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 Expert Opinion on Therapeutic Targets

ISSN: 1472-8222 (Print) 1744-7631 (Online) Journal homepage: http://www.tandfonline.com/loi/iett20

The epithelial sodium channel (ENaC) as a

therapeutic target for cystic fibrosis lung disease

Patrick J. Moore & Robert Tarran

To cite this article: Patrick J. Moore & Robert Tarran (2018) The epithelial sodium channel (ENaC)
as a therapeutic target for cystic fibrosis lung disease, Expert Opinion on Therapeutic Targets, 22:8,
687-701, DOI: 10.1080/14728222.2018.1501361

To link to this article: https://doi.org/10.1080/14728222.2018.1501361

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EXPERT OPINION ON THERAPEUTIC TARGETS
2018, VOL. 22, NO. 8, 687–701
https://doi.org/10.1080/14728222.2018.1501361

REVIEW

The epithelial sodium channel (ENaC) as a therapeutic target for cystic fibrosis lung
disease
Patrick J. Moorea and Robert Tarrana,b
a
Marsico Lung Institute, University of North Carolina, Chapel Hill, NC, USA; bDepartment of Cell Biology & Physiology, University of North Carolina,
Chapel Hill, NC, USA

ABSTRACT ARTICLE HISTORY

Introduction: Cystic fibrosis is an autosomal recessive disorder caused by mutations in the cystic Received 15 March 2018
fibrosis transmembrane conductance regulator (CFTR) gene that codes for the CFTR anion channel. In Accepted 13 July 2018
the absence of functional CFTR, the epithelial Na+ channel is also dysregulated. Airway surface liquid KEYWORDS
(ASL) hydration is maintained by a balance between epithelial sodium channel (ENaC)-led Na+ absorp- Cystic fibrosis; CFTR; ENaC;
tion and CFTR-dependent anion secretion. This finely tuned homeostatic mechanism is required to SPLUNC1; amiloride; channel
maintain sufficient airway hydration to permit the efficient mucus clearance necessary for a sterile lung activating protease;
environment. In CF airways, the lack of CFTR and increased ENaC activity lead to ASL/mucus dehydra- mucociliary clearance
tion that causes mucus obstruction, neutrophilic infiltration, and chronic bacterial infection. Rehydration
of ASL/mucus in CF airways can be achieved by inhibiting Na+ absorption with pharmacological
inhibitors of ENaC.
Areas covered: In this review, we discuss ENaC structure and function and its role in CF lung disease
and focus on ENaC inhibition as a potential therapeutic target to rehydrate CF mucus. We also discuss
the failure of the first generation of pharmacological inhibitors of ENaC and recent alternate strategies
to attenuate ENaC activity in the CF lung.
Expert opinion: ENaC is an attractive therapeutic target to rehydrate CF ASL that may serve as a
monotherapy or function in parallel with other treatments. Given the increased number of strategies
being employed to inhibit ENaC, this is an exciting and optimistic time to be in this field.

1. Introduction
1.1. CF lung disease
Cystic fibrosis (CF) is a recessive genetic disease that is com-
mon in Caucasians. In this population, CF occurs in ~1:3000
live births and affects ~70,000 individuals worldwide, with a
predicted survival age that is now close to 43 years [1]. CF is
caused by mutations in the cystic fibrosis transmembrane
regulator (CFTR) gene which codes for a cAMP/protein kinase
A (PKA)-regulated apical membrane anion channel. CF-causing
mutations lead to reduced or absent anion secretion across
affected epithelia, including the intestines, pancreas, repro-
ductive tracts, sweat glands, and lungs [2]. To date, over
1700 CFTR mutations have been found that fall into 6 classes:
defective protein production mutations (Class 1), protein pro-
cessing mutations (Class 2), gating mutations (Class 3), con-
duction mutations (Class 4), insufficient protein mutations
(Class 5), and accelerated degradation of CFTR (Class 6). The
different classes of CFTR mutations are summarized in
Figure 1 [3].
Whilst some of the first manifestations of CF occur in the
pancreas, due to altered CFTR-dependent bicarbonate secre-
tion and a failure to secrete digestive enzymes, to date, lung
disease accounts for the vast majority of CF morbidity [4]. CF
pancreatic disease occurs in utero [5]. However, CF patients
are born with normal lungs and pathology develops over time
[6]. CF lung disease is characterized by the accumulation of
dehydrated mucus that serves as a reservoir for chronic bac-
terial infections which result in unremitting cycles of chronic
infection and neutrophilia/inflammation [7,8]. Colonization of
the CF lung occurs in the first years of life with pathogens
including Staphylococcus aureus and Haemophilus influenza,
followed by the acquisition of Pseudomonas aeruginosa, typi-
cally by 5 years of age [9–11]. The dehydrated mucus present
in the CF lung is thought to enhance P. aeruginosa pathogeni-
city through promotion of biofilm formation and antibiotic
resistance [12]. Chemical factors such as high levels of reactive
oxygen species, high iron levels, and lower pH may also con-
tribute to persistent microbial infection [13–15]. Similarly, the
dehydrated mucus is hypoxic which promotes P. aeruginosa
biofilm formation [16]. Elevated levels of interleukin (IL)-8
[17,18], IL-6 [19], and tumor necrosis factor α [20–22], along
with reduced levels of anti-inflammatory cytokines, contribute
to necrosis of the lung [23]. Neutrophil elastase levels are also
elevated which can degrade host antimicrobial peptides such
as defensins, LL-37, and short palate lung and nasal epithelial
clone 1 (SPLUNC1) which may further impede bacterial clear-
ance [24,25].
Mucus rehydration is a desired therapeutic approach to
prevent mucus plugging and airflow obstruction in the CF

CONTACT Robert Tarran robert_tarran@med.unc.edu Department of Cell Biology & Physiology, University of North Carolina, 7102 Marsico Hall, 125 Mason
Farm Road, Chapel Hill, NC 27599, USA
© 2018 Informa UK Limited, trading as Taylor & Francis Group
688 P. J. MOORE AND R. TARRAN
Article highlights
● The role of ENaC in CF lung disease.
● SPLUNC1 is an allosteric modulator of ENaC activity.
● Strategies to attenuate Na+ absorption in the CF lung.
● Failure of first generation of ENaC antagonists.
● Targeting ENaC inhibition may alleviate CF symptoms.
● Small molecules and peptides demonstrate promising results for
inhibiting ENaC activity.
This box summarizes key points contained in the article.
lung. Since mucus hydration is directly proportional to mucus
clearance rates [26], such an approach is predicted to increase
mucus clearance and reduce mucus accumulation.
Rehydrating the airway surface liquid (ASL) may be achieved
by (1) direct addition of osmolytes (e.g. inhaled hypertonic
saline), (2) restoring CFTR function, (3) activating an alternate
Cl− secretory pathway, or (4) inhibiting the epithelial Na+
channel (ENaC). This review focuses on the role of ENaC in
maintaining ASL hydration and summarizes the current ther-
apeutic approaches to modulate ENaC and Na+ absorption for
the treatment of patients with CF.
1.2. ENaC structure
ENaC is expressed at the apical surface of epithelia of the lung,
colon, kidney, salivary ducts, and sweat ducts. Here, ENaC is the
rate limiting step for transepithelial Na+ absorption [27–29].
Homology studies using the crystal structure of the acid-sensing
ion channel (ASIC) have shown that ENaC is a heterotrimeric
transmembrane (TM) protein composed of α, β, and γ-subunits
that form a pore with high selectivity for Na+ and Li+ over K+ and
sensitivity to the small molecule antagonist amiloride [30]. Each
subunit is encoded by a different gene, i.e. SCNN1A (alpha),
SCNN1B (beta), and SCNN1G (gamma). These subunits contain
Figure 1. Different classes of CFTR mutations.
two membrane-spanning domains connected by a large extra-
cellular domain and intracellular N- and C-termini. The extracel-
lular, pre-M2 region near the second transmembrane domain of
α, β, and γ ENaC subunits affects the channel conductance and is
where amiloride binds [31] (Figure 2). The HG motif, so named
because it possesses a glycine and a histidine in the amino
terminus, is present in every ENaC/degenerin family member
known to conduct ions and is involved in channel opening [32].
Mutation of these residues in the β ENaC subunit causes pseu-
dohypoaldosteronism, a disease characterized by increased Na+
excretion (natriuresis) due to decreased renal ENaC activity and
increased mucus clearance rates due to decreased pulmonary
ENaC activity [33,34]. According to the ASICs and ENaC structural
models, the DEG gating is located within the highly conserved
TM2 region [30,35] and mutations of residues in the gating
region results in channel activation and subsequently an increase
in open probability (PO) [32,36]. αENaC was the first subunit to be
cloned and when αENaC is injected into Xenopus oocytes, ~2% of
current is seen relative to α, β, γ ENaC co-expression. In contrast,
the β or γ subunits alone do not produce currents when
expressed alone. When the α, β or α, γ subunit combinations
are co-injected, up to 15% of αβγENaC activity occurs [37]. A
fourth subunit, δENaC, has been shown to assemble with β and
γ-subunits and contributed to 50% of amiloride-sensitive salt
transport across primary human nasal epithelial cells [38,39].
However, its relevance to lower airways transepithelial Na+
absorption is not fully understood.
1.3. ENaC regulation
Like most plasma membrane ion channels, ENaC is regulated
by either altering PO or by altering the number of channels at
the cell surface (N). ENaC gating kinetics are typically charac-
terized by long opening and closing times. However, ENaC can
be posttranslationally modified to affect both channel density
(N) and PO. For example, three different classes of proteases

Figure 2. Structural features of the epithelial Na+ channel (ENaC). ENaC exists as a heterotrimer consisting of α, β, and γ subunits. Each subunit contains two
membrane spanning domains with intracellular N- and C-termini. The extracellular domains contain sites for proteolytic cleavage while the PY motif in the C-termini
is the site for ubquination.
have been shown to activate ENaC: (1) intracellular, conver-
tase-type proteases such as furin [40]; (2) extracellular, cell-
attached proteases, which can be membrane spanning or GPI-
anchored such as prostasin [41]; and (3) soluble/secreted pro-
teases including trypsin and neutrophil elastase [42,43]. All of
these proteases cleave the extracellular domains of α- and γ-
ENaC to increase the channel open probability. Many of these
proteases predominantly belong to a family of serine pro-
teases which target mainly R and K residues on α- and
γ-subunits of ENaC [44]. That is, cleavage occurs at multiple
sites, including at conserved basic residues located at α-K561,
β-R503, and γ-R515. However R-138 on γ-ENaC is the only
cleavage site that is required for activation [45].
ENaC is cleaved intracellularly by furin-type convertases
and can then be further cleaved by transmembrane or extra-
cellular channel activating proteases (CAPs) [40]. Furin, which
is primarily present in the trans-Golgi networks, cleaves α and
γ-ENaC subunits through a consensus sequence R/S-XX-R
where X is any amino acid. Cleavage of the α-subunit occurs
twice. A furin site is also present on γ-ENaC; however, a second
protease at a distal site to the furin site is required for ENaC
activation [40,46]. For reasons that are not fully understood,
some ENaCs can bypass the furin cleavage steps and traffic to
the plasma membrane as ‘silent’ or ‘near-silent’ channels,
which can then be fully activated by other proteases.
However, regardless of whether or not ENaC is cleaved intra-
cellularly or extracellularly, the end result of cleavage is the
EXPERT OPINION ON THERAPEUTIC TARGETS 689
transition to a high PO state [42]. Prostasin, or CAP1, is a GPI-
anchored plasma membrane protein that was the first pro-
tease to be identified that could cleave ENaC, an affect that
was reprised with trypsin [47]. Human transmembrane pro-
tease serine 4 (CAP2) and matriptase (CAP3) are also known to
regulate ENaC [48]. However, in contrast to prostasin, both
CAP2 and CAP3 must possess catalytic activity to stimulate
ENaC [49]. Serine proteases can also cleave ENaC and have
been demonstrated to increase amiloride sensitive currents in
Xenopus laevis oocytes and in airway epithelia [43,50]. Harris
et al. observed that a brief exposure of ENaC expressed in
Xenopus oocytes to neutrophil elastase generated a new frag-
ment of γ-ENaC in the surface pool, consistent with cleavage
downstream of the γ-ENaC furin site [51]. In addition, the
specific residues V182 and V193 in human γ-ENaC were iden-
tified as being essential for ENaC stimulation by neutrophil
elastase [45].
As well as being cleaved by numerous proteases, ENaC can
also be regulated by intracellular second messengers. For
example, the cAMP/PKA complex regulates ENaC by phos-
phorylation and inhibition of NEDD4-2 [52]. Stutts et al. pre-
viously studied fibroblasts stably transduced with αβγ-ENaC
subunits ± CFTR. Here, they found that the presence/absence
of CFTR changed ENaC’s sensitivity to cAMP/PKA and whilst
cAMP/PKA inhibited ENaC in the presence of CFTR, they acti-
vated ENaC in CFTR’s absence [53,54]. Whilst the nature of this
interaction is not fully understood, the intracellular domains of
690 P. J. MOORE AND R. TARRAN
CFTR were also shown to alter ENaC’s sensitivity to cAMP/
PKA [55–57]. The interaction between CFTR and ENaC may
also vary according to their location, and in the sweat glands,
where Cl− absorption rather than Cl− secretion occurs, ENaC is
activated in parallel with CFTR rather than being inactivated
[58], so more studies are required to fully understand this
phenomenon. A reduction in the intracellular Cl− concentra-
tion following stimulation of either CFTR or ClC Cl− channels
has also been reported to lower ENaC activity [57,59,60].
However, intracellular Cl− may not change so much during
absorption/secretion and the physiological relevance of
this mode of action remains to be verified [61]. Stimulation
of P2Y2 receptors on the apical membrane of airway epithelial
cells with purine nucleotides such as ATP activated
phospholipase C which in turn cleaves phosphatidylinositol
4,5-bisphosphate (PIP2) into diacyl glycerol and inositol 1,4,5-
trisphosphate (IP3) [62]. The fall in PIP2 leads to a decrease in
ENaC PO [63,64]. Unlike cAMP regulation, PIP2 depletion inhi-
bits ENaC activity in both normal lung and CF airways [65].
Both PKA and PIP2 affect ENaC Po [66]. However, ENaC N is
also precisely regulated. ENaC can be ubiquitinated on all three
subunits by the ubiquitin ligase neural precursor cell expressed
developmentally downregulated protein 4 (NEDD4-2) also
known as NEDD4L [67]. Each ENaC subunit has a PY motif (L/
PPXY) that can bind to NEDD4-2 [68]. The subsequent ubiqui-
tination of ENaC subunits leads to their rapid internalization
and degradation [34]. Butterworth et al. demonstrated that
inhibition of deubiquitinating enzymes increased ubiquitination
and decreased surface αβγENaC [69]. Interestingly, deletions of
the PY motifs cause spontaneous upregulation of ENaC and
cause Liddle’s syndrome [66], which is characterized by
increased renal ENaC activity, K+ excretion and Na+/H2O reten-
tion that lead to hypertension and metabolic alkalosis [70]. In
addition to the mutations observed in the PY motifs,
mutations in the C-terminus (β, R563Q) and the TM2 segment
(γ, N530S) were also reported to be associated with Liddle’s
syndrome [71,72]. Although these mutations are not associated
with CF-like disease, mutations in individual ENaC subunits
would likely influence the impact of posttranslational proteoly-
tic cleavage of this subunit.
1.4. SPLUNC1 and ENaC
SPLUNC1 is a ~25-kDa secreted protein that is highly abun-
dant in the airways and plays an important role in innate
defense [73]. Using its N-terminal ‘S18’-region, SPLUNC1
binds to and regulates ENaC [74]. Indeed, stable knockdown
of SPLUNC1 increases the amiloride-sensitive transepithelial
voltage and causes CF-like ASL dehydration in normal
human bronchial epithelial cultures (HBECs), indicating that
SPLUNC1 is required to limit ENaC activity and prevent ASL
dehydration [75]. SPLUNC1 binds extracellularly to ENaC and
leads to internalization of the channel. More recently,
SPLUNC1’s mechanism of action has become clear: αENaC-
GFP and βENaC-mCherry, where the subunits were labeled
on their intracellular termini, along with unlabeled γENaC
were co-expressed in HEK293T cells. Using these constructs,
we observed that extracellular SPLUNC1 binding altered intra-
cellular ENaC FRET, thus demonstrating allostery [76]. We also
found that SPLUNC1 binding led to a NEDD4.2-dependent
ubiquitination of αENaC and internalization of channel subu-
nits [76]. Thus, we propose that the information of SPLUNC1
binding extracellularly was transmitted through ENaC, leading
to a conformational change and increased accessibility of
αENaC’s intracellular termini to NEDD4-2 and ubiquitination.
Indeed, co-expression of a concatemer where α–β–γ were
linked N–C in order to constrain the channel abolished
SPLUNC1-induced ENaC internalization [76]. These data sug-
gest a previously undescribed way of regulating ENaC, where
ENaC serves as the receptor for SPLUNC1 that regulates chan-
nel density (N). Such findings lead us to postulate that
SPLUNC1’s ability to negatively regulate ENaC could be devel-
oped as a potential therapeutic.
1.5. The role of ENaC in CF lung disease
Electrolyte transport across pulmonary epithelia, together with
accompanying osmotic water flow, regulates ASL volume. In
turn, ASL volume has been shown to directly affect mucus
rheology and mucus clearance rates [26]. Simply put, the more
fluid in the lung, the more quickly mucus is cleared. For
example, patients with pseudohypoaldosteronism have
reduced ENaC activity, increased airway secretions, and incred-
ibly fast mucus clearance rates [77]. During normal mucus
clearance, ASL/mucus moves from the distal to the proximal
airways. However, with every successive generation, there is a
reduction in pulmonary surface area that leads to an abun-
dance of ASL. Thus, excess ASL needs to be absorbed in a
controlled fashion, without causing dehydration, in order to
maintain a constant ASL volume and prevent ASL pooling. In
normal airways, Cl− secretion via CFTR and Na+ absorption
through ENaC are balanced to control ASL volume homeosta-
sis and to achieve this goal. Glands can also secrete additional
ASL. However, submucosal glands are most commonly present
in large airways (i.e. the first 16–17 generations) and are less
common/absent in successive generations [78,79]. In contrast,
Na+ absorption has been detected from the nose and trachea,
to the alveolar surfaces, which highlights its importance in
lung fluid homeostasis [80,81].
In CF airways, the lack of functional CFTR and the subse-
quent increase in ENaC activity result in Cl− hyposecretion and
Na+ hyperabsorption, respectively, which combine to dehy-
drate airway surfaces (Figure 3). The importance of ENaC in
the in vivo pathogenesis of CF lung disease was first demon-
strated by Knowles, Boucher, and colleagues using electrophy-
siological techniques. Indeed, they demonstrated that
the amiloride-sensitive voltage was upregulated in CF air-
ways [82,83]. More recently, it has been hypothesized that
these observations were an artifact of the nasal potential
difference electrophysiological technique [84]. That is, when
measured under open circuit conditions, amiloride could
cause apical membrane hyperpolarization that generates an
electrical gradient for Cl− secretion via basally active CFTR in
normal but not CF tissues, which could have accounted for the
difference in amiloride sensitivity between normal and CF
subjects. However, the presence of CFTR leads to diminished
ENaC activity in cell lines, as measured by the patch clamp
technique [53], which would not be subject to this potential

Figure 3. Ion transport in the cystic fibrosis airway. In normal airways (A), the ASL is hydrated by a combination of Cl− secretion and Na+ absorption and in CF
airways (B), reduced Cl− secretion and Na+ hyperabsorption causes ASL dehydration that favor mucostasis.
artifact since the cells are voltage clamped to control the
electrical gradient and the cells are studied with intracellular
and extracellular solutions that inhibit anion channels.
Moreover, the differences in amiloride-sensitivity were also
observed in primary normal versus CF HBECs under thin film
conditions (i.e. with native ASL), even when the cultures were
pretreated with bumetanide to block Cl− secretion, which also
removed this potential artifact [85,86]. Furthermore, studies of
ASL volume homeostasis on primary airway cultures revealed
ASL volume hyperabsorption in CF compared to normal air-
ways that was ENaC-led [26,85,87,88]. Importantly, the ASL
hyperabsorption was coupled with a decrease in mucus trans-
port rates – thus linking defective ENaC with the CF clinical
manifestation of mucus dehydration/mucus stasis.
We have previously demonstrated that CF HBECs failed to
regulate ENaC over time [85]. Similar results were found by
researchers at the University of Pittsburgh [89] and this group
termed the phrase ‘ASL volume expansion,’ i.e. if you flood
HBECs and wash away native ASL and its soluble/endogenous
ENaC inhibitors such as SPLUNC1, then ENaC activity increases.
However, addition of the protease inhibitor aprotinin reduced
CF ENaC activity, suggesting that a failure of regulation had
occurred in CF airway epithelia and that ENaC was ‘fixable’ by
restoring key components in the extracellular regulatory path-
way [85,90]. Subsequently, we identified dis-regulation of
SPLUNC1 as being the cause of this increased ENaC activity.
Indeed, our data indicated that recombinant SPLUNC1 was
capable of inhibiting ENaC in normal but not CF HBECs [86].
After solving SPLUNC1’s crystal structure, we identified two
pH-sensitive salt bridges (R at position 151-D 193 and K 138-D
112) on the main body of SPLUNC1 that prevented SPLUNC1
from binding to ENaC in CF HBECs at even moderately acidic
pH (<7.0) [86]. Ablation of any of these four residues rendered
SPLUNC1 capable of binding to ENaC and regulating ASL
height in CF HBECs, indicating that restoring SPLUNC1’s
EXPERT OPINION ON THERAPEUTIC TARGETS 691
inhibitory functions would be beneficial for rehydrating CF
lungs [86]. Intracellular SPLUNC1 may be upregulated in CF
airways [91]. However, data suggest that SPLUNC1 is degraded
by neutrophil elastase over time [92,93]. Similarly, we detected
reduced coverage of SPLUNC1 using proteomic approaches in
CF versus normal sputum, suggesting that SPLUNC1 may be
part of the normal lung’s ENaC regulatory mechanism that is
absent in CF airways [74].
Whether or not CFTR directly interacts with and/or nega-
tively regulates ENaC is still under debate. However, regard-
less of the regulatory role that CFTR may exert on ENaC,
other extrinsic factors can also enhance ENaC activity in CF
airways. For example, both neutrophil elastase and cathe-
psin B can cleave and activate ENaC independently of func-
tional CFTR [42,94]. Furthermore, proteases released by
Gram-negative bacteria also cleave and activate ENaC.
Indeed, alkaline protease and metalloprotease from
Pseudomonas species resulted in cleavage and activation
of ENaC in human bronchial epithelial cells [95,96].
Finally, a number of mutations that lead to increased ENaC
activity have been found in patients with atypical CF which are
summarized below: Sheridan et al. identified 20 patients out of
a cohort of 185 who had elevated sweat chloride concentra-
tions and pulmonary disease without CFTR mutations. It was
revealed that two patients contained heterozygous mutations
in SCNN1B: one patient exhibited a missense mutation (P26L)
while the other patient had a splice mutation and two mis-
sense mutations (G294S and E539K). Both P26L and E539K
mutants demonstrated decreased activity while G294S muta-
tion increased activity [97]. In a multicenter European study,
30 ENaC mutations were found in 76 patients with atypical CF.
In two of the patients sampled, the CF-like disease could be
explained by mutations in SCNN1A (F61L and V114I) that lead
to an alteration in function [98]. Furthermore, an additional
mutation (W293R) in SCNN1A demonstrated a fourfold
692 P. J. MOORE AND R. TARRAN
increase in amiloride-sensitive currents compared to wild type
ENaC when expressed in X. laevis oocytes [98]. Similarly, a
mutation in the β-ENaC subunit, V348M, was identified
which increased ENaC PO by destabilizing the closed channel
state [99]. Another study of atypical CF phenotypes detected
three missense variants in SCNN1A (R204W, A357T, C641F) and
one missense in SCNN1B (R543Q). The authors of this study
suggested that these variants that appear at a higher inci-
dence in patients with atypical CF may be responsible for
the observed CF-like symptoms [100]. More recently, a muta-
tion in the δ subunit reduced ENaC activity indicating δENaC
as a potential genetic CF disease modifier [101]. SPLUNC1 has
also been shown to be a potential gene modifier and CF
patients who have a polymorphism on the G allele of
rs1078761 had decreased SPLUNC1 expression and increased
disease severity [102]. Taken together, these data suggest that
increased ENaC activity and/or a failure of SPLUNC1 to regu-
late ENaC might contribute to the development of CF
lung disease.
1.6. Mouse models of CF-like lung disease
Mice have fewer generations of airways than humans (13 versus
21, respectively) and CFTR−/− mice do not have a lower airways
disease phenotype [78]. However, there is a dehydration phe-
notype in the nasal tissues of these mice [103,104].
Interestingly, murine nasal epithelia are the only part of the
mouse airways that show elevated Na+ transport [105]. Since CF
mice lacked any obvious lower airway pathology, Mall et al.
overexpressed β-ENaC in a transgenic mouse, which caused
spontaneous lung disease [106]. Furthermore, this lung disease
approximated the development of the CF lung, including
mucus plugging, neutrophilia, and infection, leading to
increased mortality rates [106]. Knockout of the ubiquitin ligase
NEDD4.2 also caused an increase in ENaC surface densities and
spontaneous lung disease that was similar to that seen in the
βENaC mouse [67]. The development of the β-ENaC overexpres-
sing and NEDD4.2 knockout transgenic mice clearly demon-
strate a link between unregulated ENaC activity and the
development of pulmonary disease. Importantly, given the
aforementioned lack of phenotype of the CF mice, mice with
altered ENaC activity have served as an important test bed for
new therapies aimed at rehydrating CF lungs.
2. ENaC inhibitors
2.1. Small molecule/pore blocking ENaC antagonists
Amiloride is a first-generation ENaC antagonist that was
developed in the 1960s as a potassium-sparing compound
by Cragoe and colleagues at Merck Pharmaceuticals [107].
ENaC is highly expressed in the distal convoluted tubule of
the kidney and while this portion of the nephron is only
responsible for handling 10% of reabsorbed Na+, it plays an
important role in fine-tuning Na+ and fluid levels in the
body [108,109]. Amiloride was initially used to prevent
Na+ absorption in the kidney and to induce natriuresis/
diuresis[110]. However, inhibition of ENaC leads to potas-
sium-sparing excretion of Na+ and hyperkalemia [111,112].
Thus, whilst amiloride is an effective diuretic, it has now
been superseded by so called ‘loop diuretics’ such as thia-
zide that target the Na+/K+/2 Cl− cotransporter (NKCC2) in
the loop of Henle to block both Na+ and K+ reabsorption
which does not cause hyperkalemia [113].
Because of its affinity for ENaC, amiloride was tested as an
inhaled CF therapeutic that could reduce Na+ absorption in the
airways to rehydrate mucus and restore mucus clearance [87,114].
When added 2–4 times per day, amiloride had a moderate effect
on MCC and lung function in CF patients [111,115,116]. The lung
expresses pumps and transporters that can remove xenobiotics
from the lung and concentrate them in the bloodstream, so that
they can be metabolized by the liver and/or excreted by the
kidneys [117,118]. Amiloride is a substrate for some of these
transporters and was found to have a half-life in humans of
~40 min, and a half-life in the ASL of human airway cultures of
~10 min [116,119]. This short half-life likely accounted for its failure
to achieve significant clinical efficacy in the lung. Indeed, the
increase in mucus clearance lasted ~40 min [111]. Amiloride was
also studied in βENaC overexpressing mice: Mall and colleagues
reported that intranasal administration of amiloride to these mice,
3 times daily from the first day of life resulted in the reduction of
pulmonary mortality and significantly reduced in mucus accumu-
lation/obstruction, goblet cell hyperplasia, and pulmonary inflam-
mation [120]. These data indicated that if the poor
pharmacodynamics can be overcome, then ENaC inhibition via
inhalation could be a viable therapy for the treatment of CF [120].
Amiloride derivatives have been developed in an attempt
to improve the efficacy and potency seen with amiloride.
Parion Sciences have developed several amiloride-derivatives.
Parion-552-002 (P-552-02) is an ENaC antagonist that
was more efficacious and more selective for ENaC than amilor-
ide [119]. However, P-552-02 studies were halted after Phase II
clinical trials when hyperkalemia was observed. Parion
Sciences P-680 (Gilead Sciences, GS-9411) was a subsequent
amiloride derivative that was 100-fold more potent than
amiloride that also had a longer duration of action. P-680/
GS-9411 was retained in the ASL for up to 8 h and also
increased ASL volume in vitro. However, Phase I clinical trials
failed, after administration of GS-9411 to healthy subjects also
induced hyperkalemia [112]. Parion sciences subsequently
developed a long-acting ENaC antagonist called P-1037.
Preclinical studies demonstrated promising results when P-
1037 was used in combination with hypertonic saline and
P-1037 produced a sustained increase in ASL height over 8 h
in vitro, resulting in an eight- and fourfold increase in
volume compared to isotonic saline and hypertonic saline,
respectively [121]. In collaboration with Vertex
Pharmaceuticals, P-1037 (now VX-371) entered Phase II clinical
trials. However, following a 28-day trail, VX-371 and hypertonic
saline caused a nonsignificant increase in FEV1 of 0.1% when
added along with Orkambi (VX-770/VX-809) in ΔF508 homo-
zygous patients (https://www.businesswire.com/news/home/
20171025006319/en/). This lack of effect may have been due
to a short duration of action and/or poor bioavailability in vivo.
AstraZeneca developed ‘compound A’ which is a small
molecule ENaC antagonist that may have been amiloride-
derived [122]. Whilst this compound was more potent than
amiloride, was capable of increasing ASL height, and could

restore mucus clearance, it also showed hyperkalemia in ani-
mal models and was not taken into the clinic [122]. A second
ENaC inhibitor, AZD5634, was subsequently developed. This
compound was retained in the ASL and had a high affinity for
ENaC [123]. In primary ΔF508 homozygous HBECs, AZD5634
elicited greater changes in ASL height and mucociliary clear-
ance than benzamil, a second generation amiloride-derivative.
AZD5634 completed Phase I clinical trials and it was generally
well tolerated with no obvious hyperkalemia [124]. To date, a
Phase II study in CF patients using MCC as the readout is
ongoing. Novartis developed NVP-QBE170, a dimeric-amiloride
derivative that has shown some promising results in
sheep [125]. For example, NVP-QBE170 was able to enhance
mucus clearance in a dose dependent manner without indu-
cing hyperkalemia [125]. Similarly, Novartis and Boehringer
Ingelheim have also developed the novel ENaC antagonists,
QBW 276, and BI 443651, respectively. Both have entered
clinical testing and showed good safety profiles following
Phase I clinical trials. However, Phase II/efficacy results are
still pending.
2.2. Protease inhibitors
In healthy airways, a balance exists between the number of
proteases and their inhibitors. Whilst these proteases have
many innate defense functions, they also affect ENaC activity
and hence can influence ASL hydration [42]. In CF airways, an
imbalance between protease/protease inhibitor levels results
may cause increased proteolysis of α- and γ-ENaC subunits
that may contribute to ASL/mucus dehydration. Levels of furin,
cathepsin B, neutrophil elastase, and prostasin are all
increased in CF airways [42,126–128]. Thus, inhibition of CF
proteases represents an alternate approach to limit ENaC
hyperactivity in the CF lung that is currently being evaluated.
A novel protease inhibitor, QUB-TL1, was developed by the
Martin group at Queen’s University in Belfast. This compound
reduced mucosal protease activity on CF HBECs [129]. QUB-
TL1-depdendent inhibition of protastin, furin, and P. aerugi-
nosa exotoxin A resulted in reduced ENaC-mediated Na+
absorption in CF HBECs that lead to decreased amiloride-
sensitive currents and increased ASL volume/mucociliary clear-
ance [129]. Whilst QUB-TL1 is a broad spectrum protease
inhibitor that looks promising in preclinical development, it
has relatively low potency and this program may benefit from
a second generation compound with improved efficacy. Other
protease inhibitors have also been tested in CF patients. For
example, AZD9668 was tested and had no effect on inflam-
mation or lung function in CF patients [130]. Similarly,
recombinant α1 antitrypsin also failed to affect lung inflam-
mation [131]. Camostat, an inhibitor of protastin, induced a
potent and prolonged attenuation of ENaC in the guinea pig
tracheal potential difference (PD) model and enhanced mucus
clearance for 5 h after nebulization into sheep [125]. Using the
nasal potential difference assay, it was reported that the
amiloride-sensitive PD was significantly reduced in CF patients
treated with camostat compared to placebo, indicating that a
reduction in Na+ transport in CF airways can be achieved
though protease inhibition [132]. Three subjects experienced
serious adverse events during the clinical trial; two of which
EXPERT OPINION ON THERAPEUTIC TARGETS 693
were pulmonary exacerbations after camostat treatment and
one subject had appendicitis following placebo treatment. In
addition, six subjects had adverse events which were thought
to be drug related including epistaxis, nasal disruption, and
hematuria. Nasal sensitivity and rhinorrhea were also reported.
ENaC is highly promiscuous and is cleaved by multiple pro-
teases. Thus, a possible contribution to the failure of protease
inhibitors to induce clinically meaningful changes in lung
function in the clinic is that they tend to be relatively specific
and inhibit only one protease or one class of protease per
drug. In contrast, multiple proteases are upregulated in the CF
lung and for example if neutrophil elastase is inhibited, cathe-
psin B or another protease can still independently cleave and
activate ENaC.
2.3. Peptides and other approaches
Whilst the idea of using small molecules and protease inhibi-
tors to inhibit ENaC has been around for a while, more
recently alternate approaches to regulate ENaC are now
being evaluated. For example, the ability to specifically inhibit
the individual ENaC subunits is possible through the use of
antisense oligonucleotides (ASO). ASO are strands of nucleic
acid-based complementary messenger RNA (mRNA). There are
two classes of ASO: (1) the RNase-H-dependent oligonucleo-
tides which induce degradation of mRNA and (2) the steric-
blocker oligonucleotides which physically inhibit the splicing
translational machinery. Most of the currently available ASOs
function via the RNase-H-dependent mechanism [133].
Generally, uptake of the ASO through active transport
depends on the structure and concentration of the oligonu-
cleotide [134,135]. Recently, an ASO complimentary to SCNN1A
was administrated directly into a Nedd4L knockout mouse
model, which resulted in a 70% mRNA target knockdown
and an overall improvement in biomarkers of disease [136].
ASOs are currently in preclinical development with Ionis
Pharmaceuticals. However, consistent issues with gene-based
approaches to treating CF lung disease are poor delivery and
local inflammation [137,138]. Thus, whether or not ASOs will
be able to achieve therapeutic doses in order to decrease
ENaC levels without inducing inflammation in CF patients
remains to be determined.
The discovery of an ENaC-inhibitory domain on SPLUNC1’s
N-terminus has led to the development of novel ENaC antago-
nists that are peptides rather than small molecules. Despite
having an ENaC inhibitory domain, SPLUNC1 fails to regulate
ENaC in CF airway cultures [86]. An explanation for this was
found in SPLUNC1’s crystal structure. Indeed, SPLUNC1 possess
pH-sensitive salt bridges that prevent SPLUNC1 from binding
to and regulating ENaC in the mildly acidic CF ASL environ-
ment [86]. In contrast, unlike full-length SPLUNC1, SPLUNC1-
derived peptides that correspond to the N-terminal ENaC
inhibitory domain lack the salt bridges and can regulate ASL
hydration equally well in normal and CF HBECs [139]. However,
perceived issues with inhaled peptides for the treatment of CF
lung disease include the possibility that they will stick to mucus
and fail to reach their target and/or that they will degrade by
lung proteases. We have previously demonstrated that the
SPLUNC1-derived S18 peptide can be mixed with neutrophil
694 P. J. MOORE AND R. TARRAN
elastase and/or activated neutrophil supernatant that contains
neutrophil elastase and cathepsins and it still functions to inhibit
ENaC [74], suggesting that the stability issue can be overcome.
Indeed, due to the long duration of action induced by the
removal of ENaC subunits from the plasma membrane after
exposure to SPLUNC1-derived peptides, it is likely that the effects
persist over time even after washout. Using biophysical techni-
ques, we recently demonstrated that the S18 ENaC inhibitory
peptide does not bind to mucins and that fluorescently labeled
S18 was able to easily diffuse through dehydrated mucus that
had accumulated on CF HBECs [139]. This is consistent with
previous observations that positively charged residues bind to/
interact with mucins [140] and S18 (GGLPVPLDQTLPLNVNPA)
does not contain positive K, R, or H residues. Additionally, using
this peptide, we were able to reprise the experiments of Zhou
et al. and significantly increase survival of newly born βENaC
mice. Interestingly, while amiloride caused a ~10% loss of body
weight/day, S18 did not have these effects [139]. Further inves-
tigations revealed that S18 did not affect renal function when
injected intraperitoneally to mice and did not affect renal func-
tion when added intravenously to rats. Furthermore, when
100 mM of S18 was added intranasally to mice, only ~1 nM
peptide was detected in serum, suggesting that SPLUNC1-
derived peptides are poorly absorbed across pulmonary epithelia
and have very favorable pharmacodynamics [139].
SPX-101 is a novel, size-optimized SPLUNC1-derived pep-
tide/ENaC inhibitor that is in clinical development for the
treatment of CF lung disease. SPX-101 is more potent than
SPLUNC1/S18 and bound to ENaC to promote subunit inter-
nalization in both normal and CF HBECs, leading to a signifi-
cant reduction in the amiloride-sensitive current and an
increase in ASL height/mucus clearance [141]. A once daily
installation of SPX-101 increased survival and reduced airway
inflammation in βENaC mice [141]. The Abraham lab in Miami
recently developed a pharmacological CF ovine mucus clear-
ance model. Here, inhalation of the CFTR antagonist INH172
slows mucus clearance rates by ~50%. Using this model, SPX-
101 increased tracheal mucus velocity for extended periods,
consistent with the long duration of action seen in vitro and in
the βENaC mice [141]. These findings, coupled with its unique
mechanism of action, indicate that SPX-101 is an attractive
therapeutic peptide to reduce ENaC activity in the CF lung. A
major advantage of using intrinsically disordered peptides like
SPX-101 is that due to their inherent flexibility, they achieve
good contact with their target protein, thus maximizing their
binding efficiency. In preclinical toxicology, SPX-101 was admi-
nistered by inhalation to two species (rats and dog), was safe,
and did not alter serum electrolyte levels [142]. Similarly, SPX-
101 did not cause hyperkalemia in humans following 14-day
administration in a Phase I study [143]. To date, SPX-101 has
entered Phase II clinical trials in CF patients (NCT03229252).
Cohort 1 of this trial has now been completed and these data
were recently presented by Professor Isabelle Fajac at the
European CF Conference. Indeed, in CF patients with a percent
predicted FEV1 (ppFEV1) of 40–80%, SPX-101 increased ppFEV1
by 5.2% in a mutation-agnostic fashion relative to placebo.
However, in patients with an FEV1 greater than 55%, SPX-101
significantly increased ppFEV1 by 8.3% without causing
hyperkalemia. Thus, SPX-101 shows promise for improving
lung function by inhibiting ENaC since it is active in CF
patients and has good pharmacodynamics and a favorable
safety profile. We speculate that CF subjects with higher base-
line FEV1 may have more mucus obstruction and less bronch-
iectasis and are thus more amenable to mucus rehydration
therapies. Importantly, these data suggest that SPX-101 may
be more beneficial in these milder CF patients. Furthermore, it
was demonstrated that SPX-101 also induces ASL rehydration
in COPD-derived/tobacco smoke-exposed HBECs, suggesting
that it SPX-101 may also be efficacious in COPD patients who
also exhibit defective CFTR function and mucus dehydration
[144,145].
2.4. ENaC inhibition and other therapies
CF sputum contains large amounts of extracellular DNA that is
released by infiltrating inflammatory cells such as neutrophils.
Nebulization of recombinant human deoxyribonuclease
(rhDNase) is a commonly used method to mobilize CF sputum
[146]. Dornase alfa (Pulmozyme; Genentech) is a purified
rhDNase that cleaves extracellular DNA, thereby reducing the
viscosity of the mucus layer [146,147]. Daily treatment with
rhDNase reduces the number of pulmonary exacerbations,
improves lung function, and is well tolerated [148,149]. Use of
Dornase alfa would not affect ASL hydration or ENaC activity
directly but would likely be complementary to an inhaled ENaC
antagonist, since the latter would likely facilitate mucus hydra-
tion/clearance. Clinical trials have demonstrated that rhDNase
significantly improves lung function [147,150]. However, varia-
tions in clinical response to rhDNase have been observed in CF
patients [151,152] which have been attributed to treatment
regimens and optimal timing [153,154].
Increasing ASL hydration by influencing the osmotic gradi-
ent is another attractive therapeutic target. Mannitol is a
naturally occurring sugar alcohol which has been inhaled by
CF patients [155]. Similarly, hypertonic saline has also been
used as an osmolyte [156]. When inhaled, these compounds
create an osmotic gradient that induces water movement into
the airway lumen, thus rehydrating the ASL and enhancing
mucus clearance [157]. Two randomized multicenter, double-
blind, controlled, parallel-group Phase III studies were carried
out to assess the safety and efficacy of inhaled mannitol in CF
patients aged 6 years over a period of 6 months. Bilton et al.
and Aitken et al. observed a statistically significant improve-
ment in FEV1 within 6 weeks of treatment with mannitol was
maintained throughout the study [158,159]. Inhaled mannitol
has since been approved for use in adults with CF in the
European Union and in children over 6 years of age in
Australia. Phase III trials of Bronchitol (mannitol, Pharmaxis
Ltd.) in adults in the United States has reached its primary
end point and FDA submission is expected in 2018. The usage
of rhDNase and mannitol have been well documented in
enhancing the CF patients' quality of life. ENaC antagonists
have been shown to be synergistic with hypertonic saline in
vitro [160]. However, amiloride-pretreatment before hyper-
tonic saline resulted in a reduction in FEV1 [156], and whether

osmolytes and ENaC agonists will work synergistically remains
to be determined and may depend on the ENaC antagonist
used and the order of administration.
2.5. ENaC inhibition therapy: with or without CFTR
modulators?
Whilst some epithelia can only secrete or only absorb fluid,
airway epithelia are capable of both modalities. This is espe-
cially relevant to airways hydration since net ASL/fluid secre-
tion is the sum of Cl− secretion minus opposing Na+
absorption. That is, any given Cl− secretory event will be larger
if absorption is blocked. For example, we have previously
applied phasic shear stress to normal and CF HBECs in order
to mimic tidal breathing, which causes an increase in ATP
release, subsequent adenosine formation, and activation of
Cl− secretion via purinergic receptors [161]. Here, we found
that phasic shear stress elicited similar amounts of bumeta-
nide-sensitive transepithelial voltage, indicative of Cl− secre-
tion, in both normal and CF HBECs. However, whilst the
amiloride-sensitive voltage (a marker of ENaC activity) was
spontaneously abolished (a reduction of >90%) in normal
HBECs, it was only reduced by ~50% in CF HBECs. In parallel,
we observed an increase in ASL height to ~15 μm in normal
HBECs, but only to ~7 μm in CF HBECs, suggesting that the
persistent ENaC activity was hampering CF ASL secretion [161].
These observations highlight the importance of homeostasis
and indicate how balancing multiple facets of airway ion
transport can favor Cl− secretion.
Recent attention has justifiably been focused on the pharma-
cological rescue of mutant CFTR using novel potentiators [162]
that increase CFTR PO and correctors that increase CFTR traffick-
ing to the plasma membrane [163]. While ENaC antagonists
show efficacy when administered alone, it is likely that an ENaC
antagonist in combination with CFTR modulators would be ben-
eficial/synergistic in increasing CF ASL hydration. When VX-770
(ivacaftor, Kalydeco) is added to CFTRG551D-expressing cultures,
there is a 10–20% correction of CFTRG551D function [164]. While
this is a significant improvement, it is still 80–90% less than wild-
type CFTR function, and how much CFTR function is needed to
‘fix’ the CF lung is still under debate. Crucially, CFTRG551D, which
traffics to the plasma membrane but fails to open, with VX-770, is
currently the best case scenario for approved CFTR modulators;
yet these patients fail to eradicate bacteria from their lungs over
time [165], although we acknowledge that newer, more potent
CFTR modulators are under development which may improve on
this. For CFTRΔF508, which has (1) reduced expression, (2) trouble
escaping the endoplasmic reticulum’s quality control mechan-
isms, (3) has a reduction in Po, and (4) has a reduced residency
time in the plasma membrane, restoration of function may be
more problematic [166–169]. Thus, multiple therapies will likely
be needed to prolong patient survival and improve their quality
of life and ENaC inhibition in the face of CFTR modulation is
predicted to be synergistic. This hypothesis is born out experi-
mentally: the intracellular Cl− concentration is almost threefold
lower than that in the ASL [26]. Thus, there is no chemical
gradient for Cl− secretion. This is important, because ion chan-
nels such as CFTR are passive and only move Cl− according to the
electrochemical gradient. So, in order for Cl− to be secreted into
EXPERT OPINION ON THERAPEUTIC TARGETS 695
the ASL, ENaC needs to be inhibited, which, due to the lack
of Na+ absorption, causes apical membrane hyperpolariza-
tion [170]. This increased negative potential causes an electrical
driving force to induce Cl− secretion against the unfavorable
concentration gradient. This is why experimentally, amiloride is
added in Ussing chambers before activating CFTR, even in the
presence of Vertex-type CFTR modulators [162,163]. In the clinic,
therefore, ENaC inhibition will increase the electrical driving force
for Cl− secretion, even for wild-type or fully corrected CFTR.
Whilst CFTR potentiators and modulators would likely be
synergistic with ENaC antagonists, approximately 7% of CF
patients have Type I mutations where CFTR protein is not
made [171] (i.e. W1282X), which will never be amenable to
corrector/potentiator-type therapies. In contrast, ENaC inhibi-
tion is mutation agnostic and should be equally effective even
in the complete absence of CFTR protein (i.e. for Type I muta-
tions). Thus, whilst read-through therapies for Type I muta-
tions are being developed [172], ENaC antagonism may be the
earliest chance at normalizing ASL volume homeostasis in
these patients.
3. Conclusion
Therapeutics for the treatment of CF lung disease have been
focused on symptomatic management of the existing pulmon-
ary disease. However, in recent years, there has been a shift
with to an emphasis on pharmacotherapy for ENaC inhibition
and CFTR correction that can both contribute to ASL rehydra-
tion that may prevent lung disease from occurring. The
advances in ENaC therapeutics, especially with new and emer-
ging therapeutics such as SPX-101, make it clear that the
evolving CF pharmacotherapy is an exciting field that warrants
further investigation. ENaC antagonists can be either inhaled
or oral. However, whilst oral ENaC antagonists exist, inhalation
is the preferred route in order to maximize exposure and limit
extrapulmonary effects including K+ sparing natriuresis and
hyperkalemia [107]. Thus, a major hurdle to developing ENaC
antagonists is to constrain the compound to the lung and
prevent any renal effects. A schematic summary of the main
classes of ENaC inhibitors is depicted in Figure 4
4. Expert opinion
Due to the complex and severe nature of CF lung disease, it is
increasingly evident that the use of novel pharmacological
ENaC inhibitors to attenuate Na+ absorption is becoming an
attractive therapeutic approach to treat CF lung disease that
may be used alone or in combination with other therapies.
Early ENaC antagonists such as amiloride suffered from poor
duration of action and off target effects. Although the spec-
trum of ENaC inhibitors has expanded during the last
5–10 years with many showing increased potency over amilor-
ide, many have failed to progress past Phase II clinical trials,
most commonly due to hyperkalemia. Thus, the two greatest
hurdles to ENaC antagonists becoming viable therapies are (1)
duration of action and (2) preventing hyperkalemia. While
protease inhibitors are unlikely to cause hyperkalemia, they
may suffer from not being broad-spectrum enough for the
696 P. J. MOORE AND R. TARRAN
Figure 4. ENaC inhibition as a strategy for ASL rehydration. (1) Pharmacological inhibitors such as amiloride directly inhibit Na+ absorption by occupying the channel
pore. (2) Novel peptides such as SPX-101 reduce the number of available ENaC subunits at the plasma membrane. (3) Protease inhibitors prevent channel activating
proteases from cleaving ENaC, maintaining ENaC in an inactive form; thus, preventing Na+ absorption. (4) Antisense oligonucleotides inhibit mRNA translation and
prevent ENaC trafficking to the surface of the cell.
highly proteolytic CF lung environment. Given the previous
failures in getting foreign DNA/RNA into the lung, ASOs may
suffer from achieving sufficient ENaC knockdown.
In this review, we have highlighted the failures of small
molecule antagonists, as well as potential successes of novel
approaches that may be used in alone or in combination with
current CFTR modulators to alleviate CF symptoms. ENaC
inhibitors such as P-1037/VX-371 demonstrated high affinity
for ENaC. However, CF clinical trials with this compound were
not positive. It is important to highlight, therefore, that even
though a small molecule may be highly potent in vitro and in
animal models, the complex nature of the CF lung may reduce
the efficacy and may lead to unfavorable pharmacodynamics.
Currently, there are several potential new drugs that are in
the development pipeline, some of which are in clinical trials.
The novel peptide SPX-101 offers a key advantage over ‘tradi-
tional’ small molecule antagonists including negligible sys-
temic absorption and a long duration of action. Generating
novel peptides for therapeutic intervention presents a unique
and exciting opportunity but is not without challenges, includ-
ing the need for stability in the face of a highly proteolytic
environment. Given the number of ENaC antagonists currently
being developed that employ multiple modes of action, it is
hopeful that one of these will translate into the clinic in the
near future.
Funding
This paper was funded by the CF Foundation, USA, and Emily’s Entourage,
USA.
Declaration of interest
R. Tarran is a founder of and has equity in Spyryx Biosciences, USA. The
authors have no other relevant affiliations or financial involvement with
any organization or entity with a financial interest in or financial conflict
with the subject matter or materials discussed in the manuscript. This
includes employment, consultancies, honoraria, stock ownership or
options, expert testimony, grants or patents received or pending, or
royalties.
Reviewer of interest
One reviewer is affiliated with Enterprise Therapeutics, Sussex, UK. Other
peer reviewers on this manuscript have no relevant financial or other
relationships to disclose
References
Papers of special note have been highlighted as either of interest (•) or of
considerable interest (••) to readers.
1. Internet. Foundation NACF. 2016 North American CFF Patient
Registry Annual Data Report [cited 2018 Mar 14]. Available from:
https://www.cff.org/Research/Researcher-Resources/Patient-
Registry/2016-Patient-Registry-Annual-Data-Report.pdf.
2. Riordan J, Rommens J, Kerem B, et al. Identification of the cystic
fibrosis gene: cloning and characterization of complementary DNA.
Science. 1989;245(4922):1066–1073.
3. Vankeerberghen A, Cuppens H, Cassiman J-J. The cystic fibrosis
transmembrane conductance regulator: an intriguing protein with
pleiotropic functions. J Cyst Fibros. 2002;1(1):13–29.
4. Gibson RL, Burns JL, Ramsey BW. Pathophysiology and
Management of Pulmonary Infections in Cystic Fibrosis. Am J
Respir Crit Care Med. 2003;168(8):918–951.

5. Wilschanski M, Novak I. The Cystic Fibrosis of Exocrine Pancreas.
Cold Spring Harb Perspect Med. 2013;3(5):a009746. PubMed PMID:
PMC3633181.
6. VanDevanter DR, Kahle JS, O’Sullivan AK, et al. Cystic fibrosis in
young children: a review of disease manifestation, progression, and
response to early treatment. J Cyst Fibros. 2016;15(2):147–157.
7. Nichols D, Chmiel J, Berger M. Chronic Inflammation in the Cystic
Fibrosis Lung: alterations in Inter- and Intracellular Signaling. Clin
Rev Allergy Immunol. 2008;34(2):146–162.
8. Puchelle E, Bajolet O, Abély M. Airway mucus in cystic fibrosis.
Paediatr Respir Rev. 2002;3(2):115–119.
9. Salsgiver EL, Fink AK, Knapp EA, et al. Changing Epidemiology of
the Respiratory Bacteriology of Patients With Cystic Fibrosis. Chest.
2016 Jan 12;149(2):390–400. PubMed PMID: PMC5831653.
10. Muhlebach MS, Zorn BT, Esther CR, et al. Initial acquisition and
succession of the cystic fibrosis lung microbiome is associated with
disease progression in infants and preschool children. PLoS Pathog.
2018;14(1):e1006798.
11. Kidd TJ, Ramsay KA, Vidmar S, et al. Pseudomonas aeruginosa
genotypes acquired by children with cystic fibrosis by age 5-
years. J Cyst Fibros. 2015;14(3):361–369.
12. Landry RM, An D, Hupp JT, et al. Mucin–pseudomonas aeruginosa
interactions promote biofilm formation and antibiotic resistance.
Mol Microbiol. 2006;59(1):142–151.
13. Mathee K, Ciofu O, Sternberg C, et al. Mucoid conversion of
Pseudomonas aeruginos by hydrogen peroxide: a mechanism for
virulence activation in the cystic fibrosis lung. Microbiology.
1999;145(6):1349–1357.
14. Cornelis P, Dingemans J. Pseudomonas aeruginosa adapts its iron
uptake strategies in function of the type of infections. Front Cell
Infect Microbiology.. 2013;3:75.
15. Pezzulo AA, Tang XX, Hoegger MJ, et al. Reduced Airway Surface pH
Impairs Bacterial Killing in the Porcine Cystic Fibrosis Lung. Nature.
2012 Jul 04;487(7405):109–113. PubMed PMID: PMC3390761.
16. Worlitzsch D, Tarran R, Ulrich M, et al. Effects of reduced mucus
oxygen concentration in airway Pseudomonas infections of cystic
fibrosis patients. J Clin Invest. 2002 Feb 01;109(3):317–325. .
17. Nakamura H, Yoshimura K, McElvaney NG, et al. Neutrophil elastase
in respiratory epithelial lining fluid of individuals with cystic fibrosis
induces interleukin-8 gene expression in a human bronchial
epithelial cell line. J Clin Investig. 1992;89(5):1478–1484. PubMed
PMID: PMC443018.
18. Black HR, Yankaskas JR, Johnson LG, et al. Interleukin-8 Production
by Cystic Fibrosis Nasal Epithelial Cells after Tumor Necrosis Factor-
α and Respiratory Syncytial Virus Stimulation. Am J Respir Cell Mol
Biol. 1998;19(2):210–215. PubMed PMID: 9698592.
19. Bonfield TL, Panuska JR, Konstan MW, et al. Inflammatory cytokines in
cystic fibrosis lungs. Am J Respir Crit Care Med. 1995;152(6):2111–2118.
20. Mitola S, Sorbello V, Ponte E, et al. Tumor Necrosis Factor-α in
Airway Secretions from Cystic Fibrosis Patients Upregulate
Endothelial Adhesion Molecules and Induce Airway Epithelial Cell
Apoptosis: implications for Cystic Fibrosis Lung Disease. Int J
Immunopathol Pharmacol. 2008;21(4):851–865.
21. Norman D, Elborn JS, Cordon SM, et al. Plasma tumour necrosis
factor alpha in cystic fibrosis. Thorax. 1991;46(2):91–95. PubMed
PMID: PMC462953.
22. Greally P, Hussein MJ, Cook AJ, et al. Sputum tumour necrosis
factor-alpha and leukotriene concentrations in cystic fibrosis. Arch
Dis Child. 1993;68(3):389–392. PubMed PMID: PMC1793872.
23. Rowe SM, Miller S, Ej S. Cystic Fibrosis. New England J Med.
2005;352(19):1992–2001. PubMed PMID: 15888700.
24. Weiner DJ, Bucki R, Janmey PA. The Antimicrobial Activity of the
Cathelicidin LL37 Is Inhibited by F-actin Bundles and Restored by
Gelsolin. Am J Respir Cell Mol Biol. 2003;28(6):738–745. PubMed
PMID: 12600826.
25. É F, Humphreys H, Greene CM, et al. Potential of Host Defense
Peptide Prodrugs as Neutrophil Elastase-Dependent Anti-
Infective Agents for Cystic Fibrosis. Antimicrob Agents
Chemother. 2014;58(2):978–985.
EXPERT OPINION ON THERAPEUTIC TARGETS 697
26. Tarran R, Grubb BR, Gatzy JT, et al. The Relative Roles of Passive
Surface Forces and Active Ion Transport in the Modulation of
Airway Surface Liquid Volume and Composition. J Gen Physiol.
2001;118(2):223.
27. Garty H, Palmer LG. Epithelial sodium channels: function, structure,
and regulation. Physiol Rev. 1997;77(2):359–396. PubMed PMID:
9114818.
28. Butterworth MB. Regulation of the epithelial sodium channel
(ENaC) by membrane trafficking. Biochim Biophys Acta. 2010 Mar
27;1802(12):1166–1177. PubMed PMID: PMC2921481.
29. Hamm LL, Feng Z, Hering-Smith KS. Regulation of sodium transport by
ENaC in the kidney. Curr Opin Nephrol Hypertens. 2010;19(1):98–105.
PubMed PMID: PMC2895494.
30. Stockand JD, Staruschenko A, Pochynyuk O, et al. Insight toward
epithelial Na+ channel mechanism revealed by the acid-sensing
ion channel 1 structure. IUBMB Life. 2008;60(9):620–628.
31. Sheng S, McNulty KA, Harvey JM, et al. Second Transmembrane
Domains of ENaC Subunits Contribute to Ion Permeation and
Selectivity. J Biol Chem. 2001;276(47):44091–44098.
32. Kellenberger S, Gautschi I, Schild L. An External Site Controls Closing
of the Epithelial Na+ Channel ENaC. J Physiol. 2004;543(2):413–424.
33. Kerem E, Bistritzer T, Hanukoglu A, et al. Pulmonary epithelial
sodium-channel dysfunction and excess airway liquid in pseudo-
hypoaldosteronism. N Engl J Med. 1999 Jul 15;341(3):156–162.
PubMed PMID: 10403853.
34. Staub O, Gautschi I, Ishikawa T, et al. Regulation of stability and
function of the epithelial Na+ channel (ENaC) by ubiquitination.
Embo J. 1997 Nov 3;16(21):6325–6336. PubMed PMID: 9351815.
35. Kashlan OB, Kleyman TR. ENaC structure and function in the wake
of a resolved structure of a family member. Am J Physiology-Renal
Physiol. 2011;301(4):F684–F696.
36. Snyder PM, Bucher DB, Olson DR. Gating Induces a
Conformational Change in the Outer Vestibule of Enac. J Gen
Physiol. 2000;116(6):781.
37. Harris M, Garcia-Caballero A, Stutts MJ, et al. Preferential Assembly
of Epithelial Sodium Channel (ENaC) Subunits in Xenopus Oocytes:
ROLE OF FURIN-MEDIATED ENDOGENOUS PROTEOLYSIS. J Biol
Chem. 2008;283(12):7455–7463.
38. Ji H-L, Su X-F, Kedar S, et al. δ-Subunit Confers Novel Biophysical
Features to αβγ-Human Epithelial Sodium Channel (ENaC) via a
Physical Interaction. J Biol Chem. 2006;281(12):8233–8241.
39. Bangel-Ruland N, Sobczak K, Christmann T, et al. Characterization
of the Epithelial Sodium Channel δ-Subunit in Human Nasal
Epithelium. Am J Respir Cell Mol Biol. 2010;42(4):498–505.
PubMed PMID: 19520916.
40. Hughey RP, Bruns JB, Kinlough CL, et al. Epithelial Sodium Channels
Are Activated by Furin-dependent Proteolysis. J Biol Chem.
2004;279(18):18111–18114.
41. Vuagniaux G, Vallet V, Jaeger NF, et al. Activation of the Amiloride-
Sensitive Epithelial Sodium Channel by the Serine Protease mCAP1
Expressed in a Mouse Cortical Collecting Duct Cell Line. J Am Soc
Nephrol. 2000;11(5):828–834.
42. Caldwell RA, Boucher RC, Stutts MJ. Neutrophil elastase activates
near-silent epithelial Na+ channels and increases airway epithelial
Na+ transport. Am J Physiology-Lung Cell Mol Physiol. 2005;288(5):
L813–L819. PubMed PMID: 15640288.
43. Donaldson SH, Hirsh A, Li DC, et al. Regulation of the Epithelial
Sodium Channel by Serine Proteases in Human Airways. J Biol
Chem. 2002;277(10):8338–8345.
44. Antalis TM, Buzza MS, Hodge KM, et al. The Cutting Edge: mem-
brane Anchored Serine Protease Activities in the Pericellular
Microenvironment. Biochem J. 2010;428(3):325–346. PubMed
PMID: PMC3680374.
45. García-Caballero A, Dang Y, He H, et al. ENaC Proteolytic Regulation
by Channel-activating Protease 2. J Gen Physiol. 2008;132(5):521.
46. Bruns JB, Carattino MD, Sheng S, et al. Epithelial Na+ Channels
Are Fully Activated by Furin- and Prostasin-dependent Release
of an Inhibitory Peptide from the γ-Subunit. J Biol Chem.
2007;282(9):6153–6160.
698 P. J. MOORE AND R. TARRAN
47. Vallet V, Chraibi A, Gaeggeler H-P, et al. An epithelial serine pro-
tease activates the amiloride-sensitive sodium channel. Nature.
1997 Oct 09;389:607.
48. Vuagniaux G, Vallet V, Jaeger NF, et al. Synergistic Activation of
ENaC by Three Membrane-bound Channel-activating Serine
Proteases (mCAP1, mCAP2, and mCAP3) and Serum- and
Glucocorticoid-regulated Kinase (Sgk1) in Xenopus Oocytes. J Gen
Physiol. 2002;120(2):191.
49. Andreasen D, Vuagniaux G, Fowler-Jaeger N, et al. Activation of
Epithelial Sodium Channels by Mouse Channel Activating
Proteases (mCAP) Expressed in Xenopus Oocytes Requires
Catalytic Activity of mCAP3 and mCAP2 but not mCAP1. J Am
Soc Nephrol. 2006;17(4):968–976.
50. Chraïbi A, Vallet V, Firsov D, et al. Protease Modulation of the
Activity of the Epithelial Sodium Channel Expressed in Xenopus
Oocytes. J Gen Physiol. 1998;111(1):127.
51. Harris M, Firsov D, Vuagniaux G, et al. A Novel Neutrophil Elastase
Inhibitor Prevents Elastase Activation and Surface Cleavage of the
Epithelial Sodium Channel Expressed in Xenopus laevis Oocytes. J
Biol Chem. 2007;282(1):58–64.
52. Snyder PM, Olson DR, Kabra R, et al. cAMP and Serum and
Glucocorticoid-inducible Kinase (SGK) Regulate the Epithelial Na+
Channel through Convergent Phosphorylation of Nedd4-2. J Biol
Chem. 2004;279(44):45753–45758.
53. Stutts MJ, Canessa CM, Olsen JC, et al. CFTR as a cAMP-dependent
regulator of sodium channels. Science. 1995;269(5225):847.
54. Stutts MJ, Rossier BC, Boucher RC. Cystic Fibrosis Transmembrane
Conductance Regulator Inverts Protein Kinase A-mediated
Regulation of Epithelial Sodium Channel Single Channel Kinetics.
J Biol Chem. 1997;272(22):14037–14040.
55. Kunzelmann K, Mall M, Briel M, et al. The cystic fibrosis transmem-
brane conductance regulator attenuates the endogenous Ca2+
activated Cl- conductance of Xenopus oocytes. Pflugers Arch.
1997 Dec;435(1):178–181. PubMed PMID: 9359918.
56. Schreiber R, Hopf A, Mall M, et al. The first-nucleotide binding
domain of the cystic-fibrosis transmembrane conductance regula-
tor is important for inhibition of the epithelial Na+ channel. Proc
Natl Acad Sci U S A. 1999 Apr 27;96(9):5310–5315. PubMed PMID:
10220462; eng.
57. Ji HL, Chalfant ML, Jovov B, et al. The cytosolic termini of the
beta- and gamma-ENaC subunits are involved in the functional
interactions between cystic fibrosis transmembrane conductance
regulator and epithelial sodium channel. J Biol Chem. 2000 Sep
8;275(36):27947–27956. PubMed PMID: 10821834.
58. Reddy MM, Light MJ, Quinton PM. Activation of the epithelial Na+
channel (ENaC) requires CFTR Cl- channel function. Nature. 1999
11 18;402(6759):301–304.
59. Mo L, Wills NK. ClC-5 chloride channel alters expression of the epithelial
sodium channel (ENaC). J Membr Biol. 2004 Nov;202(1):21–37. PubMed
PMID: 15702377.
60. Bachhuber T, Konig J, Voelcker T, et al. Cl- interference with the
epithelial Na+ channel ENaC. J Biol Chem. 2005 Sep 9;280
(36):31587–31594. PubMed PMID: 16027156.
61. Willumsen NJ, Boucher RC. Intracellular pH and its relationship to
regulation of ion transport in normal and cystic fibrosis human
nasal epithelia. J Physiol. 1992;455: 247–269. PubMed PMID:
PMC1175643.
62. Hall IP. Second messengers, ion channels and pharmacology of
airway smooth muscle. Eur Respir J. 2000;15(6):1120.
63. Ma HP, Saxena S, Warnock DG. Anionic phospholipids regulate
native and expressed epithelial sodium channel (ENaC). J Biol
Chem. 2002 Mar 8;277(10):7641–7644. .
64. Yue G, Malik B, Eaton DC. Phosphatidylinositol 4,5-bisphosphate
(PIP2) stimulates epithelial sodium channel activity in A6 cells. J
Biol Chem. 2002 Apr 5;277(14):11965–11969. .
65. Mall M, Wissner A, Gonska T, et al. Inhibition of Amiloride-Sensitive
Epithelial Na+ Absorption by Extracellular Nucleotides in Human
Normal and Cystic Fibrosis Airways. Am J Respir Cell Mol Biol.
2000;23(6):755–761.
66. Knight KK, Olson DR, Zhou R, et al. Liddle’s syndrome mutations
increase Na+ transport through dual effects on epithelial Na+
channel surface expression and proteolytic cleavage. Proc Natl
Acad Sci U S A. 2006;103(8):2805.
67. Kimura T, Kawabe H, Jiang C, et al. Deletion of the ubiquitin ligase
Nedd4L in lung epithelia causes cystic fibrosis-like disease. Proc
Natl Acad Sci U S A. 2011 Feb 07;108(8):3216–3221. PubMed PMID:
PMC3044364.
68. Staub O, Dho S, Henry P, et al. WW domains of Nedd4 bind to the
proline-rich PY motifs in the epithelial Na+ channel deleted in
Liddle’s syndrome. EMBO J. 1996;15(10):2371–2380. PubMed
PMID: PMC450167.
69. Butterworth MB, Edinger RS, Ovaa H, et al. The Deubiquitinating
Enzyme UCH-L3 Regulates the Apical Membrane Recycling of the
Epithelial Sodium Channel. J Biol Chem. 2007;282(52):37885–37893.
70. Schild L, Lu Y, Gautschi I, et al. Identification of a PY motif in the
epithelial Na channel subunits as a target sequence for mutations
causing channel activation found in Liddle syndrome. EMBO J.
1996;15(10):2381–2387. PubMed PMID: PMC450168.
71. Bl R, Ep O, Ja K, et al. A new mutation, R563Q, of the beta subunit
of the epithelial sodium channel associated with low-renin, low-
aldosterone hypertension. J Hypertens. 2003;21:5.
72. Hiltunen TP, Hannila-Handelberg T, Petäjäniemi N, et al. Liddle’s
syndrome associated with a point mutation in the extracellular
domain of the epithelial sodium channel γ subunit. J Hypertens.
2002;20:12.
73. Di YP. Functional roles of SPLUNC1 in the innate immune
response against Gram-negative bacteria. Biochem Soc Trans.
2011;39(4):1051.
74. Hobbs CA, Blanchard MG, Alijevic O, et al. Identification of the
SPLUNC1 ENaC-inhibitory domain yields novel strategies to treat
sodium hyperabsorption in cystic fibrosis airway epithelial cultures.
Am J Physiol Lung Cell Mol Physiol. 2013;305(12):L990–L1001.
75. Garcia-Caballero A, Rasmussen JE, Gaillard E, et al. SPLUNC1
regulates airway surface liquid volume by protecting
ENaC from proteolytic cleavage. Proc Natl Acad Sci. 2009 Jul
7;106(27):11412–11417.
•• Important study demonstrating ENaC activity and ASL hydra-
tion is regulated by SPLUNC1.
76. Kim CS, Ahmad S, Wu T, et al. SPLUNC1 is an allosteric modulator of
the epithelial sodium channel. FASEB J. 2018;fj.201701126R.
DOI:10.1096/fj.201701126R.
77. Kerem E, Bistritzer T, Hanukoglu A, et al. Pulmonary Epithelial Sodium-
Channel Dysfunction and Excess Airway Liquid in
Pseudohypoaldosteronism. New England J Med. 1999;341(3):156–162.
PubMed PMID: 10403853.
78. Hyde DM, Hamid Q, Irvin CG. Anatomy, pathology, and physiology
of the tracheobronchial tree: emphasis on the distal airways. J
Allergy Clin Immunol. 2009;124(6):S72–S77.
79. Patwa A, Shah A. Anatomy and physiology of respiratory system
relevant to anaesthesia. Indian J Anaesth. 2015;59(9):533–541.
PubMed PMID: PMC4613399.
80. Liedtke CM. Electrolyte transport in the epithelium of
pulmonary segments of normal and cystic fibrosis lung. FASEB J.
1992;6(12):3076–3084.
81. Knowles MR, Buntin WH, Bromberg PA, et al. Measurements of
Transepithelial Electric Potential Differences in the Trachea and
Bronchi of Human Subjects in vivo. Am Rev Respir Dis.
1982;126(1):108–112.
82. Knowles M, Gatzy J, Boucher R. Increased Bioelectric Potential
Difference across Respiratory Epithelia in Cystic Fibrosis. New
England J Med. 1981;305(25):1489–1495.
83. Boucher RC, Stutts MJ, Knowles MR, et al. Na+ transport in cystic fibrosis
respiratory epithelia. Abnormal basal rate and response to adenylate
cyclase activation. J Clin Invest. 1986 Nov 01;78(5):1245–1252. .
84. Itani OA, Chen J-H, Karp PH, et al. Human cystic fibrosis airway
epithelia have reduced Cl(−) conductance but not increased Na(+)
conductance. Proc Natl Acad Sci U S A. 2011 June 06;108
(25):10260–10265. PubMed PMID: PMC3121869.

85. Tarran R, Trout L, Donaldson SH, et al. Soluble Mediators, Not Cilia,
Determine Airway Surface Liquid Volume in Normal and Cystic
Fibrosis Superficial Airway Epithelia. J Gen Physiol. 2006;127(5):591.
86. Garland AL, Walton WG, Coakley RD, et al. Molecular basis for pH-
dependent mucosal dehydration in cystic fibrosis airways. Proc Natl
Acad Sci. 2013;110(40):15973–15978.
87. Matsui H, Grubb BR, Tarran R, et al. Evidence for Periciliary Liquid Layer
Depletion, Not Abnormal Ion Composition, in the Pathogenesis of
Cystic Fibrosis Airways Disease. Cell. 1998;95(7):1005–1015.
88. Tarran R, Button B, Picher M, et al. Normal and Cystic Fibrosis
Airway Surface Liquid Homeostasis: the effects of phasic shear
stress and viral infections. J Biol Chem. 2005;280(42):35751–35759.
89. Myerburg MM, Butterworth MB, McKenna EE, et al. Airway Surface
Liquid Volume Regulates ENaC by Altering the Serine Protease-
Protease Inhibitor Balance: a mechanism for sodium hyperabsorp-
tion in cystic fibrosis. J Biol Chem. 2006;281(38):27942–27949.
90. Myerburg MM, Harvey PR, Heidrich EM, et al. Acute Regulation of
the Epithelial Sodium Channel in Airway Epithelia by Proteases and
Trafficking. Am J Respir Cell Mol Biol. 2010;43(6):712–719.
91. Bingle L, Barnes FA, Cross SS, et al. Differential epithelial expression
of the putative innate immune molecule SPLUNC1 in Cystic
Fibrosis. Respir Res. 2007;8(1):79.
92. Ahmad S, Tyrrell J, Walton WG, et al. Short Palate, Lung, and Nasal
Epithelial Clone 1 Has Antimicrobial and Antibiofilm Activities
against the Burkholderia cepacia Complex. Antimicrob Agents
Chemother. 2016;60(10):6003–6012.
93. Jiang D, Wenzel SE, Wu Q, et al. Human Neutrophil Elastase
Degrades SPLUNC1 and Impairs Airway Epithelial Defense against
Bacteria. PLOS ONE. 2013;8(5):e64689.
94. Prulière-Escabasse V, Clerici C, Vuagniaux G, et al. Effect of neutro-
phil elastase and its inhibitor EPI-hNE4 on transepithelial sodium
transport across normal and cystic fibrosis human nasal epithelial
cells. Respir Res. 2010;11(1):141.
95. Butterworth MB, Zhang L, Heidrich EM, et al. Activation of the
epithelial sodium channel (ENaC) by the alkaline protease from
Pseudomonas aeruginosa. J Biol Chem. 2012.
96. Butterworth MB, Zhang L, Liu X, et al. Modulation of the Epithelial
Sodium Channel (ENaC) by Bacterial Metalloproteases and Protease
Inhibitors. PLOS ONE. 2014;9(6):e100313.
97. Sheridan MB, Fong P, Groman JD, et al. Mutations in the beta-
subunit of the epithelial Na+ channel in patients with a cystic
fibrosis-like syndrome. Hum Mol Genet. 2005;14(22):3493–3498.
98. Azad Abul K, Rauh R, Vermeulen F, et al. Mutations in the amilor-
ide-sensitive epithelial sodium channel in patients with cystic fibro-
sis-like disease. Hum Mutat. 2009;30(7):1093–1103.
99. Rauh R, Soell D, Haerteis S, et al. A mutation in the β-subunit of
ENaC identified in a patient with cystic fibrosis-like symptoms has a
gain-of-function effect. Am J Physiology-Lung Cell Mol Physiol.
2013;304(1):L43–L55. PubMed PMID: 23087020.
100. Ramos MD, Trujillano D, Olivar R, et al. Extensive sequence analysis
of CFTR, SCNN1A, SCNN1B, SCNN1G and SERPINA1 suggests an
oligogenic basis for cystic fibrosis-like phenotypes. Clin Genet.
2013;86(1):91–95.
101. Agrawal PB, Wang R, Li HL, et al. The Epithelial Sodium Channel Is
a Modifier of the Long-Term Nonprogressive Phenotype
Associated with F508del CFTR Mutations. Am J Respir Cell Mol
Biol. 2017;57(6):711–720. PubMed PMID: 28708422.
102. Saferali A, Obeidat M, J-C B, et al. Polymorphisms Associated with
Expression of BPIFA1/BPIFB1 and Lung Disease Severity in Cystic
Fibrosis. Am J Respir Cell Mol Biol. 2015;53(5):607–614.
103. Snouwaert JN, Brigman KK, Latour AM, et al. An Animal Model for Cystic
Fibrosis Made by Gene Targeting. Science. 1992;257(5073):1083–1088.
104. Tarran R, Grubb BR, Parsons D, et al. The CF Salt Controversy: in Vivo
Observations and Therapeutic Approaches. Mol Cell. 2001;8(1):149–158.
105. Grubb BR, Vick RN, Boucher RC. Hyperabsorption of Na+ and raised
Ca(2+)-mediated Cl- secretion in nasal epithelia of CF mice. Am J
Physiology-Cell Physiol. 1994;266(5):C1478–C1483. PubMed PMID:
7515571.
106. Mall M, Grubb BR, Harkema JR, et al. Increased airway epithelial Na
+ absorption produces cystic fibrosis-like lung disease in mice [].
EXPERT OPINION ON THERAPEUTIC TARGETS 699
Nat Med. 2004Apr;10(5):487–493. http://www.nature.com/nm/jour
nal/v10/n5/suppinfo/nm1028_S1.html.
107. Cragoe EJ, Woltersdorf OW, Bicking JB, et al. Pyrazine Diuretics. II.
N-Amidino-3-amino-5-substituted 6-Halopyrazinecarboxamides. J
Med Chem. 1967;10(1):66–75.
108. Schmitt R, Ellison DH, Farman N, et al. Developmental expression of
sodium entry pathways in rat nephron. Am J Physiology-Renal
Physiol. 1999;276(3):F367–F381.
109. Loffing J, Zecevic M, Féraille E, et al. Aldosterone induces rapid
apical translocation of ENaC in early portion of renal collecting
system: possible role of SGK. Am J Physiology-Renal Physiol.
2001;280(4):F675–F682.
110. Rengo F, Trimarco B, Bonaduce D, et al. Potassium sparing effect of
amiloride in patients receiving diuretics: a quantitative study. Acta
Cardiol. 1979;34(4):259–267. PubMed PMID: 315689; eng.
• Development of amiloride for treatment of hypokalemia.
111. Em A, King M, Helfesrieder R, et al. Acute and Long-term Amiloride
Inhalation in Cystic Fibrosis Lung Disease: A Rational Approach to
Cystic Fibrosis Therapy. Am Rev Respir Dis. 1990;141(3):605–612.
112. Tg Dk O, Hodsman P, Ansede JH, et al. Acute Hyperkalemia
Associated with Inhalation of a Potent ENaC Antagonist: phase 1
Trial of GS-9411. J Aerosol Med Pulm Drug Deliv. 2014;27:3.
113. Wilcox CS. New Insights into Diuretic Use in Patients with Chronic
Renal Disease. J Am Soc Nephrol. 2002;13(3):798–805.
114. Matsui H, Randell SH, Peretti SW, et al. Coordinated clearance of
periciliary liquid and mucus from airway surfaces. J Clin Invest.
1998 Sep 15;102(6):1125–1131. .
115. Köhler D, App E, Schmitz-Schumann M, et al. Inhalation of amilor-
ide improves the mucociliary and the cough clearance in patients
with cystic fibroses. Eur J Respir Dis Suppl. 1986;146:319–326.
PubMed PMID: 3465558; eng.
•• Use of amiloride increased mucociliary clearance rates in CF
patients.
116. Knowles MR, Church NL, Waltner WE, et al. A Pilot Study of
Aerosolized Amiloride for the Treatment of Lung Disease in Cystic
Fibrosis. New England J Med. 1990;322(17):1189–1194.
117. Scheffer GL, Pijnenborg ACLM, Smit EF, et al. Multidrug resistance
related molecules in human and murine lung. J Clin Pathol. 2002
Dec 12;55(5):332–339. PubMed PMID: PMC1769658.
118. Bahadduri PM, D’Souza VM, Pinsonneault JK, et al. Functional
Characterization of the Peptide Transporter PEPT2 in Primary
Cultures of Human Upper Airway Epithelium. American Journal of
Respiratory Cell and Molecular. Biology. 2005;32(4):319–325.
119. Hirsh AJ, Zhang J, Zamurs A, et al. Pharmacological Properties of
N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-4-[4-(2,3-dihydrox-
ypropoxy)phenyl]butyl-guanidine Methanesulfonate (552-02), a
Novel Epithelial Sodium Channel Blocker with Potential Clinical
Efficacy for Cystic Fibrosis Lung Disease. J Pharmacol Exp Ther.
2008;325(1):77–88.
120. Zhou Z, Treis D, Schubert SC, et al. Preventive but Not Late
Amiloride Therapy Reduces Morbidity and Mortality of Lung
Disease in βENaC-overexpressing Mice. Am J Respir Crit Care
Med. 2008;178(12):1245–1256. PubMed PMID: 18849497.
121. Thelin WD, Ansede K, Johnson J. Poster Session Abstract 201. The
ENaC inhibitor P-1037 is CFTR Independent Therapeutic Agent That
Promotes Sustained Airways Hydration and Mucociliary Transport.
Pediatr Pulmonol. 2015;50(S41):S193–S453.
122. Abm Å, Hemmerling M, Root J, et al. Linking increased airway
hydration, ciliary beating, and mucociliary clearance through
ENaC inhibition. Am J Physiology-Lung Cell Mol Physiol.
2014;308(1):L22–L32.
123. Fortinberry H, Birket S, Astrand A, et al. ENaC Inhibitor AZD5634
Augments Airway Surface Liquid and Mucociliary Transport in
Primary Cystic Fibrosis Airway Cells. C80-A. EVERYTHING YOU
NEED TO KNOW ABOUT BRONCHOPULMONARY DYSPLASIA,
CYSTIC FIBROSIS AND DIFFUSE PARENCHYMAL LUNG DISEASES.
A6466–A6466.
124. Gardiner P, Malmgren A, Ersdal E, et al. ENaC Inhibitor AZD5634
First in Human Trial Reveals Promising Clinical Profile for the
Treatment of Cystic Fibrosis. D94. Advances in cystic fibrosis and
700 P. J. MOORE AND R. TARRAN
non-cystic fibrosis bronchiectasis. American Thoracic Society
International Conference Abstracts: American Thoracic Society.
2017. A7306–A7306.
125. Coote K, Hc A-W, Sugar R, et al. Camostat Attenuates Airway
Epithelial Sodium Channel Function in Vivo through the
Inhibition of a Channel-Activating Protease. J Pharmacol Exp Ther.
2009;329(2):764–774.
126. Ornatowski W, Poschet JF, Perkett E, et al. Elevated furin levels in
human cystic fibrosis cells result in hypersusceptibility to exotoxin A–
induced cytotoxicity. J Clin Invest. 2007 Nov 01;117(11):3489–3497. .
127. Tan CD, Hobbs C, Sameni M, et al. Cathepsin B contributes to Na+
hyperabsorption in cystic fibrosis airway epithelial cultures. J
Physiol. 2014;592(23):5251–5268.
128. Tong Z, Illek B, Vj B, et al. Prostasin, a membrane-anchored serine
peptidase, regulates sodium currents in JME/CF15 cells, a cystic
fibrosis airway epithelial cell line. Am J Physiology-Lung Cell Mol
Physiol. 2004;287(5):L928–L935. PubMed PMID: 15246975.
129. Reihill JA, Walker B, Hamilton RA, et al. Inhibition of Protease–
epithelial Sodium Channel Signaling Improves Mucociliary
Function in Cystic Fibrosis Airways. Am J Respir Crit Care Med.
2016;194(6):701–710. PubMed PMID: 27014936.
130. Elborn JS, Perrett J, Forsman-Semb K, et al. Efficacy, safety and
effect on biomarkers of AZD9668 in cystic fibrosis. Eur Respir J.
2012 Oct;40(4):969–976. PubMed PMID: 22267768; eng.
131. Martin SL, Moffitt KL, McDowell A, et al. Association of airway
cathepsin B and S with inflammation in cystic fibrosis. Pediatr
Pulmonol. 2010 Sep;45(9):860–868. PubMed PMID: 20632407; eng.
132. Rowe SM, Reeves G, Hathorne H, et al. Reduced Sodium Transport
With Nasal Administration of the Prostasin Inhibitor Camostat in
Subjects With Cystic Fibrosis. Chest. 2013;144(1):200–207.
133. Dias N, Stein CA. Antisense Oligonucleotides: basic Concepts and
Mechanisms. Mol Cancer Ther. 2002;1(5):347.
134. Loke SL, Stein CA, Zhang XH, et al. Characterization of oligonucleo-
tide transport into living cells. Proc Natl Acad Sci U S A. 1989;86
(10):3474–3478. PubMed PMID: PMC287160.
135. Vlassov VV, Blakireva LA, Yakubov LA. Transport of oligonucleotides
across natural and model membranes. Biochimica Et Biophysica
Acta (BBA) - Reviews on Biomembranes. 1994;1197(2):95–108.
136. Crosby JR, Zhao C, Jiang C, et al. Inhaled ENaC antisense oligonu-
cleotide ameliorates cystic fibrosis-like lung disease in mice. J Cyst
Fibros. 2017;16(6):671–680.
137. Grubb BR, Pickles RJ, Ye H, et al. Inefficient gene transfer by
adenovirus vector to cystic fibrosis airway epithelia of mice and
humans. Nature. 1994;371:802. .
138. Yang Y, Su Q, Wilson JM. Role of viral antigens in destructive
cellular immune responses to adenovirus vector-transduced cells
in mouse lungs. J Virol. 1996;70(10):7209–7212.
139. Terryah ST, Fellner RC, Ahmad S, et al. Evaluation of a SPLUNC1-
Derived Peptide for the Treatment of Cystic Fibrosis Lung Disease.
Am J Physiol Lung Cell Mol Physiol. 2017. DOI:10.1152/
ajplung.00546.2016.
140. Kesimer M, Sheehan JK. Analyzing the functions of large glycocon-
jugates through the dissipative properties of their absorbed layers
using the gel-forming mucin MUC5B as an example. Glycobiology.
2008;18(6):463–472.
141. Scott DW, Walker MP, Sesma J, et al. SPX-101 is a Novel ENaC-
targeted Therapeutic for Cystic Fibrosis that Restores Mucus
Transport. Am J Respir Crit Care Med. 2017..
•• This study demonstrated that SPX-101 significantly improves
ASL rehydration and mucociliary clearance rates
142. Walker MP, Cowlen M, Christensen D, et al. Nonclinical safety
assessment of SPX-101, a novel peptide promoter of epithelial
sodium channel internalization for the treatment of cystic fibrosis.
Inhal Toxicol. 2017;29(8):356–365.
143. Wheeler A, Schaberg A, Scott D, et al. Safety and Pharmacokinetics of
SPX-101 in Healthy Human Subjects. B103. CLINICAL STUDIES IN
BRONCHIECTASIS, IMMUNODEFICIENCY, AND DRUG INDUCED
LUNG DISEASE. American Thoracic Society International Conference
Abstracts: American Thoracic Society. 2017. A4736–A4736.
144. Moore PJ, Reidel B, Ghosh A, et al. Cigarette smoke modifies and
inactivates SPLUNC1, leading to airway dehydration. FASEB J. 2018;
fj.201800345R. DOI:10.1096/fj.201800345R.
145. Clunes LA, Davies CM, Coakley RD, et al. Cigarette smoke exposure
induces CFTR internalization and insolubility, leading to airway
surface liquid dehydration. FASEB J. 2012 Jul 19;26(2):533–545.
PubMed PMID: PMC3290447.
146. Shak S, Capon DJ, Hellmiss R, et al. Recombinant human DNase I
reduces the viscosity of cystic fibrosis sputum. Proc Natl Acad Sci.
1990;87(23):9188.
147. Fuchs HJ, Borowitz DS, Christiansen DH, et al. Effect of Aerosolized
Recombinant Human DNase on Exacerbations of Respiratory
Symptoms and on Pulmonary Function in Patients with Cystic
Fibrosis. New England J Med. 1994 Sep 08;331(10):637–642.
148. Quan JM, Tiddens HAWM, Sy JP, et al. A two-year randomized,
placebo-controlled trial of dornase alfa in young patients with
cystic fibrosis with mild lung function abnormalities. J Pediatr.
2001;139(6):813–820.
149. Hodson ME, McKenzie S, Harms HK, et al. Dornase alfa in the
treatment of cystic fibrosis in Europe: A report from the
Epidemiologic Registry of Cystic Fibrosis. Pediatr Pulmonol.
2003;36(5):427–432.
150. Shah PL, Scott SF, Geddes DM, et al. Two years experience with
recombinant Human DNase I in the treatment of pulmonary dis-
ease in cystic fibrosis. Respir Med. 1995;89(7):499–502.
151. Davies J, Trinda De M- T, Wallis C, et al. Retrospective review of the
effects of rhDNase in children with cystic fibrosis. Pediatr Pulmonol.
1998. [cited 1995 Aug 01];23(4):243–248. AID-PPUL1>3.0.CO;2-N.
152. Bollert FG, Paton JY, Marshall TG, et al. Recombinant DNase in
cystic fibrosis: a protocol for targeted introduction through n-of-1
trials. Scottish Cystic Fibrosis Group. Eur Respir J. 1999;13(1):107.
153. Van Der Giessen Lianne J, De Jongste Johan C, Gosselink R, et al.
RhDNase before airway clearance therapy improves airway patency
in children with CF. Pediatr Pulmonol. 2007;42(7):624–630.
154. Wilson Christine J, Robbins Lisel J, Murphy Jennifer M, et al. Is a longer
time interval between recombinant human deoxyribonuclease (dor-
nase alfa) and chest physiotherapy better?: A multi-center, rando-
mized crossover trial. Pediatr Pulmonol. 2007;42(12):1110–1116.
155. De Boeck K, Haarman E, Hull J, et al. Inhaled dry powder mannitol
in children with cystic fibrosis: A randomised efficacy and safety
trial. J Cyst Fibros. 2017;16(3):380–387.
156. Donaldson SH, Bennett WD, Zeman KL, et al. Mucus Clearance and
Lung Function in Cystic Fibrosis with Hypertonic Saline. New
England J Med. 2006;354(3):241–250. PubMed PMID: 16421365.
157. Daviskas E, Anderson SD, Brannan JD, et al. Inhalation of dry-
powder mannitol increases mucociliary clearance. Eur Respir J.
1997;10(11):2449.
158. Bilton D, Robinson P, Cooper P, et al. Inhaled dry powder mannitol in
cystic fibrosis: an efficacy and safety study. Eur Respir J. 2011;38(5):1071.
• Pooled analysis of two Phase III trials demonstrated mannitol
significantly improved FEV1 and observed a decrease in pul-
monary exacerbations.
159. Aitken ML, Bellon G, De Boeck K, et al. Long-Term Inhaled Dry
Powder Mannitol in Cystic Fibrosis. Am J Respir Crit Care Med.
2012;185(6):645–652.
160. Goralski JL, Wu D, Thelin WR, et al. The in vitro effect of nebulised
hypertonic saline on human bronchial epithelium. Eur Respir J.
2017;51(5). DOI:10.1183/13993003.02652.
161. Tarran R, Button B, Picher M, et al. Normal and Cystic Fibrosis
Airway Surface Liquid Homeostasis: THE EFFECTS OF PHASIC
SHEAR STRESS AND VIRAL INFECTIONS. J Biol Chem. 2005 Aug
08;280(42):35751–35759. PubMed PMID: PMC2924153.
162. Van Goor F, Hadida S, Grootenhuis PDJ, et al. Rescue of CF airway
epithelial cell function in vitro by a CFTR potentiator, VX-770. Proc
Natl Acad Sci. 2009;106(44):18825.
163. Van Goor F, Hadida S, Grootenhuis PDJ, et al. Correction of the
F508del-CFTR protein processing defect in vitro by the investi-
gational drug VX-809. Proc Natl Acad Sci U S A. 2011 Oct
05;108(46):18843–18848. PubMed PMID: PMC3219147.

164. Ramsey BW, Davies J, McElvaney NG, et al. A CFTR Potentiator in
Patients with Cystic Fibrosis and the G551D Mutation. New
England J Med. 2011;365(18):1663–1672. PubMed PMID:
22047557.
•• Landmark study showing that VX-770 significantly improves
lung function in CF patients with G551D mutation.
165. Hisert KB, Heltshe SL, Pope C, et al. Restoring Cystic Fibrosis
Transmembrane Conductance Regulator Function Reduces Airway
Bacteria and Inflammation in People with Cystic Fibrosis and
Chronic Lung Infections. American. J Respir Crit Care Med.
2017;195(12):1617–1628.
166. Li M, McCann JD, Liedtket CM, et al. Cyclic AMP-dependent protein
kinase opens chloride channels in normal but not cystic fibrosis
airway epithelium. Nature. 1988;331:358. .
167. Cheng SH, Gregory RJ, Marshall J, et al. Defective intracellular
transport and processing of CFTR is the molecular basis of most
cystic fibrosis. Cell. 1990;63(4):827–834.
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168. Sheppard DN, Rich DP, Ostedgaard LS, et al. Mutations in CFTR
associated with mild-disease-form CI- channels with altered pore
properties. Nature. 1993;362:160. .
169. Haardt M, Benharouga M, Lechardeur D, et al. C-terminal Truncations
Destabilize the Cystic Fibrosis Transmembrane Conductance
Regulator without Impairing Its Biogenesis: A NOVEL CLASS OF
MUTATION. J Biol Chem. 1999;274(31):21873–21877.
170. Boucher RC. Human airway ion transport. Part one. Am J Respir Crit
Care Med. 1994;150(1):271–281.
171. Watson MS, Cutting GR, Desnick RJ, et al. Cystic fibrosis population
carrier screening: 2004 revision of American College of Medical
Genetics mutation panel. Genet Med. 2004 Sep-Oct;6(5):387–391.
PubMed PMID: PMC3110945.
172. Xue X, Mutyam V, Tang L, et al. Synthetic Aminoglycosides
Efficiently Suppress Cystic Fibrosis Transmembrane Conductance
Regulator Nonsense Mutations and Are Enhanced by Ivacaftor. Am
J Respir Cell Mol Biol. 2013;50(4):805–816.