility that some of these compounds act as potentiators of ΔF508-

CFTR activity (once at the plasma membrane) cannot be excluded based on our functional

analysis. The summary of all the validation results is presented in Table 1. 18 compounds

67

rescued ΔF508-CFTR in at least one of these assays. Several compounds, such as Oxozeaenol or

SU5402, gave consistent rescue of ΔF508-CFTR in all of the assays performed.

III. Dose Response Curves of Rescue of ΔF508-CFTR in MDCK Cells Treated with

Select Kinase Inhibitors

The concentration of the compounds initially used in the screen was 10µM, which was

rather high. Therefore, to further assess the effect of increasing doses of these kinase inhibitors

on rescuing ∆F508-CFTR function, Isc analyses using Ussing chamber were performed in

MDCK cells stably expressing ∆F508-CFTR treated with an increasing concentrations covering

a range from 1nM to 10µM of our top hit inhibitors (Figure 14 and table 1). The estimated half

maximal effective concentrations (EC50) of most of the analyzed compounds were in the

nanomolar range (6 to 125nM) with the exception of SU6668 (EC50~1µM). The effects of two

other compounds, PD173074 and FPA124, could only be seen at concentration of 10µM. Due to

limited amount of HBE samples, dose-response analyses in these cells could not be performed.

68

Figure 14:Dose response curves of select kinase inhibitors for rescue of ∆F508-CFTR

expressed in MDCK cells. Average increase in short-circuit currents (∆Isc) of MDCK cell

monolayers stably expressing ∆F508-CFTR (relative to DMSO vehicle control alone) treated for

48 h with 1, 10, 20, 100, 200, 1000,and 10,000 nM of the top inhibitor compounds, indicated in

panels (A–K). Data are mean ± S.E. of (n) samples.

69

I)

70

IV. Analyses of E6201, a derivative of (5Z)-7-Oxozeaenol

1. E6201 did not rescue ∆F508-CFTR maturation and function

One of our top hits from the screen was a theTAK1/p38 and ERK inhibitor, Oxozeaenol.

However, Oxozeaenol is not useful for therapeutic utilization due to its instability in plasma

(Goto et al., 2009); thus, a derivative of it, E6201, was synthesized by the pharmaceutical

company Eisai. E6201 has now passed phase II clinical trials for the treatment of psoriasis

(Muramoto et al., 2010; Kumar et al., 2012). Therefore, we wanted to determine if E6201 can

rescue ∆F508-CFTR similar to its parental compound, Oxozeaenol. E6201 was provided to us by

Eisai. We thus tested the effect of E6201 on rescue of∆F508-CFTR using the Cellomics assay,

Immunoblotting, and using Ussing chamber as described above. Figure 15A shows structures of

both compounds with the differences encircled. The most prominent change is the N-alkyl

substitution in E6201 (Shen et al. 2010).

Unfortunately, while Oxozeaenol exhibited rescue in all of our validation methods,

treatment of cells with E6201 at several doses did not yield any significant rescue of ∆F508-

CFTR in any of the assays performed. In the Cellomics assays on HEK293GT cells, 48h

treatment with increasing concentrations of E6201 (1nM-50µM) did not yield any significant

rescue of ∆F508-CFTR activity. Lower doses of E6201 (10nM to 10μM) improved the channel

activity, but not significantly, only about 1-2%. The highest increase in activity, about 4-5%, was

observed with the treatment at 10 µM (Figure 15B). Furthermore, treatments at higher doses of

E6201 (20µM to 50µM) seemed to have an adverse effect, resulting in a decreased activity to a

level lower than basal activity. At these concentrations, the quenching of signal was even lower

than that of cells treated with DMSO control. Similar results were observed in Ussing chamber

71

Figure 15: E6201 does not rescue the function of ∆F508-CFTR.(A) Molecular structures of

(5Z)-7-Oxozeaeol (left) and its derivative E6201. The differences are highlighted (circled). The

most important change is the N-akyl substitution at C14 (Shen et al., 2010). (B) E6201 did not

rescue ∆F508-CFTR function in Cellomics assays. Average normalized fluorescence intensity

values of HEK293MSR-GT ΔF508-CFTR cells (which co-express eYFP (H148Q/I152L) that

were treated with either increasing concentrations of E6201 (1nM to 50μM) or 10μM of (5Z)-7-

Oxozeaenol for 48h at 37oC. After 48 h cells were stimulated with FIG and quenching of

fluorescence during Cl−/I

− exchange of minimum 100 cells was quantified simultaneously and

recorded. Data are mean ± SEM of three replicates, *: p<0.05 (unpaired one-tailed Student’s t-

test) [Experiments for this panel were carried out by Dr. Agata Trzcinska-Daneluti].

72

(A)

(B)

*

73

Figure 16: E6201 does not rescue the maturation and function of ∆F508-CFTR by

immunoblotting and Ussing chambers.(A) E6201 did not improve the appearance of band C in

immunoblotting. HEK293GT cells expressing ∆F508-CFTR were treated at different

concentrations of E6201 (1nM to 20µM) for 48h at 37oC then lysed and analyzed by

immunoblotting. Treatments at 27oC and (5Z)-7-Oxozeaenol at 15µM were used as positive

controls. Both led to improved appearance of band C. Below is the relative intensity of band B,

band C, and ratio of band B/ band C for each treatment. (B) Summary of the change in short-

circuit currents (ΔIsc) in MDCK cells stably expressing ΔF508-CFTR that were treated by

E6201 (relative to DMSO vehicle control alone). E6201 did not rescue the function of ∆F508-

CFTR in MDCK cells expressing ∆F508-CFTR in Isc analysis by Ussing chambers. MDCK cells

stably expressing ΔF508-CFTR were treated with either increasing concentrations of E6201

(1nM - 20μM) or 10µM (5Z)-7-oxozeaenol, and grown at 37C for 48h prior to analysis in

Ussing chambers. ENaC sodium channels were inhibited with 10 μM amiloride; non-CFTR

chloride channels were blocked with 250 μM DNDS (not shown in the graphs). CFTR currents

were stimulated with FIG at time 0 and after the indicated times (arrows) inhibited using 15 μM

GlyH-101 (Gly). Data are mean ± S.E. (n): number of experiments. *: p<0.05 (paired one-tailed

Student’s t-test)

74

(A)

(B)

* (B)

75

experiments in MDCK cells treated with increasing concentrations of E6201 (1nM-20µM).

Treatments with E6201 at 10 or 20 µM reduced the stimulated current compared to DMSO

control (ΔIsc is below 0), and stimulated currents recorded in cells treated with lower doses

(1nM to 1µM) were not significantly different from DMSO control (ΔIsc~0) (Figure 16B).

Lastly, on Western blot, E6201 also did not improve the appearance of band C in cells

expressing ∆F508-CFTR treated with 1nM to 20µM of E6201 (Figure 16A). Thus, altogether,

these results suggest that unlike the parental compound Oxozeanol, E6201 was not effective at

rescuing ∆F508-CFTR.

2. Comparison of the effect of E6201 and (5Z)-7-Oxozeaenol on different signalling

pathways

Because Oxozeaenol and its derivative, E6201, had different effects on the rescue of

∆F508-CFTR, determining the differences in the effects of both compounds on downstream

signaling pathways might provide us insight into the mechanism/pathways utilized by the

parental compound in the rescue of ∆F508-CFTR. While Oxozeaenol has been shown to inhibit

both TAK1 and the MEK/ERK pathway (even though with much lower potency for the latter

pathway) (Ninomiya-Tsuji et al, 2003), E6201 has only been shown to primarily inhibit the

MEK/ERK pathway (Goto et al, 2009) (Figure 17). This suggests that Oxozeaenol might work

through inhibiting TAK1 and the downstream signaling pathways to rescue ∆F508-CFTR.

To test this hypothesis, inhibition of phosphorylation of signaling targets downstream of

TAK1 was studied. Macrophages extracted from mouse bone marrow (BMM) and RAW264.7

cells were treated with increasing concentrations of either Oxozeaenol or E6201 (100nM to 5 or

10µM) for 1 hour prior to a 20-minute stimulation with LPS. Downstream inhibition of

76

Figure 17: Signaling pathways inhibited by (5Z)-7-Oxozeaenol and E6201. (5Z)-7-

Oxozeaenol is a potent inhibitor of TAK1 (IC50~8nM), which phosphorylates p38, JNK, NFκB

to activate downstream signaling pathways. (5Z)-7-Oxozeaenol also inhibits the MEK/ ERK

pathway, but with a lower potency (IC50~411nM). On the other hand, E6201 has only been

shown to be a strong inhibitor of MEK/ERK pathway with IC50 ~5nM (*Ninomiya-Tsuji et al.,

2003; ** Goto et al., 2009).

77

E6201

TNFR/IL1-R/TLR

TAK1

MEK1/2

ERK1/2

p38 NFκB

JNK

Oxozeaenol

IC50~411nM*

IC50~8nM*

IC50~5nM**

78

phosphorylation was determined by immunoblotting for phosphorylated p38, JNK, ERK1/2,

NFκB and total level of IκB. Both E6201 and Oxozeaenol were found to inhibit the

phosphorylation of all of the above targets. However, Oxozeaenol exhibited a 5-10 fold better

inhibition of phosphorylation compared to E6201 (Figure 18A, B). For example, Oxozeaenol

abolished phosphorylation of p38 and JNK at 500nM while this could only be observed at 5µM

of E6201. Moreover, Oxozeaenol also exhibited better inhibition of the phosphorylation of

ERK1/2 compared to E6201. This was unexpected as E6201 was proposed to be a more potent

inhibitor of the MEK/ERK pathway (IC50 of 5.1nM compared to 411nM of Oxozeaenol) (Figure

17).

The difference in inhibition of phosphorylation of NFκB was not clear; neither of the two

compounds seemed to completely inhibit NFκB phosphorylation (Figure 18A, B). However, the

re-appearance of the total level of IκB showed a significant difference between the effects of

Oxozeaenol and E6201. IκB is an inhibitor of NFκB that is targeted for degradation during

NFκB activation (Sakurai H, 2012); thus, the restoration of the total level of IκB can be used as

an indicator of the inhibition of NFκB activation. As shown in Figure 18, the level of IκB

restored with the treatment of 1µM of Oxozeaenol was much higher than that with the treatment

of E6201 at 10µM. This indicated a more than 10-fold greater potency for the inhibition of this

pathway. Nevertheless, this difference in potency cannot explain why E6201 did not rescue

∆F508-CFTR because this compound was used at much higher concentrations in both Cellomics

and Ussing chamber assays (20 and 50µM respectively). There might be other different

pathways/mechanism involved, and further investigation is therefore required.

79

Figure 18: Both (5Z)-7-Oxozeaenol and E6201 inhibit the phosphorylation of similar

downstream signaling targets. Both compounds exhibit inhibition of phosphorylation of p38,

ERK1/2, JNK, NFκB in (A) BDMM and (B) RAW264.7 cells stimulated with 100ng/mL LPS

after 1h treatment with the indicated concentrations of either (5Z)-7-Oxozeaenol or E6201. Cells

were lysed 20 min after stimulation and analyzed by immunoblotting with the indicated

antibodies.

80

(B) RAW 264.7

(A) BDMM

81

V. Validation of hits of esi-RNA screen

To complement the kinase inhibitor screen above, an esi-RNA screen was performed to

identify kinases that usually suppressed the rescue of ∆F508-CFTR. Using the same technique as

in the former screen, a library of about 900 esiRNAs targeting primarily kinases and other related

proteins (provided by L. Pelletier, Mt.Sinai, Toronto) was used to knock down target genes.

Among the hits from this screen, 21 genes (Table II) were chosen to be validated by using a

different RNAi technique, shRNA. This would also reduce the chances of having off-target

effects when knocking down target genes.

In the Cellomics assays, since fluorescence-tagged shRNAs could not be used, a panel of

about 200 different untagged TRC-shRNA constructs targeting these 21 genes (multiples clones

for each gene) was used. These constructs were transfected intoHEK293GT cells stably

expressing ∆F508-CFTR and the halide-sensitive YFP mutant, and then subjected to Cellomics

assays to determine their ability to rescue ∆F508-CFTR function. In parallel, qPCR was

performed to determine the knock-down efficiency of these constructs.

To examine the rescue of maturation of ∆F508-CFTR using Western blot analysis,

pGIPZ-shRNA (with GFP tag) constructs were used to knock down target genes in HEK293GT

stably expressing ∆F508-CFTR. The GFP allowed easy confirmation of transfection efficiency.

After transfection, cells were placed in selection media (supplemented with 8µg/mL Puromycin)

for 3 days to obtain the highest transfection efficiency possible (about 80-90% transfection

efficiency).

The TRC clones yielded varied degrees of knock down, but most of the constructs

resulted in more than 50% knock-down of target genes (Figure 19A). In the Cellomics assays, 14

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Table II: Hit genes of the siRNA screen.

Gene Symbol Description

BRAF v-raf murine sarcoma viral oncogene homolog B1

CAMK2B Calcium/calmodulin-dependent protein kinase II beta

CDK10 Cyclin-dependent kinase 10

CLK3 CDC (cell division cycle)-like kinase 3, dual specificity protein kinase

DTYMK Deoxythymidylate kinase (thymidylate kinase)

DUSP22 Dual specificity phosphatase 22

ERN1 Endoplasmic reticulum to nucleus signaling 1

FGFR1 Fibroblast growth factor receptor 1

FLJ32685 NEK10, NIMA (never in mitosis gene a)-related kinase 10

IPMK Inositol polyphosphate multikinase

MAP3K13 Mitogen-activated protein kinase kinase kinase 13

MET Met proto-oncogene (hepatocyte growth factor receptor)

PANK1 Pantothenate kinase 1

PANK4 Pantothenate kinase 4

PCK2 Phosphoenolpyruvate carboxykinase 2 (mitochondrial)

PRKAR2B Protein kinase, cAMP-dependent, regulatory type II beta

RIPK4 Receptor-interacting serine-threonine kinase 4

RPS6KC1 52 kDa ribosomal protein S6 kinase, S6K-delta-1

SHPK sedoheptulokinase

SOCS1 Suppressor of cytokine signaling 1

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Figure 19: Validation of hits of the esiRNA screen by Cellomics assays. 200 TRC-shRNA

clones were used to knock down 21 hit proteins of the esiRNA screen. For each target, result of

one representative shRNA clone is presented. (A) For Cellomics assays, average normalized

fluorescence intensity values of HEK293GT cells stably expressing ∆F508-CFTR and mutant

eYFP (H148Q/I152L) that were transfected with TRC-shRNA constructs targeting the indicated

proteins. After transfection, cells were incubated with media containing 5μg/mL Puromycin for 3

days. Cells were then used in Cellomics assays to measure rescue of ∆F508-CFTR [performed

by Agata Trzcinska-Daneluti]. (B) Degrees of knock down of the corresponding shRNA clones

determined by qPCR are shown. Data are means ± S.E. of 3 repeats.

84

(B)

(A)

85

genes, when knocked down by shRNA, produced reproducible rescue of CFTR function (15-

30% increase in channel activity).These included genes encoding for the RTK FGFR1, the Raf

kinase BRAF and the MAPK kinase kinase MAP3K13 of the Ras/Raf/Mek/Erk pathway. Other

hits included genes that encoded for the receptor interacting protein kinase RIPK4, the

thymidylate kinase DTYMK, and the S6 ribosomal protein RPS6KC1 (Figure 19A). The result

for the knock down of MET was not included because the knock down was lethal to the cells,

and the high quenching of fluorescence signal could have been due to the loss of integrity of

cells. In Western blot analysis, most of the pGIPZ constructs had lower degrees of knock down

compared to the representative TRC clones. Most of the PGIPZ constructs resulted in less than

50% of knock down of the target genes (Figure 19B, 20B). However, 9 genes, when knocked

down, still exhibited significant improvements in the appearance of band C (Figure 20A), among

which, several genes such as RPS6KC1, FGFR1, DTYMK, RIPK4, were also confirmed in the

Cellomics assays.

The results from this screen provided complementary data to the kinase inhibitors screen.

For example, from the kinase inhibitor screen, inhibitors of FGFRs showed robust rescue of

∆F508-CFTR function. From the esi-RNA screen, knocking down FGFR1 improved both the

function of CFTR in the Cellomics assays and the appearance of mature band C on Western blot.

Other hits that were confirmed with shRNA were BRAF and MAP3K13, which belonged to the

RAS/RAF/MEK/ERK pathway, one of the four major pathways inhibited by the top hits from

the inhibitors screen (Table I) (Roring and Brummer, 2012; Craig et al, 2008). Other interesting

hits were PRKAR2B gene encoding one of the four regulatory domains of PKA (Yu et al, 2012),

which is a major kinase that phosphorylates and regulate CFTR channel activity, and IPMK,

which is involved in PI3K/Akt pathway downstream of FGFR (Lee et al, 2012).

86

Figure 20: Validation of hits of the esiRNA screen by immunoblotting.HEK293GT cells

expressing ∆F508-CFTR were transfected with either scrambled shRNA or pGIPZ-shRNA

constructs targeting the indicated proteins. 48h after transfection, cells were placed under

Puromycin (8μg/mL) selection for 3 days to obtain the highest transfection efficiency. (A) Cells

were then lysed and analyzed by immunoblotting to detect the appearance of band C. Treatment

at 27oC was used as a positive control. (B) qPCR was performed in parallel to quantify the

degree of knock down of each specific construct. Results are shown as means ± S.E of 3 replica.

87

(B)

(A)

88

In summary, the work presented in this thesis demonstrates the rescue of ∆F508-CFTR

by several kinase inhibitors in HEK293GT, BHK and MDCK cells stably expressing the mutant

protein. Most importantly, these compounds also facilitated functional rescue of ∆F508-CFTR in

HBE cells obtained from CF patients. Oxozeaenol was one of the top hit compounds;

unfortunately, its derivative, E6201, which has passed phase II clinical trials for the treatment of

psoriasis, did not yield any rescue of ∆F508-CFTR. However, since some of the other top hit

compounds are clinically available or in clinical trials for the treatment of cancer or

inflammation, their use for the treatment of CF can be expedited. The esiRNA screen provided

complimentary data to the kinase inhibitors screen. Results from both screens suggested several

signaling pathways, especially FGFR1 and its downstream signaling pathways, that might be

involved in the trafficking of ∆F508-CFTR. However, the exact mechanism/pathway of the

rescue of ∆F508-CFTR remains to be explored.

89

Chapter 4

Discussion

90

I. Kinase inhibitor screen

In this thesis, I present the results of validations for the hits of a screen of kinase

inhibitors that can correct ∆F508-CFTR. The library of kinase inhibitors used in the screen was

biased towards compounds that are clinically available or in clinical trials for treatment of

cancer, inflammation, or other diseases. This approach provides us two main advantages. First,

once proven effective, these compounds can be quickly moved into clinical trials for the

treatment of ∆F508-CFTR. Secondly, since most of the targets of these inhibitors are known,

signaling pathways involved in the rescue of ∆F508-CFTR can be identified, which might lead to

compounds that yield better rescue or are better suited for treatment in patients.

Different approaches were taken to validate the hits of the kinase inhibitors screen.

Immunoblotting and flow cytometry were used to demonstrate rescues of the maturation and

trafficking of ∆F508-CFTR to the cell surface, respectively. Moreover, to validate the functional

rescue of these inhibitors, functional assays using Isc analyses by Ussing chambers were carried

out in both MDCK cells expressing ∆F508-CFTR and, most importantly, HBE cells obtained

from ∆F508-CFTR patients. The results from HBE cells revealed that the rescue of CFTR

activity (assessed as ∆Isc) in ∆F508-CFTRpatient samples treated with several of the inhibitors

was about 10-30% of that in WT-CFTR cells. This was a significant rescue because it had been

estimated that a partial rescue of ∆F508-CFTR, 10-25%, could restore the airway epithelial

function in patients (Johnson et al., 1992; Farmen et al. 2005; Zhang et al., 2009). From these

assays, however, we could not conclude if any of these inhibitors also act as potentiators of

∆F508-CFTR.

91

In addition, using the data from Isc analyses by Ussing chambers in MDCK cells, the

EC50 of most of the top hit inhibitors identified were determined to be in the nanomolar range.

This suggests that these inhibitors and their derivatives are very promising drugs to be used for

the treatment of CF. However, Rabeh et al. have recently reported that it might require a more

complex approach to give a complete rescue of ∆F508-CFTR. The authors conclude that it is

necessary to correct the stability of both NBD1 and NBD1-MSD2 interface to yield a complete

rescue of ∆F508-CFTR (Rabeh et al., 2012). Therefore, a combination of drugs might be

required to provide a better treatment for ∆F508-CFTR.

The fact that these kinase inhibitors could rescue ∆F508-CFTR suggests that the target

kinases and the downstream signaling pathways commonly inhibit the trafficking of the mutant

CFTR. Several of the compounds, such as SU5402, SU6668, PD173074, Ki8751, are inhibitors

of receptor tyrosine kinase (RTKs) (VEGFR, FGFR, PDGFR). In addition, besides being the

target of several of these inhibitors, FGFR1, when knocked down by shRNA, also led to the

rescue of ∆F508-CFTR. This further supports the possibility that FGFR1, or other RTKs, usually

play a negative role in the maturation of ∆F508-CFTR. However, further investigation is

required to determine the exact mechanism of how inhibiting these RTKs leads to the rescue of

∆F508-CFTR.

Moreover, other compounds, such as (5Z)-7-Oxozeaenol, RDEA-119, PD0325901

specifically target proteins that are downstream of these receptors, such as components of the

MAPK pathways (e.g. Raf, MEK, ERK, p38). Inhibitors of GSK3β in the Wnt and insulin

pathways, such as Kenpaullone and GSK3β Inhibitors II, and inhibitors of Akt in the

PI3K/Akt/mTOR pathway, such as FPA124 and 10-DEBC, were also identified in the screen

(table I). In summary, these inhibitors target four major pathways: 1) Ras/Raf/MEK/ERK, 2)

92

Wnt/GSK-3β, 3) TAK1/p38, and 4) PI3K/Akt/mTOR. Interestingly, the Ras/Raf/MEK/ERK

pathway has been shown recently to be inhibited by STAT1, which was shown to promote the

rescue of ∆F508-CFTR in a previous screen performed by our lab (Trzcinska-Daneluti et al.,

2009). This further supports the conclusion that at least one of these pathways normally inhibits

the trafficking of ∆F508-CFTR. These inhibitors could facilitate the rescue of ∆F508-CFTR

probably by affecting the function or expression level of certain chaperones involved in the

folding and trafficking of ∆F508-CFTR.

The first two pathways, or ERK1/2 and GSK3β to be specific, have been shown to be

involved in suppressing the activity of heat shock factor 1 (HSF1), which is a transcription factor

that induces the expression of heat shock proteins (HSPs) such as Hsp70, Hsp27, CRYAB (Bao

and Liu, 2009; Pirkkala et al., 2001). HSF1 exists in an inactive monomeric form that is

maintained by forming a complex with the chaperone Hsp90 (Zou et al., 1998). During heat

shock, HSF1 exists in a complex with Hsp70 and Hsp40, which act in a negative feedback to

maintain a proper folding state of proteins (Hayashida et al., 2006). Activation of HSF1 is

facilitated through the hyper-phosphorylation of serine residues (Kline and Morimoto, 1997).

However, phosphorylation of specific serine residues represses the transcription activity of HSF1

(Anckarand Sistonen, 2011). In this way, ERK1/2 exhibits its inhibitory role by phosphorylating

HSF1 on Ser307, which primes the transcription factor for the second phosphorylation on Ser303

by GSK3. These simultaneous phosphorylations repress HSF1 function and inhibit subsequent

expression of HSPs (Chu et al., 1996).

Inhibiting ERK1/2 and/or Gsk3β can relieve the inhibition on HSF1 and, as a

consequence, might induce elevated expression levels of HSPs. This event might, in turn, result

in proper folding and rescue of ∆F508-CFTR, since HSPs are chaperones that promote the

93

folding of proteins and prevent the formation of aggregation (Ellis, 1987). For example, Hsp70,

one of the chaperones that are upregulated following activation of HSF1 (Silver and Noble,

2012), has been shown to promote ∆F508-CFTR maturation (Meacham et al., 1999). In our

previous screen, Hsp70- related proteins such as HSPA4 and CRYAB were also identified as

correctors of ∆F508-CFTR (Trzcinska-Daneluti et al., 2009). Moreover, Calamini et al. showed

that small molecules that acted as proteostasis regulators and induced HSF1-dependent

chaperone expression could rescue ∆F508-CFTR (Calamini et al., 2011). These provide further

evidence supporting the notion that the rescue facilitated through inhibiting these two pathways

works through activating HSF1 and upregulating the expression of chaperones.

Furthermore, HSF1 can also be regulated by phosphorylation on other sites. It has been

shown to be directly phosphorylated by the MAPK-activated protein kinase 2 (MK2) on Ser121

(Wang et al., 2005). This phosphorylation inhibits transcriptional activity of HSF1 by reducing

its binding to the promoters of target genes encoded for HSPs and cytokines, and promoting the

binding between HSF1 and its repressor Hsp90. Interestingly, MK2 is one of the downstream

substrates that are directly phosphorylated and activated by p38 in the TAK1/p38 pathway, one

of the pathways identified in the screen (Freshney et al., 1994; Rouse et al., 1994; Rogalla et

al, 1999). Therefore, the rescue of ∆F508-CFTR by inhibitors of this pathway might also be

facilitated through relieving the inhibition on HSF1 by MK2.

However, the first and most notable substrate of p38/MK2 pathway to be identified is the

small heat shock protein Hsp27 (Stokoe et al., 1993), which functions as ATP-independent

chaperone that not only promotes protein folding but also targets unfolded protein for

proteasomal degradation (Jakob et al., 1993; Kostenk and Moens, 2009; Garrido et al., 2012).

The activity and cellular localization of Hsp27 are determined by its ogliomerization state, which

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is dependent on its phosphorylation (Kostenk and Moens, 2009). Unphosphorylated Hsp27 forms

large ogliomers of up to 40 monomers, while phosphorylation, mainly by MK2, favors the

formation of smaller ogliomers (Rogalla et al., 1999; Parcellier et al., 2005; Kostenk and Moens,

2009). This is a very dynamic process that is crucial for the activity of Hsp27, since the

ogliomerization status modulates the affinity of this chaperone for its substrates (Garrido et al.,

2012).

Inhibiting the p38/MK2 pathway, therefore, terminates the phosphorylations on Hsp27,

and thus favors the large ogliomeric state. Since inhibitors of p38 pathway such as Oxozeaenol

or SKF86002 promote the rescue of ∆F508-CFTR in my study, it suggests that the smaller

ogliomeric states of Hsp27 might have a negative role in the trafficking of the mutant CFTR,

probably by directing the misfolded protein for degradation. Supporting this conclusion is the

fact that it has been shown that large ogliomers of Hsp27 are essential for its chaperone activity,

while the smaller, phosphorylated ogliomers lose their chaperone function and accelerate

degradation of certain proteins (Rogalla et al., 1999; Parcellier et al., 2006). Moreover, Hsp27

was identified as a component of the CFTR interactome (Wang et al., 2006), and recently was

reported to target ∆F508-CFTR for degradation via a SUMO-dependent pathway (Ahner et al.,

2012). In this study, Ahner et al. showed that overexpressed Hsp27, in a complex with the

SUMO E2 conjugating enzyme Ubc9, preferentially promoted SUMOylation of ∆F508-CFTR,

which was then recognized and targeted for degradation by RNF4, a SUMO-targeted ubiquitin

ligase. However, the authors did not assess the effect of different ogliomer sizes and

phosphorylation status of Hsp27 on the maturation of ∆F508. This is an interesting angle to be

further explored.

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The role of the PI3K/Akt pathway in ∆F508-CFTR trafficking, or how inhibition of this

pathway leads to the rescue of the mutant CFTR, still requires further investigation. Several

reports actually suggest a possible positive role of Akt in CFTR trafficking. For example, Tuo et

al. showed a positive involvement of the PI3K/Akt pathway in trafficking of WT-CFTR to the

plasma membrane (Tuo et al., 2009). Moreover, Akt has been shown to inhibit GSK3β and thus

contribute to the activation of HSF1 (Bijur and Jope, 2000), which, as mentioned earlier, might

induce the expression of chaperones and promote ∆F508-CFTR trafficking and maturation.

However, the PI3K/Akt pathway has also been shown to induce the expression of Hsp27 (Takai

et al., 2006), and, thus, could exhibit its negative regulation of ∆F508-CFTR trafficking through

this mechanism. Moreover, PI3K/Akt actually decreases the plasma membrane availability of

aquaporin-2 and norepinephrine transporter (Jung and Kwon, 2010; Robertson et al., 2010),

suggesting a possible negative role of this pathway in the trafficking of membrane proteins.

Therefore, the exact mechanism for how the inhibitors of PI3K/Akt pathway rescue ∆F508-

CFTR requires further investigation.

II. E6201 and(5Z)-7-Oxozeaenol

Oxozeaenol was one of the top hits of the kinase inhibitors screen. It showed rescue of

ΔF508-CFTR maturation and function in all of our validation approaches (Table 1). However,

since Oxozeaenol is unstable in plasma, its therapeutic utilization is limited (Goto et al., 2009).

In 2009, the Eisai group in Japan identified a derivative of Oxozeaenol, E6201, which now has

gone to phase I trials for the treatment of cancer and phase II trials for the treatment of psoriasis

(Shen Y. et al., 2010; Muramoto K. et al, 2010). We obtained this compound from Eisai and

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tested it for correction of ΔF508-CFTR using different methods, as in the kinase inhibitors

screen. Unfortunately, it did not yield any significant rescue of ΔF508-CFTR in any of assays

performed, and seemed to have an adverse effect at higher concentrations.

We attempted to determine the possible signaling pathways used by Oxozeaenol in the

rescue of ΔF508-CFTR by examining the different effects of these two compounds on

downstream signaling pathways. Phosphoproteins analysis was performed in BMM and

RAW264.7 cells stimulated with LPS. Using this approach, Oxozeaenol was found to exhibit a

5-10 fold more potent inhibition of phosphorylation of p38, JNK, ERK1/2, and NFκB, compared

to E6201. However, at the higher doses we used, E6201, like its parental compound, should have

inhibited all of the above signaling pathways and hence promote the rescue of ΔF508-CFTR.

One possible explanation for the lack of effect of E6201 might be that at the higher doses at

which it can actually inhibit these signaling pathways, it is also toxic to cells and/or has off-

target effects. In addition, it is possible that Oxozeanol rescued ΔF508-CFTR by inhibiting

pathway(s) downstream of TAK1 that is (are) not inhibited by E6201, which would be important

to investigate. Lastly, there is a possibility that Oxozeanol might have rescued ΔF508-CFTR by

direct binding to and stabilizing this mutant CFTR protein rather than affecting downstream

signaling pathways. This can be tested by using the iodide flux assays on purified CFTR that was

recently developed by the Bear lab (Eckford et al., 2012).

III. esiRNA screen

As a complement to the kinase inhibitor screen, another screen was performed to identify

proteins that suppress the trafficking of ΔF508-CFTR to the plasma membrane. This was done

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using esiRNA to knock down kinases and related proteins in HEK293GT cells expressing the

mutant eYFP (H148Q/I152L) and ΔF508-CFTR, before subjecting the cells in Cellomics assays

to assess the rescue of ΔF508-CFTR activity in these cells. The top hits (21 genes/proteins, Table

II) were re-tested with another type of RNAi, shRNA (from the TRC collection), to ensure the

observed rescue in the original esiRNA screen was not due to off-target effects. In parallel, GFP-

tagged pGIPZ-shRNA constructs were used to test the effect of knocking down these genes on

the maturation of ΔF508-CFTR by immunoblotting. In both cases, qPCR were performed to

check the degrees of knockdown of the genes of interest. Knocking down of several of these

genes, such as FGFR1, and DTYMK, led to a rescue of up to 30% of CFTR activity in the

Cellomics assays, and an increase in the maturation of CFTR (increase in the appearance of band

C).

The results of the reevaluations performed above lend further support to the results of the

kinase inhibitor screen. Several of the hits are kinases that either are direct targets or are involved

in the signaling pathways inhibited by the kinase inhibitors in the original kinome inhibitors

screen. For example, IPMK (Inositol Polyphosphate Multikinase) is known to act as an important

PI3K that activates Akt (Lee et al., 2012). The fact that knocking down IMPK could rescue

∆F508-CFTR supports the result of the kinase inhibitors screen which suggested a negative role

of the PI3K/Akt pathway in the biogenesis of ∆F508-CFTR. Moreover, FGFR1 is also the target

of several kinase inhibitors we identified, such as SU5402, SU6668, and PD173074(Table I).

FGFR activation leads to the activation of several downstream signaling pathways such as the

Ras/Raf/MEK/ERK cascade, PI3K/Akt and p38 pathways (Javerzat et al., 2002; Turner and

Grose, 2010; Haugsten et al., 2010). As noted previously, these pathways might suppress the

trafficking and maturation of ΔF508-CFTR probably by affecting the activity of HSF1 and

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Hsp27. Therefore, inhibiting FGFR1 or other downstream effectors involved in these signaling

pathways might relieve this suppression.

Indeed, several hits, such as BRAF and MAP3K13, are directly involved in the

Ras/Raf/MEK/ERK pathways, one of the four main signaling pathways identified (Table 1).

Both of them belong to the MAPK kinase kinase family, which is the first protein to be activated

in the MAPK signaling cascade (Kim and Choi, 2010). Specifically, BRAF is one of the three

isoforms of the Raf kinase family, an important component of the Ras/Raf/MEK/ERK pathway

that acts downstream of Ras and activates ERK through MEK kinase (Craig et al, 2008;

Roskoski , 2010), while MAP3K13 plays a role in activating the JNK and NFκB but not ERK

pathways (Ikeda et al, 2001; Masaki et al., 2003).

Other hits confirmed the involvement of HSF1 and Hsp27 in the rescue of ΔF508-CFTR

by the kinase inhibitors. One notable hit gene is RPS6KC1, which belongs to the S6 ribosomal

kinase family. Another member of this family, the 90-kD ribosomal S6 kinase RSK2, is activated

downstream of ERK and plays a central role in transmitting FGFR-driven signaling pathways

(Anjum and Blenis, 2008). Moreover, RSK2 has been shown to repress the activity of HSF1

(Wang et al., 2000). However, whether RPS6KC1 also inhibits HSF1and suppresses the

maturation of ΔF508-CFTR requires further study. Another hit gene, CAMKIIB, encodes for the

beta subunit of the Ca2+

/calmodulin-dependent protein kinase (CAMKII). Following the

activation of the Wnt pathway and the release of Ca2+

from the intracellular stores, CAMKII is

known to activate TAK1 (Kuhl et al., 2000; Ishitani et al., 2003), and thus leads to

phosphorylation of Hsp27 through the TAK1/p38 pathway (Cai et al., 2008). Therefore, the

rescue of ΔF508-CFTR facilitated by knocking down this gene might work through affecting the

phosphorylation and activity of Hsp27, as discussed earlier.

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In addition to the four major pathways, results from both screens also suggest a negative

role of the NFκB pathway toward the trafficking and maturation of ΔF508-CFTR. Hsp27 not

only promotes the degradation of ΔF508-CFTR, as mentioned before (Ahner et al., 2012), but

also activates the NFκB pathway via promoting the degradation of phosphorylated IκBα, an

inhibitor protein of NFκB (Parcellier et al., 2003). Moreover, our siRNA screen identified

RIPK4, or RIP4, which belongs to the Receptor Interacting Protein Kinase family that is known

to activate NFκB (Meylan et al., 2002). In CF airways, one factor contributing to the chronic and

hyper-inflammation is the elevated NFκB-mediated IL8 signaling (Tabary et al., 1998; Tabary et

al., 2001). This elevated activity might be due to the lack of inhibitory effect of CFTR on this

pathway in CF cells (Vij et al., 2009). However, whether and how the NFκB pathway inhibits

the trafficking of ΔF508-CFTR requires further investigation.

The results from the siRNA screen not only provide confirmation to the kinase inhibitor

screen, but also suggest new novel pathways that might be involved in the maturation of ΔF508-

CFTR. Other interesting hit genes that do not seem to be involved in any of the signaling

pathways mentioned are PRKAR2B and DTYMK. PRKAR2B encodes one isoform of the

regulatory subunit II of PKA. Upon the binding of cAMP, PKA is activated by the release of its

regulatory subunit, leading to the activation of the catalytic subunit. Therefore, knocking down

one of the regulatory subunits might lead to an increase in PKA activity (Brandon et al., 1997).

Moreover, cAMP/PKA stimulation is known not only to promote CFTR channel activity, but

also to enhance its trafficking to the cell surface and decrease its endocytosis from the plasma

membrane. Whether PRKAR2B promotes rescue of ΔF508-CFTR through this mechanism

remains to be investigated. DTYMK belongs to the family of thymidylate kinase (TMPK). This

kinase family is responsible for catalyzing dTMP to dTDP and dTTP, which is used for DNA

100

synthesis and repair (Huang et al., 1994). Since it plays a very important role in cell growth, it is

often targeted in treatment of cancer (Van Calenberghet al., 2012). However, its role in protein

synthesis and trafficking, if any, is unknown and needs to be explored.

Summary statement

In summary, the work presented in this thesis reveals that several kinase inhibitors can

work as novel correctors of ΔF508-CFTR. Since some of these inhibitors or their derivatives are

clinically available or are in clinical trials for the treatment of cancer or inflammation, their use

for the treatment of ΔF508-CFTR can be accelerated. Moreover, combined with the results of the

siRNA screen, we also identified signaling pathways that might normally suppress the trafficking

and maturation of ΔF508-CFTR. These can be used as targets to design or screen for better

compounds in the future, not only for the treatment of CF but also treatment of other diseases

caused by proteins that exhibit folding defects and are thus stuck in the ER.

FUTURE DIRECTIONS

I. Testing the effect of knocking down top hit genes on rescuing ∆F508-CFTR function

using Ussing Chambers:

To further validate the top hit genes from the siRNA screen, we want to assess the effect of

knocking down these genes in either cultured cells expressing ∆F508-CFTR or, ultimately, HBE

patient cells. Since the shRNA constructs used only target human genes, we are in the process of

generating a human CF cell line by stably expressing ∆F508-CFTR in CFBE410-, a cell line that

was generated by transformation of cystic fibrosis (CF) tracheo-bronchial cells. In the meantime,

since 11 of these genes have higher than 95% identity between human and canine, and thus the

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101

shRNAs are compatible to be used in MDCK cells, we will try to knock down these in our

MDCK-∆F508-CFTR cell lines for initial testing.

II. Elucidate the mechanisms of rescuing ∆F508-CFTR by Oxozeaenol:

Both E6201 and Oxozeaenol inhibit similar pathways (at least at higher concentration), but

only Oxozeaenol rescued the mutant CFTR. Therefore, we want to further investigate the

mechanism through which Oxozeaenol rescues ∆F508-CFTR function. One possible explanation

is that Oxozeaenol can exhibit its rescue by directly binding to and promoting the folding of

∆F508-CFTR. This can be tested in collaboration with Christine Bear’s lab using a novel Iodide

flux assay developed recently by that group (Eckford et al, 2012).

III. Elucidate the pathways through which FGFR1 regulates the rescue of ∆F508-CFTR

We have found that inhibiting or knocking down FGFR1 or some of the components of its

downstream signaling pathways (i.e. RAS/RAF/MEK/ERK, PI3K/Akt pathways) can rescue

∆F508-CFTR. We hypothesize that this rescue is facilitated by affecting the chaperones that are

involved in the folding and trafficking of ∆F508-CFTR. Once we obtain HBE cells, we will treat

them for 48 hrs with SU5402, one of the top hits from our kinase inhibitors screen, which

inhibits mainly FGFR1. We will then extract RNA from these cells and use the qPCR array kit

designed to profile the expression levels of heat shock proteins and chaperones to check if there

is any chaperone that is up or down regulated. This will allow us to further investigate the

pathways involved.

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SUMMARY

To identify compounds and drugs that rescue the trafficking defect of ∆F508-CFTR, our

lab performed a screen of a kinase inhibitor library biased toward small molecules that are

clinically available or in clinical trials for the treatment of cancer and inflammation using a

recently developed high-content Cellomics functional screen. I further validated the top hits of

the screen by: immunoblotting to test the appearance of mature ∆F508-CFTR (band C); flow

cytometry to detect the presence of ∆F508-CFTR at the cell surface; short-circuit current (Isc)

analysis in Ussing chamber to show the increased ∆F508-CFTR activity in MDCK cells stably

expressing the mutant CFTR; and most importantly, Isc analysis to demonstrate improved

∆F508-CFTR activity in Human Bronchial Epithelial (HBE) cells obtained from homozygote

∆F508-CFTR patients who had undergone lung transplant.

The results indicate that several inhibitors of receptor tyrosine kinases such as SU5402

and SU6668, which target FGFRs, VEGFR, and PDGFR, exhibited strong rescue of ∆F508-

CFTR. Moreover, prominent rescue was also observed with inhibitors of four major pathways:

Ras/Raf/MEK/ERK, TAK1/p38, Wnt/GSK-3β, and PI3K/Akt/mTOR. Several of these inhibitors

rescued ∆F508-CFTR in more than one validation approaches, such as (5Z)-7-Oxozeaenol,

SU5402, with EC50 mostly falling in the nanomolar range.

The effect of E6201, one derivative of Oxozeaenol, on the rescue of ∆F508-CFTR was

also examined using similar approaches. E6201 is currently in phase II clinical trials for the

treatment of psoriasis. Unfortunately, it did not exhibit rescue of ∆F508-CFTR in any of the

assays used. The phosphoprotein analysis revealed that E6201 inhibited the phosphorylation of

the same target proteins as Oxozeaenol, but at lower potency. This suggests a possibility that

103

Oxozeaenol could have rescued ∆F508-CFTR using a different mechanism (e.g. direct

binding)or by inhibiting a different signaling pathway that was not tested.

To identify pathways involved in the rescue of ∆F508-CFTR by these inhibitors, we also

performed a complementary siRNA screens for kinases and related proteins that suppress the

rescue. The top hits from this screen were preliminarily validated by immunoblotting. The results

revealed FGFR1, a target of several kinase inhibitors identified in the former screen, and several

kinases such BRAF and MAP3K13, which are involved in the signaling pathways downstream

of FGFRs. This suggests a possible role of FGFRs in regulating ∆F508-CFTR trafficking and

maturation, probably through affecting the expression of chaperones. In addition, other proteins

identified might reveal possible novel pathways involved in ∆F508-CFTR rescue. The exact

mechanisms of the rescue remain to be explored.

CONCLUSION

The results obtained in this study identify several kinase inhibitors that can rescue

ΔF508-CFTR, and suggest that use of compounds or drugs already in the clinic or in clinical

trials for other diseases can expedite delivery of treatment for CF patients.

104

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Rescue of ∆F508-CFTR by Kinase Inhibitors - TSpace

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