Current Pharmaceutical Design - Volume 13, Issue 23, 2007

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 Gproteins. 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............

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.

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. If 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 pacemaker 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.

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.

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.

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 multidimensional role of TSPO in the host-defense response to disease and injury.

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.

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.