Current
Pharmaceutical Design
ISSN: 1381-6128
Current Pharmaceutical Design
Volume 13, Number 23, 2007
Contents
Membrane Channels as Therapeutic Targets
Executive Editor: Jean-Claude Hervé
Editorial Pp. 2323-2324
Automated Electrophysiology in Drug Discovery
Pp. 2325-2337
B.T. Priest, A.M. Swensen and O.B. McManus
[Abstract]
Molecular Regulation and Pharmacology of Pacemaker
Channels Pp. 2338-2349
P. Bois, R. Guinamard, A. El Chemaly, J.-F. Faivre and
J. Bescond
[Abstract]
Molecular Pharmacology of the Glycine Receptor Chloride
Channel Pp. 2350-2367
T.I. Webb and J.W. Lynch
[Abstract]
Purine Ionotropic (P2X) Receptors Pp. 2368-2384
L. Köles, S. Fürst and P. Illes
[Abstract]
Channel-Like Functions of the 18-kDa Translocator
Protein (TSPO): Regulation of Apoptosis and Steroidogenesis
as Part of the Host-Defense Response Pp. 2385-2405
L. Veenman, V. Papadopoulos and M. Gavish
[Abstract]
Pharmacology of Voltage-Gated Proton Channels
Pp. 2406-2420
T.E. DeCoursey and V.V. Cherny
[Abstract]
Aquaporins as Targets for Drug Discovery
Pp. 2421-2427
A. Frigeri, G.P. Nicchia and M. Svelto
[Abstract]
Abstracts
[Back to top]
Editorial: Membrane Channels as Therapeutic Targets
– Part IV
The production of new molecular entities endowed with salutary
medicinal properties is a formidable challenge that involves
several steps and requests rational target identification,
recognition and avoidance of adverse properties of therapeutics
before commitment to clinical trials, monitoring of clinical
efficacy using surrogate markers and individualized approaches
to disease treatment. The first to face up to is the initial
identification and selection of macromolecular targets upon
which de novo drug discovery programs can be initiated.
A drug target needs to answer several criteria (as known biological
function(s), robust assay systems for in vitro characterisation
and high-throughput screening) and to be specifically modified
by and accessible to small molecular weight compounds in
vivo. Membrane channels have many of these attributes
and can be viewed as suitable targets for small molecule drugs.
Channels are membrane-embedded proteins that contain one or
several integral pore(s) able to open and to close (a process
called gating), allowing ions and sometimes small molecules
to flow across the cell membrane in a regulated manner. Their
gating can be modulated by various stimuli including changes
in membrane voltage, binding of extracellular or intracellular
ligands, membrane stretch, enzymes and G-proteins. They play
critical roles in a broad range of physiological processes,
including electrical signal transduction, chemical signalling
(involving different second messengers), transepithelial transport,
regulation of cytoplasmic or vesicular ion concentration and
pH, as well as regulation of cell volume. Channel dysfunction
may lead to a number of diseases termed channelopathies, and
a number of common diseases (e.g. epilepsy, hypertension,
arrhythmia, chronic pain or type II diabetes) are primarily
treated by drugs that modulate ion channel activities.
The cell-based methods for evaluating membrane channel pharmacology
are based on several distinct techniques such as electrophysiology,
fluorescence, radioligand binding or displacement, and radiotracer
flux assays. A better understanding of membrane channel structures
and of channel functions has been achieved in recent years
by three main scientific advances, the patch-clamp technique,
the use of selective neurotoxins and the cloning and sequencing
of genes. They allowed investigating the pharmacological effects
of traditional (antiarrhythmic, antiepileptic, ...) drugs
and the development of new approaches. This issue of Current
Pharmaceutical Design, the last of four parts, for which I
have the honour to be Executive Guest Editor, addresses topical
issues to some of these channels.
The properties of ionic channels are most often investigated
by means of voltage clamp approaches, particularly the patch
clamp technique, which allows direct electrical measurement
of ion channel currents while simultaneously controlling the
cell’s membrane potential. It relies on the use of a
fine tipped glass capillary to make contact with a patch of
a cell membrane in order to form a giga-ohm seal. However,
these assays are technically challenging and notoriously low-throughput.
The recent development of several automated electrophysiology
platforms has greatly increased the throughput of whole cell
electrophysiological recordings, allowing them to play a more
central role in ion channel drug discovery. Birgit Priest,
Andrew Swensen and Owen McManus [1] present these technologies,
which promise to enable more rapid and efficient identification
of specific ion channel modulators, which will, in turn, aid
efforts to understand the functional roles of specific ion
channels and provide new therapeutic approaches to disease
states.
Pacemaking is an electrical phenomenon, based on the function
of ion channel proteins expressed on the membrane of some
types of specialized cells (either cardiomyocytes, neurons,
or smooth muscle cells), which allows them to generate repetitive
action potentials at a constantly controlled rate. The properties
of “pacemaker” currents (termed Ih
(h for hyperpolarization-activated), If
(f for funny) or Iq (q for
queer)) are deemed unique, particularly its direct regulation
by intracellular cyclic nucleotides. Patrick Bois, Romain
Guinamard, Antoun el Chemaly, Jean-François Faivre
and Jocelyn Bescond [2] describe the molecular diversity of
the Hyperpolarization-activated Cyclic Nucleotide-gated (HCN)
channels, their biophysical properties and their potential
therapeutic use.
Glycine, the simplest of the amino acids, has diverse metabolic
functions within the mammalian central nervous system; as
GABA, glycine serves as a neurotransmitter at inhibitory synapses,
where it activates strychnine-sensitive glycine receptors
(GlyRs) which, like GABAA/C
receptors, belong to the pentameric nicotinic acetylcholine
receptor (nAChR) superfamily. GlyRs, widely distributed throughout
the central nervous system, are also found in sperm and macrophages.
When glycine binds to its site on the external receptor surface,
the pore opens, allowing Cl–
to passively diffuse across the membrane, and molecules able
to increase GlyR current may have clinical potential (e.g.
as muscle relaxant and peripheral analgesic drugs). Timothy
Webb and Joseph Lynch [3] overview the current knowledge available
concerning the pharmacology of these receptors.
The nucleotide ATP is not only indispensable inside cells
but also a signalling molecule between them, and, like other
transmitters, it activates a family of metabotropic receptors
(P2Y, coupled to intracellular second-messenger systems through
heteromeric G-proteins) as well as ionotropic receptors (P2X)
which contain intrinsic pores able to switch conformation
from closed to open on binding ATP, allowing ions to flow,
to change the transmembrane potential as well as the local
ion concentrations. P2X receptors are not only present within
nervous tissues but widely expressed, not only within nervous
tissue, and progress on several fronts has revealed the diversity
of cellular signalling in very many tissues. Laszlo Köles,
Susanna Fürst and Peter Illes [4] summarize distribution
and functional properties and pharmacology of P2X receptors
and highlight the role of purines in organ systems and body
functions.
In contrast to central-type benzodiazepine receptors, which
are located primarily in neurons in the central nervous system,
peripheral-type benzodiazepine receptors, recently renamed
translocator proteins (TSPOs), are present in peripheral tissues,
particularly in steroid synthesizing tissues. In eukaryotes,
the TSPOs are primarily located in the outer mitochondrial
membrane. TSPO appears to operate as a translocator/channel
to transfer cholesterol into mitochondria where it is converted
to pregnenolone, a precursor of further steroidogenesis. The
widespread expression of TPSOs throughout the animal kingdom
suggests they may have essential functions in different domains.
Apoptosis and steroids play for example important roles in
various aspects of the host defence response. Thus, Leo Veenman,
Vassilios Papadopoulos and Moshe Gavish [5] suggest in their
review that the involvement of TSPO and its ligands in such
seemingly disparate biological functions (as immunological
responses, apoptosis, and steroidogenesis) may have a common
denominator in the multi-dimensional role of TSPO in the host-defence
response to disease and injury.
Protons are unique among cations, particularly in their tiny
size, low free but enormous total concentration and their
behaviour in bulk solutions, in particular their reactivity
with other molecules (e.g. proteins). Voltage-gated
proton channels are unique ion channels in several respects.
They are called proton channels because they behave like ion
channels and are highly selective for protons. Although protons
exist in solution almost entirely in the form of hydronium
ions, H3O+,
all proton-selective channels conduct protons as H+,
rather than H3O+.
Thomas de Coursey and Vladimir Cherny [6] summarize the current
knowledge of the functions of voltage-gated proton channels
and emphasize their interest as potential targets for pharmacological
modulation of the production of reactive oxygen species, important
not only as bactericidal agents, but also in signalling and
as possible causes of microglia-mediated self injury in diseases
such as in Alzheimer’s and HIV-1 associated dementia.
The molecular basis of membrane water-permeability remained
elusive until the discovery of the aquaporin water-channel
(AQPs) proteins. It is now evident that membrane water permeability
can be regulated independently of solute permeability, and
the fundamental importance of these proteins is suggested
by their conservation from bacteria through plants to mammals.
Most aquaporins are selectively permeated by water, although
some family members are permeated by other small molecules.
Aquaporins are present in the membrane as tetramers, but,
unlike ion channels, the channel pore does not reside at the
centre of the tetramer but each of the monomers contains a
channel. Antonio Frigeri, Grazia Paola Nicchia and Maria Svelto
[7] highlight the physiological role and the pathological
involvement of AQPs in mammals and the potential use of some
recent therapeutic approaches (e.g. RNAi and immunotherapy)
for AQP-related diseases.
Many physiological processes, ranging from gene transcription,
membrane excitability, cell proliferation to learning and
memory, contraction and secretion, are regulated by changes
in the [Ca2+]i,
and one of the main involved mechanisms is the release of
Ca2+
from intracellular stores, mediated by two subfamilies of
intracellular Ca2+-release
channels, inositol 1,4,5-trisphosphate receptors (IP3Rs) and
ryanodine receptors (RyRs). The calcium signals mediated by
RyRs and IP3Rs are very different in kinetics, amplitude and
subcellular localization. Duncan West and Alan Williams [8]
present an overview of the plethora of exogenous pharmacological
agents that been shown to interact with and to modulate intracellular
Ca2+
release channels and describe the mechanisms underlying their
ability to modify channel function.
Pulmonary arterial hypertension (PAH) is a life-threatening
disorder that refers to a group of diseases characterized
by an abnormal elevation of the blood pressure within the
pulmonary circulation due to a vasculopathy of the pulmonary
microcirculation. All forms of PAH are characterized by pulmonary
vasoconstriction and remodelling of the arterial wall. Ion
channels in pulmonary artery smooth muscle cells (PASMC) are
implicated in both phenomena. In primary PAH or in the course
of development of the other PAH types, ions channels activity
and/or expression is altered leading, on the one hand, to
membrane depolarisation, calcium influx and vasoconstriction
and, on the other hand, to proliferation, decreased PASMC
apoptosis and vascular remodelling. Christelle Guibert, Roger
Marthan and Jean-Pierre Savineau [9] describe the involvement
of the different families of ion channels in the control of
both membrane potential and resting cytosolic Ca2+
concentration, which are key parameters of PASMC in PAH, provide
evidence for an implication of these channels in not only
vasoconstriction but also in proliferation and/or decreased
apoptosis of PASMC and present examples of substances acting
on ion channels and thus potentially constituting innovative
therapeutic approaches of PAH.
Membrane channels are involved in a wide range of cellular
functions and their defects produce a clinically diverse set
of disorders that range from cystic fibrosis to some forms
of migraine, renal tubular defects, episodic ataxias but also
to several autoimmune processes. For the latter, Zoltán
Varga, Péter Hajdu, György Panyi, Rezso Gáspár
and Zoltán Krasznai [10] examine the involvement of
ion channels in three situations: (i) channels in
effector immune cells attacking other tissues and causing
autoimmune diseases, (ii) channels as direct targets
of the immune system whereby loss of channel function leads
to disease and (iii) channels whose function is modulated
in the target cells by an apoptotic signal transduction cascade.
I wish to thank all the authors and co-authors for their commitments
and the anonymous reviewers who contributed by their constructive
remarks to the excellence of this issue.
References
[1] Priest BT, Swensen AM, McManus OB. Automated Electrophysiology
in Drug Discovery. Curr Pharm Des 2007; 13(23): 2325-2337.
[2] Bois P, Guinamard R, el Chemaly A, Faivre JF, Bescond
J. Molecular regulation and pharlacology of pacemaker channels.
Curr Pharm Des 2007; 13(23): 2338-2349.
[3] Webb TI, Lynch JW. Molecular Pharmacology of the Glycine
Receptor Chloride Channel. Curr Pharm Des 2007; 13(23): 2350-2367.
[4] Köles L, S. Fürst S, Illes P. Purinergic (P2X)
receptors. Curr Pharm Des 2007; 13(23): 2368-2384.
[5] Veenman L, Papadopoulos V, Gavish M. Channel-like functions
of the 18-kDa translocator protein (TSPO): Regulation of apoptosis
and steroidogenesis as part of the host-defense response.
Curr Pharm Des 2007; 13(23): 2385-2405.
[6] de Coursey? TE, Cherny VV. Pharmacology of Voltage-Gated
Proton Channels. Curr Pharm Des 2007; 13(23): 2406-2420.
[7] Frigeri A, Nicchia GP, Svelto M. Aquaporins as targets
for drug discovery. Curr Pharm Des 2007; 13(23): 2421-2427.
[8] West DJ, Williams AJ. Pharmacological regulators of intracellular
calcium release channels. Curr Pharm Des 2007; 13(24): 2428-2442.
[9] Guibert C, Marthan R, Savineau JP. Modulation of ion channels
in pulmonary arterial hypertension. Curr Pharm Des 2007; 13(24):
2443-2455.
[10] Varga Z, Hajdu P, Panyi G, Gáspár R, Krasznai
Z. Involvement of membrane channels in autoimmune disorders.
Curr Pharm Des 2007; 13(24): 2456-2468.
Jean-Claude Hervé
Interactions et Communications Cellulaires
UMR CNRS 6187, PBS, 40 avenue du R. Pineau
86022 POITIERS Cédex
France
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Automated Electrophysiology in Drug Discovery
B.T. Priest, A.M. Swensen and O.B. McManus
Ion channels play essential roles in nervous system signaling,
electrolyte transport, and muscle contraction. As such, ion
channels are important therapeutic targets, and the search
for compounds that modulate ion channels is accelerating.
In order to identify and optimize ion channel modulators,
assays are needed that are reliable and provide sufficient
throughput for all stages of the drug discovery process. Electrophysiological
assays offer the most direct and accurate characterization
of channel activity and, by controlling membrane potential,
can provide information about drug interactions with different
conformational states. However, these assays are technically
challenging and notoriously low-throughput. The recent development
of several automated electrophysiology platforms has greatly
increased the throughput of whole cell electrophysiological
recordings, allowing them to play a more central role in ion
channel drug discovery. While challenges remain, this new
technology will facilitate the pharmaceutical development
of ion channel modulators.
[Back to top]
Molecular Regulation and Pharmacology of Pacemaker
Channels
P. Bois, R. Guinamard, A. El Chemaly, J.-F. Faivre and
J. Bescond
The spontaneous activity of cardiac tissue originates in specialized
pacemaker cells in the sino-atrial node that generate autonomous
rhythmic electrical impulses. A number of regions in the brain
are also able to generate spontaneous rhythmic activity to
control and regulate important physiological functions. The
generation of pacemaker potentials relies on a complex interplay
between different types of currents carried by cation channels.
Among these currents, the hyperpolarization-activated current
(termed If, cardiac pacemaker
“funny” current, and Ih
in neurons) is the major component contributing to the initiation
of cardiac and neuronal excitability and to the modulation
of this excitability by neurotransmitters and hormones. Ih
is an inward current activated by hyperpolarization of the
membrane potential and by intracellular cyclic nucleotides
such as cAMP.
The identification at the end of the 1990s of a family of
mammalian genes that encode for four Hyperpolarization-activated
Cyclic Nucleotide-gated channels, HCN1-4, has made analysis
of the location of these channels and the study of their biophysical
properties an obtainable goal. As a result, specific agents
have been developed for their ability to selectively reduce
heart rate by lowering cardiac pace-maker activity where f-channels
are their main natural target. These drugs include alinidine,
zatebradine, cilobradine, ZD-7288 and ivabradine. Recent data
indicate that pharmacological tools such as W7 and genistein,
which have been used to identify some intracellular pathways
involved in ionic channel modulation, also have the ability
to inhibit If directly. This opens new perspectives for the
future development of other specific rhythm-lowering agents.
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Molecular Pharmacology of the Glycine Receptor Chloride
Channel
T.I. Webb and J.W. Lynch
The glycine receptor (GlyR) Cl- channel belongs to the cysteine-loop
family of ligand-gated ion channel receptors. It is best known
for mediating inhibitory neurotransmission in motor and sensory
reflex circuits of the spinal cord, although glycinergic synapses
are also present in the brain stem, cerebellum and retina.
Extrasynaptic GlyRs are widely distributed throughout the
central nervous system and they are also found in sperm and
macrophages. A total of 5 GlyR subunits (α1-4
and β)
have been identified. Embryonic receptors comprise α2
homomers whereas adult receptors comprise predominantly α1β
heteromers in a 2:3 stoichiometry. Notably, the α3
subunit is present in synaptic GlyRs that mediate inhibitory
neurotransmission onto spinal nociceptive neurons. These receptors
are specifically inhibited by inflammatory mediators, implying
a role for α3-containing
GlyRs in inflammatory pain sensitisation.
Because molecules that increase GlyR current may have clinical
potential as muscle relaxant and peripheral analgesic drugs,
this review focuses on the molecular pharmacology of GlyR
potentiating substances. Of all GlyR potentiating substances
identified to date, we conclude that 5HT3R
antagonists such as tropisetron offer the most promise as
therapeutic lead compounds. However, one problem is that that
virtually all known GlyR potentiating compounds, including
tropisetron analogues, lack specificity for the GlyR. Another
is that almost nothing is known about the pharmacological
properties of α3-containing
GlyRs, which is the subtype of choice for targeting by novel
antinociceptive agents. These issues need to be addressed
before GlyR-specific therapeutics can be developed.
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Purine Ionotropic (P2X) Receptors
L. Köles, S. Fürst and P. Illes
Purinergic signaling is involved in the proper functioning
of virtually all organs of the body. Although in some cases
purines have a major influence on physiological functions
(e.g. thrombocyte aggregation), more often they are just background
modulators contributing to fine tuning of biological events.
However, under pathological conditions, when a huge amount
of adenosine 5'-triphosphate (ATP) can reach the extracellular
space, their significance is increasing. ATP and its various
degradation products activate membrane receptors divided into
two main classes: the metabotropic P2Y and the ionotropic
P2X family. This latter group, the purine ionotropic receptor,
is the object of this review. After providing a description
about the distribution and functional properties of P2X receptors
in the body, their pharmacology will be summarized. In the
second part of this review, the role of purines in those organ
systems and body functions will be highlighted, where the
(patho)physiological role of P2X receptors has been suggested
or is even well established. Besides the regulation of organ
systems, for instance in the cardiovascular, respiratory,
genitourinary or gastrointestinal system, some special issues
will also be discussed, such as the role of P2X receptors
in pain, tumors, central nervous system (CNS) injury and embryonic
development. Several examples will indicate that purine ionotropic
receptors might serve as attractive targets for pharmacological
interventions in various diseases, and that selective ligands
for these receptors will probably constitute important future
therapeutic tools in humans.
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Channel-Like Functions of the 18-kDa Translocator
Protein (TSPO): Regulation of Apoptosis and Steroidogenesis
as Part of the Host-Defense Response
L. Veenman, V. Papadopoulos and M. Gavish
Due to its channel-like properties, the peripheral-type benzodiazepine
receptor (PBR) has been renamed the translocator protein (TSPO).
In eukaryotes, the TSPO is primarily located in the outer
mitochondrial membrane. In prokaryotes, it is found in the
cell membrane. A broad spectrum of functions has been attributed
to the TSPO, including various host defense responses, developmental
processes, and mitochondrial functions. In the present review,
we focus on the role of TSPO in immunological responses, apoptosis,
and steroidogenesis, to determine whether these functions
may be governed by a common denominator including TSPO. At
physiological concentrations (nM range), the TSPO specific
ligands, PK 11195 and Ro5-4864, appear to be anti-apoptotic.
Knockdown of TSPO by genetic manipulation, resulting a reduction
by more than 50% in [3H]PK
11195 binding, was reported to show anti-apoptotic effects,
suggesting a potential pro-apoptotic function of TSPO. However,
a reduction of more than 70% of TSPO abundance was found to
cause cell death, possibly due to impairment of other essential
cell functions. The pro-apoptotic function of TSPO may involve
the modulation of the channel formed by the mitochondrial
voltage-dependent anion channel (VDAC) and the adenine nucleotide
transporter (ANT) [i.e., the mitochondrial permeability transition
pore (MPTP)]. The frequently reported pro-apoptotic effects
of PK 11195 and Ro5-4864 may be due to sites with low-affinity
binding for these specific TSPO ligands, and not directly
related to VDAC and ANT. Also at concentrations in the nM
range, PK 11195 and Ro5-4864 appear to stimulate steroidogenesis.
For this function TSPO by itself appears to suffice i.e. no
involvement of VDAC and ANT. TSPO appears to operate as a
translocator/channel to transfer cholesterol into mitochondria
where it is converted to pregnenolone, a precursor of further
steroidogenesis. Apoptosis and steroids play important roles
in various aspects of the host defense response. Thus, our
review suggests that the involvement of TSPO and its ligands
in such seemingly disparate biological functions as immunological
responses, apoptosis, and steroidogenesis may have a common
denominator in the multi-dimensional role of TSPO in the host-defense
response to disease and injury.
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Pharmacology of Voltage-Gated Proton Channels
T.E. DeCoursey and V.V. Cherny
Voltage-gated proton channels are highly proton selective
ion channels that are present in many cells. Although their
unitary conductance is 1000 times smaller than that of most
ion channels, detection of single-channel currents supports
their identification as channels rather than carriers. Proton
channels are gated by membrane depolarization, but their absolute
voltage dependence is also strongly regulated by the pH gradient,
ΔpH
(pHo - pHi).
A model of this behavior postulates regulatory protonation
sites that are alternately accessible to external or internal
solutions. Consequently, proton channels open only when the
electrochemical gradient is outward, and serve to extrude
acid from cells. No “classical” blockers of proton
channels that bind to and physically occlude the channel have
been identified. A number of weak bases that inhibit proton
currents probably act indirectly, perhaps by changing local
pH. The best known and most potent inhibitors are polyvalent
cations, especially Zn2+
and Cd2+. These cations are
coordinated at two or more external protonation sites, most
likely His residues where they compete with protons and interfere
with gating. In phagocytes, proton channels are required to
compensate for the electrogenic action of NADPH oxidase. During
the “respiratory burst,” i.e., when NADPH oxidase
is active, proton channels in these cells adopt an “activated”
gating mode. Recently, two labs identified a gene that codes
for either the proton channel itself or a protein that is
essential for proton channel activity. Expression of this
protein results in currents with many similarities to the
native channel.
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Aquaporins as Targets for Drug Discovery
A. Frigeri, G.P. Nicchia and M. Svelto
The intracellular hydric balance is an essential process of
mammalian cells. The water movement across cell membranes
is driven by osmotic and hydrostatic forces and the speed
of this process is dependent on the presence of specific aquaporin
water channels. Since the molecular identification of the
first water channel, AQP1, by Peter Agre’s group, 13
homologous members have been found in mammals with varying
degree of homology. The fundamental importance of these proteins
in all living cells is suggested by their genetic conservation
in eukaryotic organisms through plants to mammals.
A number of recent studies have revealed the importance of
mammalian AQPs in both physiology and pathophysiology and
have suggested that pharmacological modulation of aquaporins
expression and activity may provide new tools for the treatment
of variety of human disorders, such as brain edema, glaucoma,
tumour growth, congestive heart failure and obesity in which
water and small solute transport may be involved. This review
will highlight the physiological role and the pathological
involvement of AQPs in mammals and the potential use of some
recent therapeutic approaches, such as RNAi and immunotherapy,
for AQP-related diseases. Furthermore, strategies that can
be developed for the discovery of selective AQP-drugs will
be introduced and discussed.
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