Perspective

pubs.acs.org/jmc

Ion Channels as Therapeutic Targets: A Drug Discovery Perspective

Sharan K. Bagal,† Alan D. Brown,† Peter J. Cox,‡ Kiyoyuki Omoto,† Robert M. Owen,† David C. Pryde,*,†
Benjamin Sidders,‡ Sarah E. Skerratt,† Edward B. Stevens,‡ R. Ian Storer,† and Nigel A. Swain†
†
Worldwide Medicinal Chemistry, Pfizer Neusentis, The Portway Building, Granta Park, Great Abington, Cambridge, CB21 6GS,
U.K.
‡
Pfizer Neusentis, The Portway Building, Granta Park, Great Abington, Cambridge, CB21 6GS, U.K.

ABSTRACT: Ion channels are membrane proteins expressed in almost all living
cells. The sequencing of the human genome has identified more than 400 putative
ion channels, but only a fraction of these have been cloned and functionally tested.
The widespread tissue distribution of ion channels, coupled with the plethora of
physiological consequences of their opening and closing, makes ion-channel-
targeted drug discovery highly compelling. However, despite some important
drugs in clinical use today, as a class, ion channels remain underexploited in drug
discovery and many existing drugs are poorly selective with significant toxicities or
suboptimal efficacy. This Perspective seeks to review the ion channel family, its
structural and functional features, and the diseases that are known to be
modulated by members of the family. In particular, we will explore the structure
and properties of known ligands and consider the future prospects for drug
discovery in this challenging but high potential area.

■ INTRODUCTION
Ion Channels as Therapeutic Targets. Ion channels are
benzodiazepine diazepam (5), which binds to the γ-aminobutyric
acid class A (GABAA) channel (Figure 1).
transmembrane proteins that create a gated, water-filled pore to
help establish and control voltage potential across cell
membranes through control of the active flow of ions between
the intracellular and the extracellular environments.1,2 The ion
channel family is intimately involved in almost all aspects of
physiology and plays a critical role in diverse processes such as
nerve and muscle relaxation, cognition, sensory transduction,
regulation of blood pressure, and cell proliferation. Their
modulation has been linked to a wide range of diseases that
include cardiac disorders, neurological indications, kidney failure,
the perception of pain, and blindness. More than 60
“channelopathies” have been identified as human diseases that
are brought about through mutations in ion channels. Given the
central functional role that the ion channel superfamily plays in Figure 1. Example ion channel ligands.
human physiology, its membrane localization, and the diverse
tissue distribution of different members of the family, it
represents an attractive potential target class for drug discovery. An examination of the ion channel-based research landscape
The human genome comprises several hundred genes that reveals many opportunities to exploit the family in a drug
encode for pore-forming ion channels within plasma membranes, discovery context, not least of all due to the surprisingly large
broadly classified as either voltage or ligand gated depending on number of family members for which there have been no ligands
the primary factors that lead to channel opening and closing. identified to date. For a similarly large number of other family
Members of both subfamilies are known to be modulated by a members, some ligands have been found that are weak and/or
range of small molecule agents. For example, the low molecular nonselective, thereby limiting their utility as pharmacological
weight local anesthetics lidocaine (1) and bupivacaine (2) act on tools or as drug discovery starting points.
voltage-gated sodium channels (Nav). The transient receptor This Perspective is intended to offer a broad overview of the
potential (TRP) family recognizes a diverse array of ligand ion channel drug discovery landscape and introduce the reader to
structural types from pungent natural substances like capsaicin the members of the family, their classification, and the range of
(3) (TRP vanilloid 1 channel, TRPV1) and menthol (4) (TRP
melastatin 8 channel, TRPM8). Several well-known pharma- Received: August 3, 2012
ceutical agents act at certain ligand-gated ion channels such as the Published: November 2, 2012

© 2012 American Chemical Society 593 dx.doi.org/10.1021/jm3011433 | J. Med. Chem. 2013, 56, 593−624
Journal of Medicinal Chemistry
diseases that are reported to originate in channel dysfunction. We
review the structural features that underpin ion channel function
and how ion channels are modulated by ligands. Finally we
discuss the recent advances in ligand identification, trends in
their molecular properties, and the prospects for the drug
discovery industry being able to translate interesting ion channel
targets into the next generation of drug therapies.
The scope of this article is, for the most part, limited to the
approximately 200 major pore-forming human ion channels
according to the International Union of Basic and Clinical
Pharmacology (IUPHAR) classification3 and does not include,
for example, the anion channels or other transport proteins such
as the connexins or aquaporins. The interested reader is referred
to several online curated databases of the entire ion channel
family, for example, the Guide to Pharmacology database,4 for
details on these other channels. In some cases, the modulation of
regulatory subunits of channel proteins is described where their
inclusion offers the reader a useful insight into alternative means
of altering channel function beyond direct targeting of the pore-
forming domains.
How Ion Channels Function. In the most basic terms, ion
channels are a collection of protein domains that together create
a water-filled pore to allow the passage of ions through a cell
membrane in response to chemical stimuli, temperature changes,
or mechanical forces.5 These pore-forming domains vary
enormously in sequence and topology but are typically
composed of four or five helices that fit together to form a
barrel-like structure. The helices are made up of repeat domains
of a single subunit or of several different subunits. In order to
fulfill the function of selectively filtering ions through a biological
membrane, most ion channels also possess a pore-loop region
called the ion or selectivity filter that regulates which ions are
permitted through the pore (Figure 2). The channel features a
gating mechanism that controls the opening and closure of the
pore through conformational changes to the channel protein,
combined with a sensor mechanism that detects and responds to
Figure 2. Cartoon representation of the basic structural components of
an ion channel featuring a pore region, ion, or selectivity filter and gate.
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Perspective
stimuli. Further intracellular and extracellular modules provide
binding sites for ligands and accessory proteins, adding further
complexity to the system. Ion channels freely and rapidly sample
a range of states from the open to the closed states on a rapid time
scale, and the modulation of channel function by a ligand or
external trigger manifests in a stabilization of the channel
structure in one particular state.
Ion flux through the pore when in the open state is an
incredibly fast process driven by an electrochemical gradient.
Flux through the channel can be as fast as some 108 ions per
second,2 leaving in the order of nanoseconds for the channel to
interact with each ion, thereby approaching the theoretical limits
described by Ohm’s law and Fick’s law. Despite the brevity of this
contact time, potassium channels allow potassium ions to pass
with a fidelity of approximately 10000:1 versus the smaller
sodium ion6 through a careful spatial arrangement of carbonyl
oxygen atoms within the selectivity filter that can accommodate
two potassium ions in the pore at any one time.7,8 These contacts
compensate for the energy penalty of desolvating ions as they
enter the filter by providing favorable compensatory interactions
with the ion. Depending on the identity of the ion, this energetic
compensation varies relative to the energy of desolvation,
resulting in an energy-driven selectivity.9 From the perspective of
a single channel recording, the trace shown in Figure 3 depicts
Figure 3. An example single channel recording of an ion channel that
cycles between the open and closed state in response to a current gating
trigger applied repeatedly.
the gating of a single ion channel that starts at a resting potential
in the closed state, and at the time points indicated by the arrows,
a change in membrane potential creates a series of conforma-
tional changes in the protein that open the channel for a time
before it closes again.
This simple gating underpins all of the physiological
consequences that are controlled by ion channels, but the
detailed elucidation of precisely how channels gate is only just
beginning to emerge. Ion channels have been discovered across
species, and in particular the prokaryotic bacterial channels have
been extremely useful to the structural biology community in
providing research tools for high resolution X-ray crystallography
and understanding of how channels function.10 More recently
there has been significant progress in the elucidation of
mammalian structures of ion channels, most notably in the
voltage-gated family of potassium channels by crystallogra-
phy11,12 and nuclear magnetic resonance13 methods. Further
discussion of our structural understanding of ion channels is
presented later.
■ PHARMACOLOGY/DISEASES
Ion channels are expressed in all tissues and cell types in the
human body, so it is unsurprising that these proteins cause, and
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are modulated by, many diseases. The availability of selective and Table 1. Selected Channelopathies Arising through Channel
nonselective pharmacological tools and the ability to genetically Function Impairmenta
manipulate channels in situ are increasing our knowledge of their
ion channel
function and provide indirect evidence of contributions of ion family channel G/L disease
channels to normal function and the pathophysiology of disease.
Kir Kir1.1 L Bartter’s syndrome
The most compelling evidence for a role in disease has come
Kir2.1 L Andersen’s Syndrome
from studies of the inheritance of relatively rare monogenic
Kir6.2 L congenital hyperinsulinism
diseases caused by mutations that affect the function of specific
G neonatal diabetes
ion channels.14 Loss and gain of function mutations in the voltage
SUR2 L dilated cardiomyopathy
gated sodium channel Nav1.7 cause profound pain insensitivity15
Kv Kv1.1 L episodic ataxia type 1
and sensitivity,16 respectively, in carriers of these mutations. A
KCNQ1 L long QT syndrome
number of Nav1.7 blockers have now entered clinical develop- G short QT syndrome
ment,17 and it will be fascinating to learn if small molecule KCNQ2 L benign neonatal febrile convulsions
exogenous channel blockers can mimic the insensitivity-to-pain KCNQ4 L nonsyndromic deafness
state of the individuals that carry the genetic loss of function hERG L long QT syndrome
mutations. A loss of function mutation in the gene that encodes G short QT syndrome
the twin pore potassium (K2P) channel TWIK-related spinal TRP TRPP2 polycystic kidney disease
cord potassium channel (TRESK) renders the channel nonfunc- TRPA1 G familial episodic pain syndrome
tional and has recently been linked to familial migraine.26 There TRPC6 G focal segmental glomerulosclerosis
is extensive evidence for mutations in voltage gated calcium CNG CNGA1 L retinitis pigmentosa
channels (Cav) affecting central functions that give rise to KCa BK G epilepsy
conditions such as ataxia and epilepsy. These are a few examples Nav Nav1.1 G epilepsy
of disease associations that exist in every ion channel subfamily.18 L severe myoclonic epilepsy
Just some of the myriad diseases and disorders that have been Nav1.5 G long QT syndrome
linked to ion channels include Cav channels and retinal disease,19 Nav1.6 L cerebellar ataxia
Nav channels and epilepsy, cardiac arrhythmias and pain,20 Kv Nav1.7 G erythromelalgia, paroxysmal extreme
channels and seizures,21 voltage gated potassium channel, pain disorder
subfamily Q (KCNQ) channels and deafness and epilepsy,22 L congenital indifference to pain
TRP polycystic (TRPP) channels and renal cysts,23 inward Nav2.1 G benign familial neonatal seizures
rectifier potassium (Kir) channels and kidney transport and Cav Cav1.2 G timothy syndrome
hypoglycaemia,24 TRP mucolipin (TRPML) channels and Cav2.1 L episodic ataxia type 2
Niemann−Pick disease,25 and K2P channels and migraine and glycine GLRA1 L stiff baby syndrome
cardiac arrhythmias.26 Table 1 presents a summary selection of receptors
just some of these “channelopathies”, human diseases that are GABA GABAA L juvenile myoclonic epilepsy
brought about through mutations to specific ion channels.27 AChR CHRNA4 L autosomal dominant nocturnal frontal
lobe epilepsy
Large scale population genetic studies, focusing on common a
variants of ion channels, are also revealing new and unexpected G/L: gain or loss of function. Adapted from Ashcroft.27b
functions28 that would not have been predicted based on current
knowledge of their function and distribution. For example, four
large scale genome wide association studies have linked a
common single nucleotide polymorphism (SNP) in the Nav1.8
gene with a small increase in human cardiac conduction velocity,
yet this channel was considered to be expressed specifically in
pain sensing neurones.29 A functional effect of this SNP on
Nav1.8 biophysical properties has not been demonstrated.
Intensive sequencing efforts are also revealing that the repertoire
of ion channel variants that an individual expresses may be an
important determinant of health.
Relatively nonspecific ion channel modulators are a potential
source of additional information on ion channel function;
however, it can be difficult to draw firm conclusions because of
poorly characterized polypharmacology. As more selective ion
channel drugs become available, their use in the general human
population is likely to provide clearer insight into the functional Figure 4. Relative size of the ion channel and other protein families.
roles that ion channels play, in addition to providing better drugs
for the treatment of diseases with ion channel etiology.

■
A phylogenetic tree of the ion channel superfamily arranged by
voltage or ligand gated families is shown in Figure 5. The largest
PHYLOGENY branch of the family covers the voltage gated Kv potassium
In the human genome, the pore-forming ion channel family is channels, and overall the potassium channels form the largest
larger than the nuclear hormone receptor family, approximately proportion of the whole ion channel proteome. There is also a
half the size of the kinase and protease families, and accounts for marked difference between members of the same subfamily.
approximately 1% of the estimated 20 000 human protein coding Members of the Kv branch of the family are quite distinct in
genes (Figure 4). sequence and topology to other potassium channels, for example,
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Journal of Medicinal Chemistry
Figure 5. Ion channel phylogenetic tree.
the K2P or the Kir channels. Between different branches of the
tree, proteins are even more dissimilar, for example comparing
the Nav channels to the Cav or Kv subfamilies. Within the ligand-
gated family of proteins, the structural and functional divergence
is even more acute and subfamilies cluster as almost distinct
collections of channels. Perhaps not surprisingly, the most
conserved regions of ion channels tend to cluster around the pore
helices or the selectivity filter regions.
The phylogenetic tree of Figure 5 also demonstrates the sheer
number of members of the family. By surveying all of the patent
and publication landscape for ion channel research, we estimate
that of all of the channels depicted in Figure 5, approximately
40% of the family has no known ligands reported against them.
Of the remaining 60%, many of these are nonselective, reactive,
peptidic or occupy very poor physicochemical space for oral drug
discovery programs. There appear to be several reasons for this.
First, biological reagents have not been made available (or
developed) for many of the family members, making screening
campaigns to identify ligands impossible. In some cases, reagents
are available, but screening efforts have merely identified poor
quality leads, which may be a consequence of many large
compound collections having been built around more traditional
target space, for example, G-protein-coupled receptors
(GPCRs), or screening campaigns having been conducted
using much smaller sets of low structural diversity and
questionable druglikeness. There are parallels to the early stages
of kinase drug discovery in which a small subset of targets
received a large amount of biopharmaceutical industry attention,
which eventually began to bear fruit after significant investment.
Significant attention has been devoted to certain TRP, Nav, and
Cav channels which has yielded more potent, selective, and
druglike compounds, and it can be anticipated that those
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Perspective
additional targets with a compelling biology will stimulate
increased interest from the biomedical research community to
ultimately identify better ligands.
The vast majority of mammalian ion channels appear to have
had their origins in prokaryotes30 but have undergone substantial
evolution to produce the eukaryotic channels apparent today.
This is perhaps driven by an increasing need for neuronal
signaling or an evolved response to external environmental
factors such as temperature or chemosensing. Comparisons of
gene numbers, however, do not indicate an exact correlation with
anatomical complexity, as the crude numbers of channel genes in
humans and nematodes is surprisingly similar.31
■ SCREENING
Ion channel screening assays can be broadly divided into ligand
binding, ion flux assays, fluorescence readouts, flash lumines-
cence assays, and automated electrophysiology.
High-throughput ion channel binding assays have been
configured for filter binding assays, scintillation proximity assays,
and fluorescence polarization assays.32 The technology is high-
throughput and low cost and has been adapted to a range of ion
channels, for example, N-methyl-D-aspartate (NMDA) subtype
glutamate receptors and Kv11.1.33 However, the technology is
limited by availability of a suitable high-affinity ligand, provides
no functional information, and only explores a single binding site.
Functional cell based assays using ion flux, fluorescent dye
assays, or flash luminescence assays report mechanistic effects of
compounds, identifying activators, inhibitors, and allosteric
modulators. Ion flux assays use a radioactive species in
combination with a scintillation counter (e.g., 86Rb for potassium
channels and 14C-guanidinium for sodium channels)34 or use a
tracer ion measured by atomic absorption/emission spectrom-
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etry (e.g., Rb+ for potassium channels and Li+ for sodium Table 2. Some Common Ion Channel-Targeting Drugsa
channels). Radiometric flux assays are hindered by safety and
target year of first clinical
disposal challenges, while nonradiometric flux assays are limited drug channel disease target usage
by lengthy read times of atomic absorption spectrometers. Cell
verapamil L-type Cav hypertension 1982
based assays using fluorometric dyes have been developed that diltiazem L-type Cav hypertension 1982
either respond directly to changes in concentration of the ions amlodipine L-type Cav hypertension 1990
(e.g., calcium, halide, and thallium dyes) or indirectly monitor nifedipine L-type Cav hypertension 1977
ion channel activity by measuring their contribution to cell gabapentin Cav (α2δ) pain 1994
membrane potential using voltage-sensitive dyes (e.g., sodium pregabalin Cav (α2δ) pain 2004
channels, potassium channels, and GABAA).35 Next generation sotalol hERG arrhythmia 1992
fluorescence plate readers with built-in field stimulation flecainide Nav1.5 arrhythmia 1982
capabilities, suitable for activating voltage-gated ion channels, ziconotide Cav2.2 severe pain 2004
avoid pharmacological methods to open ion channels (e.g., lidocaine Nav local anesthetic 1949
scorpion toxins and veratrine for activating sodium channels) bupivacaine Nav local anesthetic 1987
and potentially provide better correlation with gold-standard lamotrigine Nav epilepsy, bipolar 1994
manual patch clamp data.36 Luminescence assays use bio- riluzole Nav amyotrophic lateral 1995
luminescent calcium-sensitive photoprotein (e.g., aequorin or sclerosis
mitochondria-targeted MitoPhotina) as a reporter protein that is phenytoin Nav epilepsy 1953
co-transfected with the target ion channel. Flash luminescence lacosamide Nav seizures and pain 2008
technology can provide lower cost and higher assay performance carbamazepine Nav epilepsy 1963
compared to fluorescence methods and has been adapted to a varenicline nAChR smoking cessation 2006
range of channels permeating calcium ions (e.g., Cav channels flupirtine KCNQ2/3 epilepsy 1984
and purinoreceptor (P2X) channels).37 retigabine KCNQ2/3 epilepsy 2011
Automated electrophysiology provides the ideal platform for diazepam GABAA depression 1963
a
supporting ion channel programs because of the high data quality Adapted from Clare.40
achievable. Currently the technology is based around two core
technologies.38 Planar patch is where a hole is fabricated in a chip discovered in the 1950s and progresses to a modern drug
in the horizontal plane and requires compound addition through discovery example that was launched within the past few years.
a robot arm, while lateral patch recordings involve holes Carbamazepine (6) is a first generation anticonvulsant
fabricated in the side of a microchannel. Current planar patch developed by J. R. Geigy (now Novartis) in the 1950s for the
clamp technology such as QPatch, PatchXpress, and Patchliner/ treatment of epilepsy, trigeminal neuralgia, and mania.41 In the
Synchropatch which provide high-quality gigaseal resistance early era of drug discovery when carbamazepine was first
recordings are ideal platforms for developing structure−activity launched, drugs were often discovered using empirical means,
relationships or screening focused libraries. Higher throughput often in isolated tissue preparations or in vivo efficacy models
screening (10−50000 compound screening libraries) can be designed to mimic some component of the clinical condition
performed using a loose-seal recording configuration from being targeted. The definitive characterization of the molecular
multiple cells in parallel (population patch clamp) using target of carbamazepine as voltage-gated sodium channels came
Ionworks Quattro/Barracuda planar patch clamp technologies.39 much later.42 Carbamazepine inhibits sustained repetitive firing
Recent developments in lateral patch technology giving rise to by blocking use-dependent sodium channels. Pain relief is
machines with a small footprint and a lower cost chip believed to be a consequence of blockade of synaptic
consumable unit are likely to lead to a higher throughput transmission in the trigeminal nucleus, and seizure control is
technology with lower costs. associated with reduction of potentiation of synaptic trans-

■
ION CHANNEL DRUGS
A cursory glance over approved drugs would suggest that the ion
channel family has yielded a significant number of important
drugs across several subfamilies. Table 2 summarizes some
common ion channel drugs and the indications for which they
were approved. Upon closer inspection, the number of discrete
channels that has been successfully drugged is relatively small,
and drugs for cardiac, neurophysiological, or local anesthetic
application are most common. It is also notable how few drugs
have gained approval in the past decade, a likely consequence of
clinical failures due to efficacy and safety issues and the apparent
difficulty in identifying clinical candidate quality chemical matter
across the ion channel family.
Many of the compounds depicted above were discovered over
a decade ago, with most possessing low levels of selectivity. To
illustrate how ion channel drug discovery has evolved through
the decades, we have chosen three examples to illustrate an
increasing level of sophistication in the ability to identify potent
and selective ligands. This starts with a very early example
597
mission in the spinal cord. Also somewhat characteristic of ion
channel modulators identified in this early era of drug discovery
was a general lack of ion channel selectivity. Carbamazepine is
known to block sodium channels, calcium channels, and GABA
receptors at high micromolar levels of potency. This pan-ion-
channel inhibition profile is likely reflected in carbamazepines'
broad pharmacological properties that include anticholinergic,
antiarrhythmic, antidepressant, sedative, and neuromuscular-
blocking effects (Figure 6).
Interestingly, the structurally related oxcarbazepine42 (7) was
launched nearly 3 decades later in 1990 by Novartis for the
treatment of epilepsy and partial seizures. It is a prodrug that is
activated to S-(+)-licarbazepine (8) in the liver along with equal
amounts of the less active R-enantiomer. Eslicarbazepine
acetate43 (9) is a more recent prodrug that predominantly
generates the active enantiomer 8 and was launched in 2009 for
epilepsy. In this example, a detailed understanding of molecular
target and metabolite structure led to rationally designed
improvements in safety and tolerability via small structural
changes and/or prodrugs to identify two further drugs of clinical
utility.
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Figure 6. Drugs based on the first generation sodium channel blocker carbamazepine.
Another ion channel drug wherein subsequent drug
generations have shown improvements through pharmacoki-
netic modification is gabapentin. Gabapentin (10) is a lipophilic
GABA analogue developed by Parke-Davis (now Pfizer) over 10
years ago as an adjunctive treatment of partial seizure associated
with epilepsy (Figure 7). It was designed to increase passage
Figure 7. Gabapentin and analogues.
across the blood−brain barrier, which indeed it does, but via
recognition by the system L amino acid transporter. During
patient studies and clinical use, gabapentin was observed to show
efficacy in the treatment of neuropathic pain and postherpetic
neuralgia and subsequently demonstrated efficacy in a number of
in vivo pain models.44 Gabapentin has no effect on GABA
receptors, and the actual mechanism of action involves binding to
a high affinity binding site on the α2δ subunit of voltage gated
calcium channels which reduces calcium currents by impairing
the trafficking function of the subunits.45
DepoMed is currently developing a once-daily, extended-
release tablet formulation of gabapentin using its gastric
retention technology.46 Gabapentin enacarbil (11) was approved
in 2011 by the FDA for the once-daily oral treatment of
moderate-to-severe restless legs syndrome.47 Pregabalin (12) is
another GABA analogue, launched in 2004 by Pfizer for the
treatment of peripheral neuropathic pain, epilepsy, postherpetic
neuralgia, diabetic peripheral neuropathy, generalized anxiety
disorder, and fibromyalgia. All of these agents were designed to
improve the limited and variable absorption rates of gabapentin,
giving distinct pharmacokinetic advantages over the prototype
agent.48
The α2δ example illustrates how a focused medicinal
chemistry and screening effort, along with a carefully executed
clinical trial program, ultimately led to the discovery of an
important class of ion channel targeted drugs. Their develop-
ment was dependent on efficacy readouts in animal models, from
which the pharmacological target of the drug class was eventually
identified.
Varenicline tartrate (14, Chantix) was launched by Pfizer in
2006 for use in smoking cessation and represents the product of a
modern day drug discovery program.49 The drug is specifically
designed to partially activate the nicotinic receptor and reduce
the severity of the smoker’s craving and the withdrawal
symptoms from nicotine. Varenicline was discovered by
modification of a natural product (−)-cytisine49 (13, Figure 8)
that was known to have partial agonist activity at the α4β2
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Perspective
Figure 8. Varenicline and the starting point for its discovery.
nicotinic receptor using a high throughput binding assay to assess
binding to the α4β2 nicotinic receptor along with subtype
selectivity. Functional electrophysiology measurements were
then used to assess the desired partial agonism pharmacology
profile, which was confirmed through several in vivo models that
evaluated the ability of varenicline to alter the central
dopaminergic response to nicotine.
This approach was rather more typical of a modern drug
discovery effort, in which high throughput targeted screening was
used to advance structure−activity relationships and lower
throughput functional assays were then used to hone in on the
desired pharmacology profile.
■
ANESTHETICS
For over a century, local anesthetics such as novocaine have been
used to locally block sensory perception and thereby allow
surgical procedures to be carried out on peripheral tissues, to
reverse acute pain, or to treat chronic pain.50 These local
anesthetics are weak affinity amphipathic molecules that are
administered at relatively high doses to primarily block voltage
gated sodium channels through binding of the inner pore region.
Local anesthetics are also known to block some potassium and
calcium channels.51 Later generations of anesthetics including
lidocaine (1) and bupivacaine (2) offered duration of sensory
block or safety advantages over the earlier agents. It is the safety
aspects of these agents (central nervous system (CNS) effects
such as tinnitus and faintness or cardiac effects secondary to
respiratory depression are among the more common), coupled
with their weak, nonselective action at ion channels that limit
their use.
■
VENOMS, TOXINS, AND BIOLOGICS
The venoms of various snakes, scorpions, spiders, insects and
sea-dwelling animals are largely mixtures of neurotoxic peptides
that these animals have evolved for prey capture or as a defense
against predators. They act, often with exquisite potency and
high selectivity at Kv, Cav, and Nav ion channels and some ligand-
gated ion channels (e.g., nicotinic acetylcholine receptors), to
take advantage of the neuronal and muscle contractility effects of
channel modulation.52 Optimized versions of some of the huge
number of venomous peptides and toxins are being pursued by
researchers,53 while mixtures of fractionated venoms or
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individual toxins are available commercially as research tools
from several sources. Some common toxins are shown in Figure 9
Figure 9. Some common toxins. For the structure of protoxin 1, see the
Protein Data Bank, structure code 1KVD.
that can be both small molecule, for example, saxitoxin (15) and
tetrodotoxin (16), or small peptides, ranging from 8 to 70 amino
acids such as protoxin 1 (17). These peptides are highly compact
and are mainly stabilized by disulfide bonds, although some are
stabilized by hydrogen bonds made from post-translation
modified amino acids.
An alternative modality of targeting ion channels has been the
use of functional antibodies. Typically, ligand-gated ion channels
have been more susceptible to this approach, perhaps because of
smaller and less defined binding sites on the extracellular
domains of voltage-gated channels. While research into this
approach is making progress and could provide important
alternatives to small molecule approaches,54 it will not be covered
any further in this article.
Figure 10. Ion channels in clinical development from the ligand and voltage gated families, broken down by decade and ion channel subfamily.
599
■
Perspective
ION CHANNEL MODULATORS IN CLINICAL
DEVELOPMENT
Analysis of drugs that modulate ion channels and have reached
clinical development reveals some interesting trends. Figure 10
shows the classes of drug targets that have been pursued
according to the decade of their first disclosure in the patent
literature. In particular, both families show large spikes in activity
over the past 5 decades for certain classes of channels. For ligand-
gated targets, this occurred between 1980 and 2000 and can be
largely attributed to a significant industry effort targeting the
glutamate, nicotinic, and serotonin receptor subtype 3 (5-HT3)
channels, as described in the sections below. For voltage gated
channels this increase in activity occurred between 1980 and
1990 and corresponds to increased activity in the areas of calcium
channels blockers such as the dihydropyridine class of
antagonists and α2δ modulators, as well as an increased interest
in potassium channel modulators. While there has been an
overall drop in clinical activity around ion channels, interest in
ion channel targets has remained strong with a clear increase in
the past decade (2000−2010) in the areas of the P2X, TRP, and
sodium channel research.
Of the 606 compounds contained in this analysis, 28% have
been successfully launched as drugs with roughly an equal
proportion of launches between the ligand and voltage-gated
families (26% and 29%, respectively). In the context of drug
approvals, the increases in activity for certain classes of targets
mentioned previously did not necessarily lead to an increase in
approved drugs. For example, from the 1970s to the 1980s, the
number of calcium channel modulators increased from 19 to 69
compounds in clinical development (Figure 11). However
despite this increase, the percentage of approved drugs targeting
calcium channels fell significantly in the same period, from 65%
to 22%. In addition, this latter value is closer to the percentage of
approved drugs that targeted calcium channels in the 1990s
(13%), during which time only 16 compounds reached the clinic.
Although a wide range of reasons could have contributed to this
trend, it does point to the fact that increased clinical activity in the
field does not necessarily translate to an increased success rate for
drug launch.
These data also demonstrate an increase in focus toward more
selective agents entering clinical trials (Figure 12). For both
ligand and voltage gated targets, the most recent decade has
shown a large increase in clinical compounds that target single
channel subtypes within a family (for example, selective Nav1.7
modulators). It is worth noting that for ligand gated channels the
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Figure 11. Translating clinical activity into launched products. Compounds marked as “Not Launched” were part of the clinical data set derived from
Prous but were not flagged with a highest phase of “Launched” in the database.

Figure 12. Pursuit of selective ion channel modulators over time.

decade of 1980−1990 constituted something of an anomaly. Table 3. Physicochemical Space Comparisons across the Ion
During this decade, the industry focused a great deal on 5-HT3 Channel Familiesa
selective modulators. While these receptors are ion channels, the
family (no.) clogP PSA MW HBD rotatable bonds ring count
chemical matter that modulates them shares a great deal of
similarity to compounds that modulate the other members of the voltage (441) 3.7 72 393 1 6 3
5-HT family, which are GPCRs. If those compounds are ligand (311) 2.5 62 333 1 3 3
removed from the analysis, only 2% of the compounds that target rotatable ring
subfamily (no.) clogP PSA MW HBD bonds count
ligand gated receptors can be classed as subtype selective (from
the 29% value shown). This trend appears to have begun in the nicotinic (32) 1.8 34 242 1 1 3
1990s and is consistent with a move away from in vivo/tissue glutamate (99) 2.0 70 309 2 4 3
GABA (112) 2.9 62 346 0 3 3.5
based drug discovery approach as highlighted above in the
5HT (41) 2.4 57 333 1 2 4
carbamazepine example toward programs driven by an increased
ASIC (6) 3.5 55 337 3 3 3.5
understanding of the existence and physiological role of ion
calcium (123) 4.5 92 461 1 9 3
channel subtypes in human disease (Nav1.5 vs Nav1.7, for
nucleotide (7) 3.9 52 334 1 6 3
example) facilitated by the advances in molecular biology and
potassium (129) 2.9 75 335 1 4 3
genetics at this time. The intriguing aspect of the analysis is
P2X (23) 2.9 95 419 3 6 4
whether this change in strategy will be successful.
sodium (118) 3.3 66 365 2 5 3
We also examined these data for trends in physicochemical
TRP (49) 4.5 66 410 1 6 3
properties. To bolster the analysis to include more recent
other (13) 3.7 54 392 0 5 3
compounds that have not yet entered clinical evaluation, we a
included a set of manually curated ion channel ligands drawn The numbers in parentheses denote the number of compounds used
from the literature. Analysis of the physicochemical properties of to generate the corresponding median values. The compounds used
for this analysis were drawn from compounds that target ion channels
both ligand and voltage gated channel ligands showed that the and had progressed to the clinic and from a manually curated set
compounds lay squarely in the middle of druglike chemical space. drawn from the literature and described in the section Methods. All
Interestingly, the ligand gated modulators tended to be smaller, properties were calculated using Pfizer internal software packages.
less lipophillic, more rigid and have reduced polar surface area
(Table 3). This difference in properties may be explained in part
by the fact that a larger number of ligand gated targets are located values of their family, compounds that modulated the nicotinic
in the CNS, which has driven the property space toward receptors tended to be smaller and of lower polar surface area.
compounds that are more freely brain penetrant.55 A more Similarly, calcium channel modulators stood out relative to their
detailed analysis of compound properties at the subfamily level other family members in that they were more lipophilic, larger,
also revealed some interesting trends. Relative to the median contained more rotatable bonds, and were of higher polar surface
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Journal of Medicinal Chemistry
Figure 13. A voltage gated Nav channel and the most common binding sites identified for ligands. The four subunits of the channel are represented in
different colors.
area. Although the reasons behind these differences are unclear, it
does suggest that ion channels cannot be viewed as a
homogeneous population that prefer compounds in a particular
physicochemical space. Instead, like aminergic and peptidic
GPCRs,56 different classes within the ion channel family may be
biased toward compounds in different physiochemical space.
Analysis of compounds that were classified as more subtype
selective did not show any definitive differences in their
physicochemical properties relative to the non-subtype-selective
compounds, which could suggest that obtaining subtype
selectivity has been a direct result of improved screening
methods.
■ ION CHANNEL STRUCTURE AND MODULATION BY
LIGANDS
In an earlier section, we introduced the basic structural features
that allow ion channels to sense environmental factors,
selectively filter ions, and gate. Figure 13 shows an example of
a voltage gated ion channel and highlights the most common
regions that have been reported as supporting ligand binding.
Much of this evidence has been gathered through site directed
mutagenesis, and while there are now several apo crystal
structures of ion channels, to date there are no cocrystal
structures of any ligand bound in a channel.
TTX is known to bind to a site toward the top of the pore
region. Other toxins have been described to bind to other regions
of Nav channels spanning almost all protein domains, including
the pore. The pore module tends to be the most conserved
region of protein across subfamilies, with pore blockers tending
to support the least selectivity. This includes many of the
nonselective drugs shown in Table 2. A hydrophobic fenestration
site in the side of the pore module has also been proposed to
support small molecule binding.10b Local anesthetics bind to a
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Perspective
transmembrane region of the α-subunit of Nav channels, while
several small molecules have been proposed to bind to the
voltage sensor domain.57 Examples of ligands that bind to all of
these sites are described in the sections below.
In the case of voltage gated channels, the large channel protein
comprises four subunits that create a central ion conducting pore
with a constricted selectivity filter at the top, surrounded by four
voltage sensors. The voltage-sensing domain (VSD) is made up
of the S1−S4 segments, which are linked to the S5 and S6
segments that form the pore, and a P-loop joins the segments
together. The VSD responds to membrane depolarization and
initiates opening of the central pore through pulling on the S4−
S5 linker and opening of the gate at the base of the pore through
concerted movements of all four subunits. In this way, the ion
channel moves from one state (for example, closed) to another
(open) through concerted movements and then through to an
inactivated state as the channel ceases to conduct ions. Of course,
ligands will possess a different affinity for each of these states, and
an attractive strategy to achieve functional selectivity is to target a
high affinity activated state of high frequency firing channels
characteristic of disease states but not the low frequency firing of
the normal somatosensory system. Several compounds have
been reported to possess such “state-dependent” properties.
It is also possible to modulate ion channel function through
ligand binding to auxiliary subunits. For example, the mechanism
of action of gabapentin has already been highlighted, which elicits
its analgesic effects through binding to the α2δ accessory subunit
of Cav channels, thereby impairing channel trafficking to the cell
surface.58 Ionotropic glutamate receptors (iGluRs) are known to
interact with transmembrane bound regulatory proteins and
affect receptor trafficking and proper localization at the cell
surface. These regulatory proteins are also known to affect
channel conductance, activation, and densensitization. Other
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post-translational regulation pathways have also been inves-
tigated for iGluRs, for example, the multiple phosphorylation
sites on AMPA receptors mediated by protein kinase C and
protein kinase A.59
■ ION CHANNEL MODULATOR CHEMOTYPES
In the below sections, we survey the prevalent chemotypes that
have been described as targeting the various subfamilies within
the ion channel superfamily according to one of the modalities
described above. We have organized this survey into five sections
according to the topology of the subfamilies (Figure 14): 6-
Figure 14. Cartoon representation of the five topologies of ion channels.
Note that this representation depicts the situation where the subunits
are heteromeric for clarity to distinguish the subunits involved in
creating the channel protein, but in some cases they may be homomeric.
transmembrane channels based on four subunits and an
appended transmembrane helix voltage sensor domain, 4-TM
Cys-loop channels that form pores as pentamers, glutamate
receptors with four subunits, the ASIC/P2X channels that
contain 2-TM domains and function as trimers, and the K2P/Kir
channels that have a 2TM subunit topology comprising either a
functional tetramer or two pairs of subunits.
We relate the cartoon representations for each topology to an
exemplar protein structure in each sectioǹ to orient the reader to
the structure and relative complexity of each topology. For
further detailed description of the structural aspects of each
individual subfamily member, the reader is referred in each
section to literature papers and reviews.
■
6-TRANSMEMBRANE (6TM) TOPOLOGY ION
CHANNELS (Nav, Kv, Cav, KCa, TRP)
Medicinal chemistry programs aimed at developing modulators
of 6TM voltage-gated Nav, Kv, Cav and ligand-gated TRP
channels have been widely reported.20c,61 Structural studies on
the α1-subunit of 6TM topology ion channels date back to the
late 1980s.62 These channels are composed of approximately
2000 amino acids across four structurally distinct subunits with
each 6TM subunit contributing two helices to the pore domain
responsible for ion conduction and four helices to the voltage
sensor domain (Figure 15). Recently, structural understanding of
6TM topology has been improved by the publication of several
crystal structures: a Kv1.2/2.1 chimera crystal structure by
Mackinnon et al.63 and the bacterial channel NavAb crystal
structure from the Catterall group.10b In addition, computational
molecular dynamics has been used to understand the opening/
closing mechanism of voltage-gated channels. A publication from
Jensen et al.,64 which describes microsecond order molecular
dynamics for the Kv1.2/2.1 crystal structure, suggests that the
channel closes via hydrophobic collapse of a pore residue,
followed by movement of the voltage-sensor domain toward the
pore. In the opening process, the voltage sensor moves outward,
which pulls the pore domain open. The exact nature of small
molecule binding sites is less defined, as no cocrystal structures
have been reported. However, site-directed mutagenesis studies
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Perspective
Figure 15. Structure of Kv1.2, a 6TM ion channel (PDB code 3LUT60).
have proposed three binding sites: a pore binding site, a cleft in
the voltage sensor, and a fenestration site.65
Although no crystal structures are available for ligand-gated
TRP channels, electron microscopy and sequence analyses
suggest that the topology for these channels is similar to voltage-
gated channels.66 Homotetramers of four subunits comprise two
domains: a sensor region (S1−S4) and a pore domain (S5−S6).
The discovery of small molecule TRP channel modulators dates
back to the 1990s; however, a binding site was not identified until
2002, when Julius proposed a key role for two residues located at
the intracellular loop between S2 and S3 of the sensor region.67
In addition, an alternative pore domain binding site has also been
suggested for recognition of cholesterol and menthol by
TRPA1.68 This binding site is close to the fenestration binding
site of the voltage-gated channels and is thought that cholesterol
stabilizes the channel’s inactive state to suppress ion conduction
in the pore.
Voltage-Gated Sodium Channels (Nav). Sodium channels
comprise a family of nine members Nav1.1 to Nav1.9.69 They
have been identified as regulators of cellular excitability,
contributing to the generation and propagation of action
potentials in neurons, muscle, and heart tissues. Consequently,
sodium channels are precedented drug targets as antidysrhyth-
mics, anticonvulsants, and anesthetics.
A number of natural toxins such as TTX (16) bind to all states
of sodium channels. Toxins of this class bind to occlude the
extracellular channel pore, and the sensitivity of the nine sodium
channels to TTX blockade has been used to divide the family into
two classes: (i) TTX-S (sensitive), IC50 < 30 nM against Nav1.1,
-1.2, -1.3, -1.4, -1.6, -1.7 and (ii) TTX-R (resistant), IC50 > 30 nM
against Nav1.5 and -1.8). This affinity difference is proposed to be
due to a key cysteine residue in the TTX-binding site.70
A variety of approved drugs are modulators of sodium
channels for the treatment of clinical conditions associated with
abnormal cell excitability (Figure 16).71 Several are used to treat
CNS disorders such as anticonvulsants (lamotrigine (18) and
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Figure 16. Nav channel modulator chemotypes.
epilepsy drugs phenytoin (19) and carbamazepine (6)) by acting
on sodium channels expressed in brain neurones. Antidysrhyth-
mics (mexiletine (20), flecainide (21), and tocainide (22))
rectify cardiac rhythm by targeting Nav channels in the heart.
Local anesthetics (lidocaine (1), benzocaine (23), and
bupivacaine (2)) have been used for many years to modulate
peripheral nerves as injectable or topical agents at the site of
action. On the whole these compounds lack subtype selectivity
across sodium channels, leading to side effects that preclude their
chronic use. From a physicochemical and structural basis all of
these compounds are either weakly basic or neutral and have
been suggested to bind to a site on the intracellular side of the
channel pore.72 As this region of the channel shows a high degree
of sequence conservation across the subtypes, it seems
reasonable that compounds that bind to this region do so
without imparting appreciable levels of subtype selectivity.
More recently several newer agents with general Nav activity
have been taken into clinical development (Figure 17). These
Figure 17. Recent Nav channel modulators.
include ralfinamide (24), a state and use dependent Nav
modulator from Newron that is reported to act through
Nav1.3, Nav1.7, Nav1.8 and the voltage-gated calcium channel
Cav2.2.73 Lacosamide (25) from UCB, also based around a
benzylamino headgroup, is proposed to bind to a novel Nav
binding site that enhances slow inactivation.74 This compound
also possesses some Nav subtype selectivity toward TTX-S
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Perspective
channels with Nav1.3 IC50 = 415 nM, Nav1.7 IC50 = 182 nM, and
Nav1.8 IC50 = 16 μM.75 Lacosamide (25) was approved in 2008
for the treatment of partial onset seizures and diabetic
neuropathic pain.
Nav1.7 has received considerable attention driven by the
genetic data described above. The resulting industry-wide effort
has led to a diverse array of chemotypes (Figure 18) with several
companies moving compounds with Nav1.7 activity into clinical
trials including the proline derivative 26 from GSK/Con-
vergence.76 A structurally very different neutral spiro-oxindole
template exemplified by XEN402 (27) has been advanced to the
clinic by Xenon, and this compound has been shown to be
effective at treating pain in patients with congenital erythrome-
lalgia.77 Benzazepinone 28 has been reported by Merck to be a
state-dependent inhibitor of Nav1.7 (IC50 = 90 nM) and Nav1.8
(IC50 = 680 nM) while displaying ∼10-fold use-dependent
selectivity over Nav1.5.78 Pfizer, in collaboration with Icagen, has
published several patents in recent years based on acidic and
zwitterionic series with numerous examples (e.g., 29) reported to
possess Nav1.7 potency in the single digit nanomolar range.79
Recently Pfizer also advanced a Nav1.7 compound into clinical
trials for the treatment of pain.80 AstraZeneca has published
several patents that cover a diversity of chemical series for sodium
channel inhibition such as the chromane 30 with Nav1.7 IC50 =
66 nM, Nav1.5 IC50 = 13 μM, Nav1.2 IC50 > 33 μM.81
Vertex has published several patents in the area of TTX-S
selective channel modulators based on a series containing weakly
acidic N-heterocylic sulfonamides (estimated pKa ≈ 7),
suggesting Nav1.1 and Nav1.3 to be preferred targets for this
series with compound 31 having Nav1.1 and Nav1.3 IC50 < 2 μM
(Figure 19).82 Icagen and a collaboration between Pfizer and
Icagen have also filed patents in which N-heterocylic
sulfonamides have been reported to modulate TTX-S channels
with examples such as compound 32 having Nav1.3 IC50 = 30 nM
and >1000-fold selectivity over the cardiac channel Nav1.5.83
Pfizer reported a 6,6-biaryl series optimized for Nav1.8
exemplified by 33 which has a whole cell Nav1.8 IC50 of 260
nM and selectivity of >20-fold over Nav1.1, Nav1.5, and Nav1.7
(Figure 20). Pfizer has since reported entering clinical trials with
a Nav1.8 selective compound.20c,84 A collaboration between
Abbott and Icagen has resulted in 6,6- and 6,5-biaryls with an
alternative amide geometry. Substituted pyrazine 34 showed
Nav1.8 IC50 of 30 nM and has good selectivity over the TTX-S
channel Nav1.2 and TTX-R channel Nav1.5. Interestingly,
alternative cores were tolerated and had similar profiles, for
example, the furan compound A-803467 35.61c
Although significant progress has been made recently to define
the first subtype selective sodium channel series, there is still a
need for more research in this area to deliver alternative selective
chemical substrate.
Voltage-Gated Potassium Channels (Kv). Voltage-gated
potassium channels are found in all excitable cells. They open on
membrane depolarization, allowing potassium ions to exit the
cell, and thereby repolarize the cell.85
Researchers at Wyeth disclosed compounds of structure 36
(Figure 21) that disrupt Kv1.1 N-type inactivation and may be
useful for reducing neuronal hyperexcitability in diseases such as
epilepsy and neuropathic pain.86 Kv1.3 is widely regarded as a
promising new target for immunosuppression. Psora4 (37) is
reported to block Kv1.3 in a use-dependent manner and exhibit a
17- to 70-fold selectivity for Kv1.3 over several closely related
Kv1-family channels. It also selectively suppresses the prolifer-
ation of human and rat myelin-specific effector memory T cells.87
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Figure 18. Nav1.7 targeted modulators.
Figure 19. Nav1.3 targeted modulators.
Figure 20. Nav1.8 selective compounds.
Figure 21. Kv channel modulators.
Merck scientists have recently disclosed compounds such as
RY79688 (38) that potently inhibit Kv2.1 and another member of
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Perspective
the Kv2 family, Kv2.2 (IC50 100−200 nM) and are >10-fold
selective over Nav and other Kv channels. At clinically relevant
concentrations, the selective serotonin reuptake inhibitor
fluoxetine (39) and its major metabolite norfluoxetine (40)
inhibit Kv3.1, with IC50 of 13.1 and 0.80 μM, respectively.89
KCNQ channels play a central role in regulating several critical
functions, such as regulation of the heart beat or modulation of
neuronal activity.61b Potent and selective KCNQ1 inhibitors
have been reported by Merck90 that function as antiarrhythmic
agents both in vitro and in vivo (41, Figure 22). A number of
KCNQ2/3 channel openers have been reported. An example of
this class is ICA-2724357 42. Researchers at Icagen investigated
the location and nature of the binding site for this selective
KCNQ2/3 selective activator, and unlike nonselective KCNQ
activators including retigabine91 (43) and flupirtine92 (44), 42
reportedly binds to a novel voltage sensor domain site.57
The human ether-a-go-go-related gene (hERG, KV11.1)
potassium channel plays a significant role in cardiac action
potential repolarization. Inhibitors of this channel can induce
long QT syndrome. hERG is a promiscuous channel and binds to
a structurally diverse set of small molecules and has resulted in
many drugs being removed from the market or terminated
during clinical development because of cardiac side effects. These
include terfenadine (45), astemizole (46), and cisapride (47)
(Figure 23). It is now common practice to assess the hERG
blocking liability of compounds before they are taken to the
clinic.93 hERG channel blockers that are administered as
treatments for cardiac arrhythmias include sotalol (48). hERG
channel activators are considered potential therapeutics for
antiarrhythmia including PD-118057 49,89 an acid-containing
compound that contacts the pore domain of hERG channels to
attenuate inactivation and enhance K+ conductance. Another
example is NS3623 50 which incorporates an acidic tetrazole
group and also induces hERG inactivation.89
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Journal of Medicinal Chemistry
Figure 22. Kv7 channel modulators.
Figure 23. Modulators of the hERG channel.
Figure 24. L-type Cav channel blockers.
Voltage-Gated Calcium Channels (Ca v). In many
physiological systems, Cav channels are the molecular link
between membrane potential and intracellular calcium signaling
pathways. As a result, they have become drug targets for a range
of neurological and cardiovascular diseases.61c The principal
protein that forms Cav channels is the α1-subunit which shares
similar topology with the equivalent Nav and Kv channel
subunits. However, there are three other Cav auxiliary subunits
that also modulate the function of the α1 subunit: a cytoplasmic
β-subunit, a membrane anchored extracellular α2δ subunit, and
an integral membrane γ subunit. The 10 α1 subunits are divided
into 3 subfamilies: (1) Cav1.1−Cav1.4 (L-type); (2) Cav2.1 (P/
Q-type), Cav2.2 (N-type), Cav2.3 (R-type); (3) Cav3.1−Cav3.3
(T-type), with all three classes involved in neuronal signaling. To
date the L-type (Cav1) channels have been the most frequently
targeted by inhibitors for the treatment of angina and
hypertension, although compounds reported to date have not
been able to readily achieve subtype selectivity (Figure 24). The
three main classes of drugs are phenylalkylamines such as
verapamil (51), benzothiazepines such as diltiazem (52), and
605
Perspective
dyhydropyridines such as amlodipine (53) and nifedipine (54).
Each drug class has been established to bind to different sites on
the α1 subunit.94
Selective Cav2.1 and Cav2.3 inhibitors have not been disclosed
to date. Small molecule N-type Cav2.2 selective compounds,
however, have been identified, driven largely by the established
clinical link of this subtype to neuropathic pain.95 Ziconotide
(Figure 25) is an approved analgesic peptide but requires
intrathecal administration and is associated with some significant
side effects.96
Efforts have been made to develop systemically efficacious
small molecule N-type inhibitors to overcome these limitations.
Several groups have disclosed a variety of series including Purdue
Pharma 55, Merck 56, which has Cav2.2 IC50 = 150 nM and
Cav1.2 IC50 = 910 nM, Ionix 57 which has Cav2.2 IC50 = 730 nM
and Cav1 IC50 = 23 μM, and Ajinomoto Pharma 58 which has
Cav2.2 IC50 = 720 nM and Cav1 IC50 = 44 μM (Figure 26).97
Notably compounds patented by GSK have been progressed by
Convergence Pharmaceuticals, e.g., CNV2197944 (structure not
disclosed), into phase II clinical studies in 2012 for neuropathic
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Journal of Medicinal Chemistry
Figure 25. Structure of the ω-conotoxin ziconotide. For a detailed
structure of ziconotide, see the Protein Data Bank, structure code
1DW5.
pain. On the basis of the patents assigned to Convergence, the
clinical compound appears to be related to the piperazinesulfo-
namide 59.98 Also, compound 60,99 originally from Neuromed,
has been reformulated and progressed into phase I by Zalicus in
2012.
Currently, selective T-type Cav3 inhibitors have not been
identified, although several nonselective compounds have been
proposed, at least in part, to elicit their antihypertensive efficacy
through Cav3 inhibition.61c
The gabapentanoid drugs gabapentin and pregabalin are used
to treat epilepsy and neuropathic pain through Cav channel
modulation via binding to the α2δ-1 and α2δ-2 subunits, as
described above.
Calcium Activated Potassium Channels. This family
contains eight members: big potassium (BK) channel (KCa1.1),
small conductance (SK) channels (KCa2.1, KCa2.2, KCa2.3),
intermediate conductance (IK) channel (KCa3.1), voltage gated
potassium channel, subfamily T (KCNT) (KCa4.1, KCa4.2), and
high conductance (Slo) (KCa5.1). Most of these channels are
activated by intracellular calcium except for the KCa4 and KCa5
channels which are activated by intracellular sodium and
chloride, respectively, and the KCa1 channels are both calcium-
Figure 26. N-type Cav channel blockers.
606
Perspective
and voltage-gated. All are tetrameric channels with α1 subunit
topology similar to that of voltage-gated channels.
Several launched drugs are known to impart some of their
efficacy via opening of the BK (KCa1.1) channel (Figure 27).
Figure 27. KCa1.1 channel modulators.
These include the thiazides hydroflumethiazide saluron100 (61)
and chlorothiazide diuril101 (62), which work as diuretics
through inhibition of the NaCl co-transporter in the kidney but
also have additional antihypertensive properties suggested to be
due to BK channel activity. Flindokalner102 (63) is a positive
modulator of KCa1.1 (EC50 = 352 nM) but also a positive
modulator of Kv7.2−7.5 (EC50 = 2.4 μM) and a negative
modulator of the Kv7.1 channel. Flindokalner was developed as a
first in class compound for the treatment of stroke but did not
achieve clinical efficacy over placebo.103
A variety of unselective IK/SK channel openers are known,
such as NS309104 64 (Figure 28). Some SK channel selective
compounds are also known, such as the Neurosearch compound
CyPPA (65) (SK2 EC50 = 14 μM, SK3 EC50 = 5.6 μM, no activity
at SK1 or IK channels).105 GSK also published GSK542573X 66
as the first selective KCa2.1 subtype selective opener via binding
to a novel site on the channel.106 Several recent patents have
disclosed SK channel openers with data for KCa2.3 provided but
no further selectivity data. Neurosearch disclosed the imidazo-
pyrimidine 67 with an EC50 of 20 nM.107 As these channels are
expressed in the CNS, there is a suggestion that modulators
could be beneficial in a number of neurological diseases such as
epilepsy or for enhancing learning and memory. However, more
selective modulators will likely be required in the future.108
Senicapoc/ICA-17043 (68) currently owned by Pfizer is a
selective dual inhibitor of KCa3.1 (IC50 = 6 nM) and the Gardos
channel KCCN4 (IC50 = 11 nM). It has been reported that this
compound has IC50 > 1 μM for Kv1.5, hERG, TTX-S Nav's, IK,
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Journal of Medicinal Chemistry
Figure 28. IK/SK channel modulators.
Kv7.1, and Nav1.5. Senicapoc has been progressed to phase II
clinical trials for sickle cell anemia and for asthma.109
TRP Channels. TRP channels constitute a family of 28
mammalian cation channels with biological functions ranging
from pain perception and thermosensation to calcium and
magnesium absorption. Consequently, TRP channels are
believed to play a role in the pathogenesis of several diseases.110
TRP channels are classified into six subfamilies: TRPA (ankyrin),
TRPV (vanilloid), TRPM (melastatin), TRPC (canonical),
TRPP (polycystin), and TRPML (mucolipin) and are activated
by a range of chemical and thermal stimuli.111 TRPV1, TRPV3,
TRPV4, TRPA1, and TRPM8 are the most advanced in terms of
availability of potent and selective chemical modulators.112
TRPV1 is activated by numerous stimuli including heat (>42
°C), vanilloids, lipids, and protons/cations. Capsaicin 3 and
resiniferatoxin RTX (69) are potent small molecule TRPV1
agonists. Several selective TRPV1 antagonists have advanced
into clinical development for the potential treatment of pain and
have been covered in recent reviews.113 These include
AMG517114 70, SB-705498115 71, ABT-102116 72, and MK-
2295117 73 (Figure 29), which cover a range of chemical classes.
Crucially, reports from these clinical studies have shown TRPV1
blockade to have effects on core body temperature, which has
halted much activity on this channel.
TRPA1 is activated by a variety of ligands including exogenous
electrophiles such as cinnamaldehyde, acrolein, allyl isothiocya-
Figure 29. TRPV1 channel modulators.
607
Perspective
nate, and the endogenous ligand 4-hydroxynonenal (4-
HNE).111,113b Electrophilic ligands have been shown to activate
TRPA1 through covalent binding to cysteine residues present in
the cytoplasmic N-terminus of TRPA1 in in vitro systems.118
Nonelectrophilic compounds that activate TRPA1 include
flufenamic acid (FFA), thymol, and 2-tert-butyl-5-methylphenol.
Recombinant TRPA1 is activated by noxious cold (<17 °C).
Hydra Biosciences has disclosed several TRPA1 inhibitors such
as HC-030031119 74 (IC50 = 6.2 μM) and the related TRPA1
ligand HC-068559120 75 that have demonstrated efficacy in
preclinical pain models (Figure 30). Other known TRPA1
selective inhibitors include AP18 76 and AMG9090 77.111,113b
TRPV3 is activated by natural products such as camphor (78)
and carvacrol (79) and by 2-APB (80) and warm temperatures
(>32 °C).121 Several classes of TRPV3 antagonists have been
disclosed over recent years (Figure 31). These include
benzothiazole 81121a and quinazolin-4-one 82.121b GRC15300
(structure not disclosed), a Glenmark TRPV3 modulator for the
potential treatment of osteoarthritis and neuropathic pain, has
recently entered phase I clinical trials in partnership with
Sanofi.122
TRPV4 is activated by heat (27−34 °C) and various small
molecules (Figure 32) including anandamide (ANA, 83), 5,6-
epoxyeicosatrienoic acid (5,6-EET, 84), and GSK1016790A
85.123 In recent years, a number of apparently selective TRPV4
antagonists have been disclosed such as RN-1734 (86, IC50 = 2.3
μM)124 from Renovis and pyrrole HC-067047 87 from Hydra
which has been reported to be efficacious in preclinical models of
bladder cystitis.125 Interestingly, Renovis also reported on a
structural analogue RN-1747 (88, EC50 = 770 nM) that is an
agonist of the TRPV4 channel.124
TRPM8 is activated in vitro by cool temperatures (10−23 °C)
and cooling agents such as icilin (89) and menthol (4).121a,126
The Dendreon TRPM8 agonist D-3263127 90 advanced to
clinical trials in 2009 (Figure 33). A number of TRPM8
antagonists such as the GlaxoSmithKline compound AMTB (91)
and the Janssen carboxylic acid 92 have been disclosed, with the
latter compound (TRPM8 IC50 = 4 nM) showing efficacy in
preclinical models of pain.128
Although there is much potent and selective substrate for the
modulation of TRP channels, to date no compounds have
successfully progressed to launched drug status.
■ Cys-LOOP CHANNELS (GABAA, nAChR, 5-HT3,
GlyR, GluR)
Cys-loop channels are ligand-gated channels that control fast
synaptic transmission. The Cys-loop family comprises GABA-A
receptors, nAChR (nicotinic acetylcholine receptor), 5-HT3
receptors, glycine receptors, and glutamate receptors.
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Figure 30. TRPA1 channel modulators.
Figure 31. TRPV3 channel modulators.
Figure 32. TRPV4 channel modulators.
Cys-loop channels adopt pentameric structures (Figure 34)
with C5 symmetric assembly of five subunits, each of which
consists of two domains: an extracellular N-terminal domain and
a transmembrane domain. The transmembrane domain is
composed of four helices, which are connected to each other
by extracellular and intracellular loops.130 A number of cocrystal
structures of nicotinoids and AChBP (a homologous protein of
nAChR) have been disclosed.131 According to these structures,
the binding site lies in a cleft at the interface between the
subunits, which is located in the middle of the N-terminal
domain.
GABA channels. GABAA is a ligand-gated ion channel whose
endogenous ligand is γ-aminobutyric acid (GABA, 93), a major
neurotransmitter in the CNS. The subunits are grouped into
eight families (α1−6, β1−3, γ1−3, δ, ε, π, θ, and ρ).132 A limited
number of other ligands are known to bind to the GABA binding
608
Perspective
Figure 33. TRPM8 channel modulators.
site, including muscimol (94), THIP (95), and 4-PIOL (96)
(Figure 35).
Benzodiazepines were introduced into clinical practice in the
1960s and proved successful in the treatment of anxiety and
insomnia. They bind to a site that is separate from the GABA
binding site, and a diverse number of ligands have been reported
(Figure 36) that bind to this benzodiazepine site including
diazepam (5) (GABAA α5β3γ2 EC50 = 17 nM),133 flumazenil
(97) (GABAA α3β3γ2 Kd = 0.45 nM),134 flavonoids such as
genistein (98), and β-carbolines such as CCP (99).135
The effects of benzodiazepines on GABAA receptors are
dependent on subunit composition. The α1 subtype is associated
with sedation and addiction, while α2 and α3 subtypes are
associated with anxiolysis and muscle relaxation effects. A
number of clinical compounds with higher affinity to α1
containing GABAA receptors, such as zolpidem (100) and
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Figure 37. α1 selective GABAA ligands.

Figure 34. Structure of an anion-selective Cys-loop receptor (GluCl,

PDB code 3RHW129).
Figure 38. α2/3 selective GABAA ligands.

The α5 subunit has reported links to cognition.141 Merck

developed L-655708 105 (α1, Ki = 49 nM; α2, Ki = 27 nM; α3, Ki
= 25 nM; α5, Ki = 0.45 nM) and MRK-016 106 (α1, Ki = 0.8 nM,
23% enhancement over baseline; α2, Ki = 0.8 nM, 4%
enhancement; α3, Ki = 0.8 nM, −15% enhancement; α5, Ki =
Figure 35. GABAA ligands that are known to bind to the GABA binding 1.4 nM, −96% enhancement) (Figure 39), both partial inverse
site. α5 agonists selective over α2, α3, α1, and α6142 which enhance
cognition in animals without anxiogenic or convulsive side
effects.
zaleplon (101) (subtype selectivity GABAA α5β3γ3 EC50 > 1
μM, GABAA α1β2γ2 EC50 = 0.29 nM, GABAA α3β3γ2 EC50 > 1
μM, GABAA α2β3γ2 EC50 = 1.63 μM),136 are used for the
treatment of insomnia (Figure 37).
More recently research has focused on the delivery of α2 and/
or α3 functionally selective ligands to deliver nonsedating
anxiolytics.137 SL651498 102 (Figure 38) is a full positive
allosteric modulator (PAM) of α2 but a partial PAM of α1
subtypes.138 L-838417 103 is a Merck partial PAM of α2-, α3-,
and α5-containing receptors (α2, Ki = 0.7 nM, 34% enhancement
over baseline; α3, Ki = 0.7 nM, 39% enhancement; α5, Ki = 2.3
nM, 36% enhancement) that does not enhance the GABA Figure 39. α5 selective GABAA ligands.
response at α1 receptors139 and exhibits a nonsedating anxiolytic
effect in non-human primates. MRK-529 104 exhibits functional AChR Channels. Acetylcholine (ACh) 107 (Figure 40) is a
selectivity for α2 and α3 over α1 (α1, Ki = 1.3 nM, 0% neurotransmitter and the endogenous ligand for the nicotinic
enhancement; α2, Ki = 0.6 nM, 37% enhancement over baseline; acetylcholine receptor (nAChR). Molecular biology of nAChR
α3, Ki = 0.5 nM, 45% enhancement) and is reported to be has shown this functional receptor to consist of five subunits.143
efficacious in animal models of anxiety with no sedation or A limited number of subunit combinations have so far shown
ethanol potentiation effects.140 therapeutic promise, including α7 and α4β2, which are expressed

Figure 36. Benzodiazepine binding site ligands of the GABAA channel.

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Figure 40. α7 and α4β2 AChR ligands.

in the brain and known to be involved in several biological

processes.144 Nicotine 108 is an agonist of these receptors and
has been reported to improve cognition and attention in rodents,
primates, and humans.145 This, coupled with the observation that
nAChRs have reduced expression in the brain of schizophrenia
and Alzheimer's disease (AD) patients, has driven a search for α7
and α4β2 receptor agonists for the treatment of cognitive
impairment disorders. A number of α7 and α4β2 receptor
agonists and partial agonists have been reported, including the
pyridyl ethers A85380146 109 (α4β2 agonist, EC50 = 700 nM, Ki
= 50 pM) and AZD-3480147 110 (α4β2 agonist, EC50 = 106 nM),
the piperidine derivative GTS-21148 111 (α7 weak agonist EC50
= 81 μM), and the piperidine derivative PNU-282987149 112 (α7
agonist EC50 = 150 nM). Preclinical studies have confirmed their
efficacy in a wide range of indications, including the use of the
partial agonist varenicline (14) for smoking cessation which is
described above (binding Ki = 60 pM at rat cortex α4β2, with
∼30−60% the in vivo efficacy of nicotine with block of the in vivo
nicotine response).150
5-HT3 Channel. 5-HT3 is expressed in the CNS, the
peripheral nervous system, and the gut and is associated with
excitatory synaptic transmission. Its endogenous ligand is
serotonin (113), a monoamine neurotransmitter (Figure 41).
■ IONOTROPIC GLUTAMATE RECEPTOR CHANNELS
Ionotropic glutamate receptors (iGluRs) are ligand gated ion
channels that mediate excitatory neurotransmission and play a
key role in the development and function of the mammalian
central nervous system. Many human neurological disorders
such as epilepsy, stroke, and Alzheimer’s disease can be linked to
overstimulation or underactivity of iGluRs.157 Because of a
wealth of structural biology, they also represent one of the best
understood classes of ion channel.158 These findings have aided
development of iGluR agonists, antagonists, and modulators as
research tools and potential therapeutic agents.159
The iGluRs function as a complex of four individual subunits
(Figure 42). The AMPA and KA receptors can exist as homo-
and heterotetramers that bind glutamate, whereas NMDA are
heterotetramers with subunits that are activated by glycine (N1
and N3) and glutamate (N2). The iGluRs are subdivided into
three categories based on selective activation by small molecule
agonists (Figure 43); AMPA 119 (α-amino-3-hydroxyl-5-

Figure 41. 5-HT3 channel modulators.

Drug discovery approaches toward this channel have been very

successful, with several launched drugs having been identified,
many based on the structure of 113. Antagonists of the 5-HT3
channel have proved clinically effective in the treatment of
chemotherapy-induced nausea and vomiting and irritable bowel
syndrome. Some of the approved 5-HT3 antagonists include
indisetron151 (114), ramosetron152 (115), ondansetron153 Figure 42. Structure of an AMPA-subtype glutamate receptor (GluA2,
(116), palonosetron154 (117), and azasetron155 (118).156 PDB code 3KG2160).

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Figure 43. Agonists of glutamate receptors.

methyl-4-isoxazolepropionic acid), NMDA 120 (N-methyl-D-

aspartate), and KA 121 (kainic acid). Molecular cloning
identified four receptor subunits for AMPA (GluA1−4), five
for KA (GluK1−5), and seven for NMDA (GluN1, GluN2A−
GluN2D, GluN3A, GluN3B).
Agonists of the iGluRs are typically derived from glutamate,
AMPA, NMDA, or KA and feature or mimic the α-amino acid
moiety of the neurotransmitter. However, small structural
changes alter selectivity across subtypes and receptor families.
For instance AMPA has negligible effects on KA receptors but
replacement of the isoxazole oxygen with a sulfur atom and
increasing steric bulk at the 5-position provide analogues such as
thio-ATPA 122 which invert potency and selectivity for GluK1
over the AMPA receptors (GluA1 EC50 = 5.2 μM, GluK1 EC50 =
0.1 μM).161 Agonist activity for a series of substituted willardiines
has been compared between kainate preferring receptors in
dorsal root ganglion (DRG) neurons and AMPA preferring
receptors in the hippocampal (HPC) neurons.162 (S)-5-
Fluorowillardiine 123 shows high selectivity for AMPA receptors
(HPC EC50 = 1.5 μM, 46-fold selective over DRG neurons). (S)-
5-Chlorowillardiine 124 demonstrates similar potencies across
both neurons (EC50 of 2−7 μM), whereas (S)-5-iodowillardiine
125 provides greater agonist activity and selectivity for the KA
receptors (DRG EC50 = 0.14 μM, 137-fold selective over HPC
neurons). This preference for KA receptor agonist activity was
rationalized on steric arguments analogous to the AMPA/thio-
ATPA observations. A large, lipophilic C4 substituent on
glutamate derived LY-339434 126 also provides agonist
selectivity for GluK1 over AMPA receptors and other KA
receptor subtypes (GluK1 EC50 = 2.5 μM, GluK2 EC50 > 100
μM, GluA EC50 > 300 μM).163 Agonists of NMDA receptors that
bind to the glutamate site are typically derivatives of glutamic acid
or NMDA such as homoquinolinic acid 127. Similarly, agonists
that bind to the glycine site are usually amino acid derivatives
such as cyclopropyl analogue 128.
Most of the competitive antagonists of glutamate receptors are
expanded versions of agonists often incorporating additional
acidic moieties or with negatively charged non amino acid ends.
AMPA and KA receptor antagonists ATPO (129) and willardiine
derivative 130 are typical exemplifications of this pharmacophore
(Figure 44).164 Similarly, tezampanel (131), which represents a
ring expanded and homologated version of KA, inhibits both
AMPA and GluK1 receptors with similar affinity (IC50 of 0.5−2.5
μM).165 Quinoxalinediones, such as CNQX164 (132), represent
an alternative structural class of AMAP/KA receptors. These
classical antagonists are typically unable to differentiate
611
Figure 44. Competitive antagonists of glutamate receptors.
pharmacologically between AMPA and KA receptor families
with similar affinities at both. 5,7-Dichlorokynurenic acid166 133
and AP-V167 (134) represent antagonists targeting the glutamate
(GluN2) or glycine (GluN1, GluN3) site of NMDA receptors,
respectively.
Clinically, agonists and competitive antagonists of iGluRs
typically exhibit many severe CNS-mediated side effects. It is
widely anticipated that allosteric inhibition of iGluRs will display
a smaller number of side effects at therapeutic doses. However,
they are less well studied than competitive inhibitors and the
limited structural information to guide design has impeded
development of such therapeutic agents.
Not surprisingly, noncompetitive antagonists and allosteric
modulators of iGluRs show a much wider structural variety than
agonists and competitive antagonists. Benzodiazepine GYKI
52466 135 is the prototypical noncompetitive antagonist at the
AMPA receptor and has been used as a lead compound to
develop analogues with improved potency and biopharmaceutics
(Figure 45).168 Benzothiadiazine derivatives, such as cyclo-
thiazide (136), and sulfonamide analogues, such as LY-404187
137169 from Lilly, are positive allosteric modulators of AMPA
receptors. Modulators generally bind to the ligand-binding
domain and stabilize the agonist-bound conformation, slowing
receptor desensitization and/or deactivation. An increased
structural understanding of glutamate receptors has helped
lead research into design of subtype selective blockers in an
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Figure 45. Noncompetitive antagonists and allosteric modulators of glutamate receptors.
attempt to further separate efficacy from adverse side effects
associated with pan-receptor blockade. In particular, subtype
selective NMDA-GluN2B receptor antagonists have attracted
considerable attention in the pharmaceutical industry for the
targeting of CNS disorders including stroke and Parkinson’s
disease.170 Ifenprodil171 (138) is the prototype GluN2B
antagonist that served as a template for the design of more
selective derivatives, such as traxoprodil (139),172 which has
progressed into clinical trials for a number of indications
including stroke and neuropathic pain.
■ ASIC AND P2X CHANNELS
The acid-sensing ion channels (ASICs) and purinergic P2X
receptors are trimeric ion channels whereby each subunit
contains two transmembrane domains (Figure 46), a large
extracellular loop, and relatively short intracellular amino and
carboxyl termini.
Figure 46. Structure of a P2X receptor (P2X4, PDB code 4DW1173).
612
Perspective
ASIC Channels. There are several known subunits of the
acid-sensing ion channels (ASICs)174 (ASIC1a/b, ASIC2a/b,
ASIC3 and ASIC4) that are susceptible to activation by changes
in pH, implying a role in conveying pain from tissue acidosis.175
Channels containing the ASIC3 subunit are highly expressed in
nociceptors and have received significant interest from the
pharmaceutical industry as targets for blocking chronic
inflammatory pain.176 Diuretic agent amiloride (140) is the
prototype weak nonselective blocker of ASIC channels (IC50 of
10−50 μM) (Figure 47).177 Amidine A-317567 141 was the first
Figure 47. Examples of ASIC inhibitors.
non-amiloride small molecule blocker of ASIC demonstrating
increased potency over amiloride in vitro (ASIC3 IC50 = 9.5 μM)
and in vivo.178 The potency of amidine containing molecules has
been optimized further by several groups including an indole
series from Merck represented by compound 142 (ASIC3 IC50 =
133 nM).179 The acylguanidine or arylamidine is seen as the key
pharmacophore for ASIC activity. This motif is implicated in
potential off-target effects and poor bioavailability, hampering
development of either tools or potential drug candidates.
Attempts to explore non-amidine containing inhibitors at the
ASIC3 channel led to identification of amine 143.180 However,
this compound also demonstrated polypharmacology across
numerous targets and CNS related side effects through increased
brain penetration. Further research is required to identify potent
selective ASIC inhibitors to fully elucidate their role in pain and
neurological disease states.
P2X Channels. P2X receptors are purinergic cell surface ion
channels gated by extracellular ATP.181 Seven receptor subtypes
(P2X1−7) have been characterized in mammalian cells which
assemble as homotrimeric and/or heterotrimeric functional
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Figure 48. Examples of P2X3 and P2X2/3 receptor antagonists.
Figure 49. Examples of P2X7 receptor antagonists.
channels (depending on subtype). P2X3, P2X2/3, P2X4, and P2X7
receptors have received the most attention from the
pharmaceutical industry as potential targets to treat a variety of
conditions including chronic pain, arthritis, and bladder
malfunction.182
The discovery of A-317491 144 provided the first potent and
selective, non-nucleotide, small molecule dual P2X3/P2X2/3
antagonist (Figure 48).183 While this compound delivered
significant in vivo activity in pain and overactive bladder models,
the presence of the three carboxylic acids severely limited its oral
bioavailability. Since this discovery, many companies have
explored alternative lead matter to identify P2X 3/P2X2/3
antagonists in more clinically viable oral drug space.182c
Following extensive SAR investigation on a diaminopyrimidine
series derived from an HTS hit, researchers at Roche identified
RO-4 (145, rP2X3 IC50 = 13 nM, hP2X2/3 IC50 = 25 nM)
followed by the more potent analogue RO-51 (146, rP2X3 IC50 =
2 nM, hP2X2/3 IC50 = 5 nM) for the treatment of pain.184 The
incorporation of the diol moiety in 146 could potentially be a
strategy to impart some CNS restriction, although this is not
discussed by the authors and no data relating to this are disclosed.
Biarylbenzamide derivatives are also described as potent P2X3
and P2X2/3 receptor antagonists by a number of companies
including Roche (compound 147, hP2X2/3 IC50 = 8 nM)185 and
Merck (compound 148, hP2X2/3 IC50 = 8 nM).186 An interesting
example from AstraZeneca’s pyrrolopyrimidinone series dem-
onstrated that subtype selectivity can be achieved across
homotrimeric and heterotrimeric channels. Quinoline derivative
613
Perspective
149 is potent at the hP2X3 receptor (IC50 = 24 nM) and selective
over rP2X2/3 (IC50 = 8580 nM), but incorporation of a methyl
substituent (150) at the benzylic carbon resulted in an almost
equipotent inhibitor at the hP2X3 (IC50 = 21 nM) and rP2X2/3
(IC50 = 74 nM) receptors.187
Several groups have disclosed P2X7 receptor antagonists based
on lipophilic benzamides (Figure 49).188 AstraZeneca described
a series of adamantane containing benzamides whereby the
physicochemical properties could be significantly manipulated
through variation of the 5-phenyl substituent.189 Incorporation
of basic groups was well tolerated (e.g., compound 151, hP2X7
pIC50 > 5) providing improved solubility and metabolic stability.
Pfizer researchers described multiparameter optimization of a
high throughput screening hit to optimize potency and
physicochemical and pharmacokinetic properties within an
azauracil series, culminating in clinical candidate CE-224,535
(152, IL-1β IC 50 = 1 nM). 190 The ortho-substituted
arylbenzamide remained a key structural feature of numerous
P2X7 receptor antagonists described by Evotec (formerly
Renovis). Of particular interest are the oxoisoquinoline
benzamides which demonstrated exquisite potency in an IL-1β
inhibition assay (e.g., compound 153, IL-1β IC50 = 0.01 nM).191
A small number of series described in the literature do not
possess the benzamide pharmacophore typified by the majority
of disclosed P2X7 receptor antagonists. For example, workers at
GlaxoSmithKline have recently reported a series of pyrazolea-
cetamides with impressive ligand and ligand lipophilic
efficiencies (e.g., compound 154, hP2X7 pIC50 = 7.7).192
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Evotec, Pfizer, and AstraZeneca have all progressed P2X7
receptor antagonists to clinical trials, although the lack of efficacy
in treatment of rheumatoid arthritis for CE-224535 and
AZD9056 is likely to focus future industry efforts on potential
alternative indications such as chronic obstructive pulmonary
disease and pain.193
■ TWIN-PORE AND INWARDLY RECTIFYING
POTASSIUM CHANNELS (K2P AND Kir)
K2P Channels. Twin-pore potassium channels contain two
pore forming P-loop domains and four transmembrane segments
(M1−M4) that organize to form a single transmembrane pore
(Figure 50) selective for potassium.195 The class contains 15
Figure 50. Structure of a Kir potassium channel (KirBac3.1, PDB code
2WLK194).
members that are widely distributed, voltage-insensitive, non-
inactivating background channels expressed in neurons, muscle
cells, and endocrine cells. They are commonly named according
to their historic origins. These channels act to dampen electrical
activity and thereby regulate cell functions including secretion of
neurotransmitters and hormones. Activators (openers) are
anticipated to have potential as therapeutics to treat neurological
and cardiac diseases.196 Currently there are relatively few
compound classes that have been shown to modulate a subset
of K2P channels.
Figure 51. K2P channel modulators.
614
Perspective
It has been reported that caffeic acid derived esters such as
CDC (155) and CAPE (156) act as openers of the native TREK-
1 (K2P2.1) channel at micromolar concentrations (Figure 51).196
Acidic tetrazole compound BL-1249 157 has been shown to be
an opener of TREK-1 with micromolar activity in both binding
and electrophysiology assays.197 Riluzole (158), an established
neuroprotective agent with anticonvulsant, sedative, and anti-
ischemic properties, has also been reported to be a micromolar
affinity activator of TREK-1 and TRAAK (K2P4.1). Interestingly,
while activation of TRAAK was sustained, the activation of
TREK-1 was transient and followed by inhibition thought to be
mediated by a protein kinase A dependent inhibition of TREK-1.
TRAAK was permanently activated by riluzole, as this channel
lacks the negative regulation pathway.198
In addition to openers of the K2P channels, several
compounds have been reported as blockers. Interestingly,
dihydropyridine calcium channel antagonists have been shown
to have activity for the inhibition of TREK-1. Amlodipine (53)
has pronounced submicromolar activity at TREK-1 IC50 = 400
nM.199 The local anesthetic lidocaine (1) has also been shown to
inhibit TREK-1, albeit with weak activity (IC50 = 180 μM).200
Similarly, lamotrigine 18 inhibits TRESK (K2P18.1) but not
TREK-1.201 Sanofi-Aventis patented a series of compounds that
inhibit TASK-1/2 with sulfonamide 159 having TASK-1 IC50 of
100 nM in Xenopus oocytes and a patch clamp TASK-3 IC50 of 1
μM.202 Merck has reported an alternative TASK-1/3 inhibiting
series, compound 160, which has TASK-1 IC50 of 300 nM and
TASK-3 IC50 of 35 nM and has also been shown to be active in
preclinical rodent sleep pattern pharmacology models.203
The inward rectifier family of potassium (Kir) channels
comprises at least 16 members that have broad tissue distribution
and are implicated in a variety of functional roles. There is a
paucity of potent and selective small-molecule modulators that
target specific family members. However, considerable work has
been undertaken to deliver selective Kir1.1 (ROMK) inhibitors
(Figure 52), as Kir1.1 is thought to play a central role in the
regulation of salt and potassium homeostasis.204 A class of
selective small molecule Kir1.1 inhibitors has been described by
Merck and exemplified by piperazine 161, which shows a narrow
window over the hERG channel (ROMK IC50 = 50 nM, hERG
IC50 = 170 nM).205 Additionally, a Kir2 selective inhibitor has
been disclosed, ML133 (162) (Kir2.1 IC50 = 1.8 μM).206
Benzimidazoleimine analogues such as R70 163 were reported
by Vanderbilt University, TN, to selectively inhibit Kir2.3 (IC50 =
24 μM) over other Kir channels.207
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Figure 52. Kir channel modulators.
■ RECENT TRENDS
In a survey of the range of ion channel targeted chemotypes, it is
apparent that the breadth of ion channels being targeted in drug
discovery programs has greatly expanded in the past decade. This
is especially true of those channels for which a clear link to a
specific disease phenotype has been established, for example, the
voltage-gated sodium channels. As the drug discovery
community has increased their activity in these areas, the
number of clinical agents across the ion channel cohort has also
increased, and the field has begun to see the disclosure of more
clinical data that in some cases has raised warning flags about the
target. For example, core body temperature changes with TRPV1
blockers have limited their further progression, while for other
channels the full clinical utility of the target has not been fully
elucidated, as with the P2X ligands currently undergoing clinical
evaluation.
It is interesting that the majority of the currently launched ion
channel modulators in clinical use today are relatively unselective
and in many cases state-independent modulators of ion channel
function. This may imply that many of these compounds bind to
somewhat conserved regions of the channel pore domains. To
address some of the efficacy and/or safety shortcomings of some
of these agents, there has been a definite trend toward targeting
more selective agents. Non pore domain binding compounds
have been implicated in providing greater selectivity, for example,
through binding to the subunits of voltage gated calcium
channels, and even compounds that achieve their selectivity
profile through selectively targeting a particular channel state are
all being pursued. However, the structural knowledge of ion
channels is still at such an early stage that we have not noted any
truly predictive methods that can be applied to any chemotype to
increase selectivity against a particular channel. Rather, the
realization of more selective agents appears to have been driven
by a greater availability of quality high throughput assays that
companies and organizations have been able to screen their
proprietary compound collections against. This is consistent with
a move away from recognizable derivatives of natural products
such as menthol, camphor, capsaicin, and GABA to more
traditional small molecule chemotypes. Commensurately, the
diversity of chemotypes reported as active against members of
the ion channel superfamily has greatly increased in the past
decade. We note that several companies are now describing ion
channel targeted screening subsets based at least in some cases
on known ion channel chemotypes. It is also notable that in many
cases the definition of selective appears to be based on a relatively
small number of channel homologues from the same subfamily,
and there are very few reports of broad spectrum ion channel
screening to bring greater definition to just how selective
compounds may be, much in the same way that kinase selectivity
screens are reported now.
615
Perspective
It is also striking how similar templates/fragments appear quite
often as ligands for different channels. Small amide substituted
heterocycles have appeared as Kv7 channel openers, Kv2 channel
blockers, and Nav channel blockers, and templates such as these
may well offer a very useful basis for building homology models
for specific channels and increase our understanding of the
structural features that relate to a particular selectivity profile.
Small fragment-like compounds such as riluzole have also been
reported as ligands for several different channels.
Finally, as the field provides more human clinical data, this will
critically inform pharmacokinetic−pharmacodynamic relation-
ships and thereby the relationship between in vitro measures of
potency, compound ADME properties, and even selectivity.
While the drug discovery community has moved toward more
potent and selective ion channel agents, it remains to be seen if
this is matched by improved human safety and efficacy.
■
FUTURE PERSPECTIVE
Over the past few years, there have been significant develop-
ments in the field of ion channel drug discovery. Key highlights
include major advances in ion channel cloning and screening
capabilities and an increased awareness of the various ion
channels and their subtypes. This, coupled with clinical data from
non selective ion channel modulators, has increased under-
standing of the role that ion channels play in disease and
underpinned research interest in ion channels as therapeutic
targets.
More recently, the ability to screen ligands against a wide range
of high quality functional ion channel assays has become possible,
including the capability of screening against a number of those
known to elicit CNS and cardiovascular side effects. This,
coupled with the emergence of strong genetic linkages of specific
ion channels to certain disease states, has focused delivery on
highly selective ligands. Furthermore, several ground breaking
publications have begun to elucidate ion channel structure and its
relationship to function.
In the future, we anticipate continued advances in the
development of higher throughput, physiologically relevant ion
channel screens. We also anticipate significant advances in the
field of structural biology with the ability to generate cocrystal
structures of bound ligands in specific channel states coupled
with a capability to characterize ion channel−ligand interactions
in biophysical assays. This will increase our understanding of
ligand-binding events and their functional relevance which,
coupled with improved screening, should enhance our ability to
design safe and selective ligands. The availability of selective
ligands, associated chemical biology probes, and clinical
candidates, along with additional genetic data, will further aid
understanding of the role of specific ion channels in disease.
Currently, similarities exist between chemotypes across numer-
ous ion channels. This may be a function of cross-ion channel
dx.doi.org/10.1021/jm3011433 | J. Med. Chem. 2013, 56, 593−624
Journal of Medicinal Chemistry
pharmacophores or indicative of similar screening collections.
These chemotypes sometimes offer little in the way of ion
channel selectivity, and therefore, a challenge to the drug
discovery community may be to generate additional ion channel
chemical space for renewed opportunity in this area. Other areas
that may be of benefit are an increased understanding of ion
channel trafficking and how ion channels interact with accessory
proteins.
This is an exciting time for ion channel research. In a similar
way compared to the kinase field more than a decade ago, we
anticipate that the next decade will bring a significant growth in
the ability to design and deliver safe and selective ion channel
modulators to the clinic by harnessing advances in functional
screening, biophysical characterization, and structure-based
design techniques. This step−change in our ability to design
selective ligands should rapidly broaden our understanding of the
role of ion channels and increase the impact of ion channel
modulators in the treatment of human disease.
■ METHODS
The clinical compound analysis was conducted as follows. All ion
channel targeted compounds (voltage and ligand gated) from the Prous
Integrity database that had a highest phase above preclinical were
selected. All combination drug products that contained more than one
active ingredient were removed. Compounds with a molecular
mechanism that was not strongly linked to an ion channel were
removed. Dates were established from patent priority date of compound
determined from PatBase and manual searching of compound structure
in SciFinder. For those compounds without a structure in Prous, the
date was set to be the earliest milestone date recorded in Prous.
Selectivity designations were made as follows: multiple families
(molecular mechanism designation in Prous that covered at least two
target families, e.g., Na and Ca channels), single family (molecular
mechanism designation in Prous that covered only one target family,
e.g., AMPA), subtype selective (molecular mechanism designation in
Prous that covered a single member of a given family, e.g., Nav1.8). All
molecular properties were calculated using standard Pfizer in-house
software. PSA values were calculated using the method of Ertl et al.208
and clogP values using methodology from BioByte (www.biobyte.com).
Compounds with MW > 700 were removed from the analysis. Analysis
was focused on all available data up to 2010. Family designation was
made based on the first molecular mechanism designation or the first ion
channel designation if the first was not an ion channel. For the manually
curated set of compounds used in the analysis, reviews were conducted
of the patent and journal literature using PatBase and Scifinder, and
small molecule compounds were selected that were the most potent
and/or selective from the hits identified.
The phylogenetic tree was constructed as follows. The primary
sequence of proteins classified as ion channels in the IUPHAR database3
were retrieved from Uniprot and aligned using clustalw2. Protdist and
Kitsch from the Phylip suite of programs were used to calculate distances
and build a tree from the alignments. Voltage and ligand gated channels
were analyzed separately. The final representation is artistic and has
been produced from alignments of single families and larger subgroups
of proteins to aid separation and visualization. The tree may be
downloaded from http://www.neusentis.com/IonChannels.php.
■
AUTHOR INFORMATION
Corresponding Author
*Phone: +44 (0) 1304 643687. E-mail: David.Pryde@pfizer.
com.
Notes
The authors declare no competing financial interest.
Biographies
Sharan K. Bagal is a medicinal chemist based at Pfizer Neusentis in the
U.K. She earned her MChem in Chemistry from the University of
616
Perspective
Oxford, U.K. (2001), and Ph.D. from the University of Oxford under the
supervision of Professor Jack E. Baldwin and Dr. Robert M. Adlington in
the field of biomimetic total synthesis (2004). This was followed by
postdoctoral research with Professor Samir Z. Zard at É cole
Polytechnique, France, on xanthate based radical chemistry method
development before joining Pfizer in 2006.
Alan D. Brown is Head of Chemistry at Pfizer Neusentis. He earned his
Ph.D. under the supervision of Dr. Ernie Colvin at the University of
Glasgow, U.K., in the field of asymmetric synthesis before joining Pfizer
as a medicinal chemist in 1990.
Peter J. Cox is a biologist at Pfizer Neusentis in the U.K. He has 14 years
of experience in drug discovery aimed at identifying treatments for
chronic pain conditions. Prior to joining Pfizer, Peter completed
postdoctoral studies in Neurosciences at Cambridge University, U.K.,
and INSERM, Paris, France. He has a Ph.D. in Molecular Virology from
the University of Glasgow, U.K., and a B.Sc. (Hons) degree in
Microbiology from the National University of Ireland.
Kiyoyuki Omoto is a computational scientist based at Pfizer Neusentis
in the U.K. He gained his Ph.D. from Kyoto University, Japan, on
molecular orbital theory of organic reactions. He joined Pfizer Nagoya
Labs in 2002 before moving to the U.K. in 2007. His research interests
include lead discovery through HTS triage, 2D and 3D virtual screening
methods, and structure-based drug design.
Robert M. Owen is a medicinal chemist based at Pfizer Neusentis in the
U.K. He earned his B.A. in Chemistry from St. Olaf College, MN (1997),
and Ph.D. from the University of Wisconsin, Madison, under the
supervision of Professor Laura Kiessling, developing methods to
understand and explore multivalency in biological systems (2003),
and carried out postdoctoral research with Professor William Roush at
the University of Michigan on the total synthesis of peloruside A before
joining Pfizer in 2006.
David C. Pryde is a medicinal chemist based at Pfizer Neusentis in the
U.K. He earned his B.Sc. in Pure and Applied Chemistry from the
University of Strathclyde, U.K. (1991), and Ph.D. from the University of
Nottingham, U.K., under the supervision of Professor Gerry Pattenden
in the field of biomimetic radical cyclizations (1994) and carried out
postdoctoral research with Professor Albert Meyers at Colorado State
University on stereoselective metalation chemistry before joining Pfizer
in 1997.
Benjamin Sidders is a bioinformatician at Pfizer Neusentis in
Cambridge, U.K. He has a B.Sc. in Molecular Microbiology from the
University of Surrey, U.K., and a Ph.D. in Bioinformatics from the
University of London, U.K., where he undertook transcriptional analysis
of Mycobacterium tuberculosis with Professor Neil Stoker in order to
define diagnostic peptides. He joined Pfizer in 2008 and has worked in
both the antiviral and pain research units.
Sarah E. Skerratt is a medicinal chemist based at Pfizer Neusentis in the
U.K. She earned her Masters in Chemistry from the University of
Sheffield, U.K.(1998), and Ph.D. from the University of Nottingham,
U.K., under the supervision of Professor Jim Anderson on the
preparation of unnatural α,α-disubstituted α-amino acid building blocks
(2002) and carried out postdoctoral research with Professor Paul
Wender at Stanford University, CA, on the design and synthesis of
(−)-laulimalide analogues for biological evaluation (2003) before
joining Pfizer in 2004.
Edward B. Stevens is a biologist at Pfizer Neusentis with over 20 years
experience in electrophysiology and ion channel biology. He obtained a
B.Sc. (Hons) degree and Ph.D. from Imperial College London, U.K. He
held postdoctoral positions at the University of Cambridge, U.K., and
Parke-Davis Neuroscience Research Centre, Cambridge, U.K. He then
dx.doi.org/10.1021/jm3011433 | J. Med. Chem. 2013, 56, 593−624
Journal of Medicinal Chemistry
worked as a Section Head at BioFocus and then as Chief Operating
Officer of NeuroSolutions Ltd., Warwick, U.K., before joining Pfizer.
R. Ian Storer earned his MSci MA in Natural Sciences from the
University of Cambridge, U.K. (1999), where he remained to undertake
Ph.D. research on the application of solid-supported reagents to the total
synthesis of natural products with Professor Steven V. Ley (2003). This
was followed by a postdoctoral research fellowship at California Institute
of Technology, working with Professor David W. C. MacMillan on the
development of new methods for enantioselective organocatalysis
before joining Pfizer as a medicinal chemist in 2006.
Nigel A. Swain is a medicinal chemist based at Pfizer Neusentis in the
U.K. He earned his MChem from the University of Southampton, U.K.
(1999), where he remained to undertake Ph.D. research under the
supervision of Professor Richard C. D. Brown, working in the field of C−
H insertion chemistry in natural product synthesis (2002) before joining
Pfizer in 2003.
■
ABBREVIATIONS USED
TRESK, TWIK-related spinal cord potassium channel; TWIK,
tandem of P-domains in a weakly inward rectifying potassium
channel; CNS, central nervous system; SNP, single nucleotide
polymorphism; PAM, positive allosteric modulator; TRP,
transient receptor potential; KCNQ, voltage gated potassium
channel, subfamily Q; Kir, inward rectifier potassium channel;
K2P, twin-pore potassium channel; SUR, sulfonylurea receptor;
hERG, human ether-a-go-go-related gene; CNG, cyclic nucleo-
tide gated; BK, big potassium channel; GLR, glycine receptor;
GABA, γ-aminobutyric acid; CHRNA, cholinergic receptor,
nicotinic, α; AChR, acetylcholine receptor; NMDA, N-methyl-D-
aspartate; P2X, ATP-activated purinoreceptor; 5-HT, serotonin
receptor; ASIC, acid-sensing ion channel; SK, small conductance
calcium-activated potassium channel; IK, intermediate con-
ductance calcium-activated potassium channel; KCNT, voltage
gated potassium channel, subfamily T; Slo, high conductance
potassium channel; iGluR, ionotropic glutamate receptor; Kv,
voltage-gated potassium channel; Nav, voltage-gated sodium
channel; Cav, voltage-gated calcium channel
■
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■ NOTE ADDED AFTER ASAP PUBLICATION

After this paper was published online November 29, 2012,
changes were made to references 123 and 124. The corrected
version was reposted December 3, 2012.

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