Screening Technologies for Inward Rectifier Potassium Channels: Discovery of New Blockers and Activators.

K+ channels play a critical role in maintaining the normal electrical activity of excitable cells by setting the cell resting membrane potential and by determining the shape and duration of the action potential. In nonexcitable cells, K+ channels establish electrochemical gradients necessary for maintaining salt and volume homeostasis of body fluids. Inward rectifier K+ (Kir) channels typically conduct larger inward currents than outward currents, resulting in an inwardly rectifying current versus voltage relationship. This property of inward rectification results from the voltage-dependent block of the channels by intracellular polyvalent cations and makes these channels uniquely designed for maintaining the resting potential near the K+ equilibrium potential (EK). The Kir family of channels consist of seven subfamilies of channels (Kir1.x through Kir7.x) that include the classic inward rectifier (Kir2.x) channel, the G-protein-gated inward rectifier K+ (GIRK) (Kir3.x), and the adenosine triphosphate (ATP)-sensitive (KATP) (Kir 6.x) channels as well as the renal Kir1.1 (ROMK), Kir4.1, and Kir7.1 channels. These channels not only function to regulate electrical/electrolyte transport activity, but also serve as effector molecules for G-protein-coupled receptors (GPCRs) and as molecular sensors for cell metabolism. Of significance, Kir channels represent promising pharmacological targets for treating a number of clinical conditions, including cardiac arrhythmias, anxiety, chronic pain, and hypertension. This review provides a brief background on the structure, function, and pharmacology of Kir channels and then focuses on describing and evaluating current high-throughput screening (HTS) technologies, such as membrane potential-sensitive fluorescent dye assays, ion flux measurements, and automated patch clamp systems used for Kir channel drug discovery.

Thermostable potassium channel-inhibiting neurotoxins in processed scorpion medicinal material revealed by proteomic analysis: Implications of its pharmaceutical basis in traditional Chinese medicine.

The neurotoxins of venomous scorpion act on ion channels. Whether these neurotoxins are retained in processed Buthus martensii Karsch scorpions used in traditional Chinese medicine materials is unknown. Comprehensive mass spectrometry-based proteomic characterization of functionally active toxins in the processed medicinal scorpion material revealed 22 full-length and 44 truncated thermostable potassium channel-modulatory toxins that preserved six conserved cysteine residues capable of forming the three disulfide bonds necessary for toxicity. Additionally, a broad spectrum of degraded toxin fragments was found, indicating their relative thermal instability which enabled toxicity reduction. Furthermore, the suppression of interleukin-2 (IL-2) production in Jurkat cells and the reduced delayed-type hypersensitivity (DTH) response demonstrated that the extracts have immunoregulatory activity both in vitro and in vivo. Our work describes the first "map" of functionally active scorpion toxins in processed scorpion medicinal material, which is helpful to unveil the pharmaceutical basis of the processed scorpion medicinal material in traditional Chinese medicine. BIOLOGICAL SIGNIFICANCE: Scorpions have been used as medicinal materials in China for more than one thousand years. This is an example of the well-known "Combat poison with poison" strategy common to traditional Chinese medicine. In the past 30 years, extensive investigations of Chinese scorpions have indicated that the neurotoxins in the scorpion venom are the main toxic components and they target various ion channels in cell membranes. However, whether these neurotoxins are retained in processed Buthus martensii Karsch scorpions used for traditional Chinese medicine remains unknown. Our study described the thermal stability and instability of potassium channel-modulatory neurotoxins in processed scorpions and helps to understand the pharmaceutical basis underling the strategy of "combat poison with poison to cure diseases".

Three Minor Diterpenoids with Three Carbon Skeletons from Euphorbia peplus.

Euphorbia peplus has been used in traditional medicine to treat asthma and psoriasis. Three highly modified diterpenoids, namely, pepluacetal (1) and pepluanol A-B (2-3), have been isolated and identified from this plant. Compounds 1-3 exhibit unprecedented 5/4/7/3, 5/6/7/3, and 5/5/8/3 ring systems, respectively. Their structures with absolute configurations were determined by spectroscopic analyses, X-ray crystallography, and electronic circular dichroism calculations. Since Kv1.3 is a validated target for the treatment of autoimmune diseases, such as multiple sclerosis, type-1 diabetes, asthma, and psoriasis, Kv1.3 was studied in terms of its response to the new compounds. All three compounds inhibit Kv1.3, with compound 3 being the most effective with an IC50 value of 9.50 µM.

HPLC-Based Activity Profiling for hERG Channel Inhibitors in the South African Medicinal Plant Galenia africana.

The human ether-a-go-go-related gene channel is a voltage-activated K(+) channel involved in cardiac action potential. Its inhibition can lead to QT prolongation, and eventually to potentially fatal arrhythmia. Therefore, it is considered a primary antitarget in safety pharmacology. To assess the risk of human ether-a-go-go-related gene channel inhibition by medicinal plants, 700 extracts from different parts of 142 medicinal plants collected in Southern Africa were screened on Xenopus laevis oocytes. A CH2Cl2 extract from the stems and leaves of Galenia africana (Aizoaceae) reduced the peak tail human ether-a-go-go-related gene current by 50.4 ± 5.5 % (n = 3) at a concentration of 100 µg/mL. By means of high-performance liquid chromatography-based activity profiling, nine flavonoids were identified in the active time windows. However, the human ether-a-go-go-related gene channel inhibition of isolated compounds was less pronounced than that of extract and active microfractions (human ether-a-go-go-related gene inhibition between 10.1 ± 5 and 14.1 ± 1.6 at 100 µM). The two major constituents, 7,8-methylenedioxyflavone (1) and 7,8-dimethoxyflavone (13), were quantified (4.3 % and 9.4 %, respectively, in the extract). Further human ether-a-go-go-related gene inhibition tests for compounds 1 and 13 at 300 µM showed a concentration-dependent inhibitory activity (33.2 ± 12.4 and 30.0 ± 7.4, respectively). In a detailed phytochemical profiling of the active extract, a total of 20 phenolic compounds, including six new natural products, were isolated and identified.

Neurotoxic and neuroactive compounds from Cnidaria: five decades of research .and more.

Cnidarians are numbered among the most venomous organisms. Their venoms are contained in intracellular capsules, nematocysts, which inject the content into preys/attackers through an eversion system resembling a syringe needle. Several cnidarian venoms have activity against the nervous system, being neurotoxic, or affect other systems whose functioning is under nerve control. Besides direct damage to nerve cells, the activity on ionic conductance, blockade of neuromuscular junctions, and influence on action potentials and on voltage-gated channels have been described. Therefore, cnidarians can be a useful source of nervous system-targeted compounds which could have, in perspective, a role in the therapy of some nervous system diseases. Following this idea, this article aims to review the existing data about the neuroactive properties of cnidarian venoms and their possible usefulness in tackling some neurological diseases as well as neurodegenerative age-related diseases whose incidence is expected to raise in the next decades owing to the increase of life expectancy.

Scorpion venom components as potential candidates for drug development.

Scorpions are well known for their dangerous stings that can result in severe consequences for human beings, including death. Neurotoxins present in their venoms are responsible for their toxicity. Due to their medical relevance, toxins have been the driving force in the scorpion natural compounds research field. On the other hand, for thousands of years, scorpions and their venoms have been applied in traditional medicine, mainly in Asia and Africa. With the remarkable growth in the number of characterized scorpion venom components, several drug candidates have been found with the potential to tackle many of the emerging global medical threats. Scorpions have become a valuable source of biologically active molecules, from novel antibiotics to potential anticancer therapeutics. Other venom components have drawn attention as useful scaffolds for the development of drugs. This review summarizes the most promising candidates for drug development that have been isolated from scorpion venoms.

Identification of diterpene alkaloids from Aconitum napellus subsp. firmum and GIRK channel activities of some Aconitum alkaloids.

Diterpene alkaloids neoline (1), napelline (2), isotalatizidine (3), karakoline (4), senbusine A (5), senbusine C (6), aconitine (7) and taurenine (8) were identified from Aconitum napellus L. subsp. firmum, four (2-4, 6) of which are reported for the first time from this plant. The structures were determined by means of LC-MS, 1D and 2D NMR spectroscopy, including (1)H-(1)H COSY, NOESY, HSQC and HMBC experiments. Electrophysiological effects of the isolated compounds, together with nine diterpene alkaloids previously obtained from Aconitum toxicum and Consolida orientalis were investigated on stable transfected HEK-hERG (Kv11.1) and HEK-GIRK1/4 (Kir3.1 and Kir3.4) cell lines using automated patch clamp equipment. Significant blocking activity on GIRK channel was exerted by aconitine (7) (45% at 10 µM), but no blocking activities of the other investigated compounds were detected. The tested compounds were inactive on hERG channel in the tested concentration. The comparison of the previously reported metabolites of A. napellus subsp. firmum and compounds identified in our experiment reveals substantial variability of the alkaloid profile of this taxon.

Diterpene alkaloids from Aconitum anthora and assessment of the hERG-inhibiting ability of Aconitum alkaloids.

A new norditerpene alkaloid, 10-hydroxy-8- O-methyltalatizamine (1), was isolated from the whole plant of ACONITUM ANTHORA L. besides the known isotalatizidine (2) and hetisinone (3). The structures were determined by means of HR-ESI-MS, 1D and 2D NMR spectroscopy, including ¹H-¹H COSY, NOESY, HSQC and HMBC experiments, resulting in complete ¹H and ¹³C chemical shift assignments for 1- 3, and revision of some earlier ¹³C-NMR data. The effects of the isolated compounds, together with twenty-one other ACONITUM alkaloids with different skeletal types and substitution patterns, on hERG channels were studied by the whole-cell patch clamp technique, using the QPatch-16 automated patch clamp system. At 10 µM, aconitine, 14-benzoylaconine 8- O-palmitate, songoramine, gigactonine and neolinine demonstrated significant hERG K+ channel inhibition; all other compounds exerted only low (6-21%) inhibitory activity.

A potent potassium channel blocker from Mesobuthus eupeus scorpion venom.

Scorpion venom-derived peptidyl toxins are valuable pharmacological tools for investigating the structure-function relationship of ion channels. Here, we report the purification, sequencing and functional characterization of a new K(+) channel blocker (MeuKTX) from the venom of the scorpion Mesobuthus eupeus. Effects of MeuKTX on ten cloned potassium channels in Xenopus oocytes were evaluated using two-electrode voltage-clamp recordings. MeuKTX is the orthologue of BmKTX (α-KTx3.6), a known Kv1.3 blocker from the scorpion Mesobuthus martensii, and classified as α-KTx3.13. MeuKTX potently blocks rKv1.1, rKv1.2 and hKv1.3 channels with 50% inhibitory concentration (IC(50)) of 203.15 ± 4.06 pM, 8.92 ± 2.3 nM and 171 ± 8.56 pM, respectively, but does not affect rKv1.4, rKv1.5, hKv3.1, rKv4.3, and hERG channels even at 2 µM concentration. At this high concentration, MeuKTX is also active on rKv1.6 and Shaker IR. Our results also demonstrate that MeuKTX and BmKTX have the same channel spectrum and similar pharmacological potency. Analysis of the structure-function relationships of α-KTx3 subfamily toxins allows us to recognize several key sites which may be useful for designing toxins with improved activity on hKv1.3, an attractive target for T-cell mediated autoimmune diseases.

Inhibitory effects of isoliquiritigenin and licorice extract on voltage-dependent K(+) currents in H9c2 cells.

The effect of isoliquiritigenin (ISL), a component of licorice, on the voltage-dependent, ultra-rapidly activating delayed-rectifier K(+) current (IKur) was examined in H9c2 cells, a cell-line derived from rat cardiac myoblasts. IKur was recorded using the whole-cell patch clamp method with a pipette solution containing 140 mM K(+). Depolarizing voltage pulses of 200-ms duration were given with 10-mV steps every 10 s from -40 mV holding potential. ISL inhibited IKur in a concentration-dependent manner. The median inhibitory concentration (IC(50)) of ISL was approximately 0.11 microM and the Hill coefficient was 0.71. Using CHO cells expressing Kv1.5 IKur channels, ISL also inhibited Kv1.5 IKur, but less potently than the IKur current in H9c2 cells. Furthermore, in H9c2 cells, the licorice extract itself inhibited IKur in a manner similar to ISL. We conclude that ISL, one component of licorice, is a potent inhibitor of K(+) channels, which specifically in H9c2 cells could be Kv2.1, and that this inhibition may be involved in various pharmacological effects of licorice.

Immobilization effect of Ruta graveolens L. on human sperm: a new hope for male contraception.

AIM OF THE STUDY: Contraceptive plants which were introduced by folk in traditional remedies are investigated worldwide. In this study, the contraceptive effects of Ruta graveolens L., which has been mentioned for male contraceptive in Iranian traditional folk medicine, was experimented on human sperm. MATERIALS AND METHODS: Different doses of lyophilized aqueous extract of Ruta graveolens L. were added to an amount of fresh semen, containing 10(6) cells in a 1:1 volumic ratio. Motility and viability of cells, DNA status, mitochondrial activity and sperm revival tests were carried out. RESULTS: The sperm immobilization effects of the extract appeared immediately in a does-dependent manner and 100% of the sperms became immotile at a concentration of 100mg/ml but other parameters were intact. After washing the sperms, motility was returned in 30.8+/-3.2% of the sperms, besides coiled tails in 38.6+/-5.5% of the treated cells, in comparison to 12.5+/-2.0% of the control group (p=0.001). The part of the extract, responsible for immobilization of the sperms was stable upon boiling. CONCLUSIONS: As the cells were alive and immotile, probably some ionic currents are blocked by a thermostable component of the plant which can be promising as a new male channel blocker contraceptive.

Cloning and characterization of BmK86, a novel K+ -channel blocker from scorpion venom.

Scorpion venom represents a tremendous hitherto unexplored resource for understanding ion channels. BmK86 is a novel K+ -channel toxin gene isolated from a cDNA library of Mesobuthus martensii Karsch, which encodes a signal peptide of 22 amino acid residues and a mature toxin of 35 residues with three disulfide bridges. The genomic sequence of BmK86 consists of two exons disrupted by an intron of 72 bp. Comparison with the other scorpion toxins BmK86 shows low sequence similarity. The GST-BmK86 fusion protein was successfully expressed in Escherichia coli. The fusion protein was cleaved by enterokinase and the recombinant BmK86 was purified by HPLC. Using whole-cell patch-clamp recording, the recombinant BmK86 was found to inhibit the potassium current of mKv1.3 channel expressed in COS7 cells. These results indicated that BmK86 belongs to a representative member of a novel subfamily of alpha-KTxs. The systematic number assigned to BmK86 is alpha-KTx26.1.

Novel neuroprotective K+ channel inhibitor identified by high-throughput screening in yeast.

Discovery of K+ channel modulators is limited by low-throughput capacity of existing K+ channel assays. To enable high-throughput screening for novel pharmacological modulators of K+ channels, we developed an assay based on growth of yeast that functionally expresses mammalian Kir2.1 channels. Screening of 10,000 small molecules from a combinatorial chemical library yielded 42 potential Kir2.1 inhibitors. One compound, 3-bicyclo[2.2.1]hept-2-yl-benzene-1,2-diol, was confirmed to inhibit K+ channels in patch-clamp measurements in mammalian cells with EC50 values of 60 and 1 microM for Kir2.1 and Kv2.1 channels, respectively. Inhibition of Kv2.1 channels decreased in the presence of the external pore blocker tetraethylammonium (TEA) and depended on a residue required for extracellular TEA action, suggesting that the identified compound targets the external mouth of the channel. Furthermore, at the nontoxic concentration of 3 microM, the identified compound completely abolished in vitro neuronal apoptosis mediated by Kv2.1 channels. Therefore, yeast-based screening has identified a novel uncharged neuroprotective mammalian K+ channel inhibitor.

Virtual screening on natural products for discovering active compounds and target information.

Natural products, containing inherently large-scale structural diversity than synthetic compounds, have been the major resources of bioactive agents and will continually play as protagonists for discovering new drugs. However, how to access this diverse chemical space efficiently and effectively is an exciting challenge for medicinal chemists and pharmacologists. While virtual screening, which has shown a great promise in drug discovery, will play an important role in digging out lead (active) compounds from natural products. This review focuses on the strategy of virtual screening based on molecular docking and, with successful examples from our laboratory, illustrates the efficiency of virtual screening in discovering active compounds from natural products. On the other hand, the sequencing of the human genome and numerous pathogen genomes has resulted in an unprecedented opportunity for discovering potential new drug targets. Chemogenomics has appeared as a new technology to initiate target discovery by using active compounds as probes to characterize proteome functions. Natural products are the ideal probes for such research. Binding affinity fingerprint is a powerful chemogenomic descriptor to characterize both small molecules and pharmacologically relevant proteins. Therefore, this review also discusses binding affinity fingerprint strategy for identifying target information from the genomic data by using natural products as the probes.

BmTx3B, a novel scorpion toxin from Buthus martensi Karsch, inhibits delayed rectifier potassium current in rat hippocampal neurons.

AIM: To examine the effect of BmTx3B, a novel short-chain peptide isolated from the venom of Asian scorpion Buthus martensi Karsch, on voltage-gated potassium channels. METHODS: Two types of voltage-dependent potassium currents were recorded from dissociated hippocampal neurons of neonatal rat in whole-cell voltage-clamp mode, and separated based upon their kinetic properties. RESULTS: BmTx3B (10-100 micromol/L) selectively inhibited the delayed rectifier potassium current (I(K)), without affecting the fast transient potassium current (I(A)). The inhibition of the peptide on I(K) was reversible, concentration-dependent and voltage-independent. BmTx3B did not affect the steady-state activation and inactivation kinetics of the current. CONCLUSION: The short-chain scorpion peptide BmTx3B selectively blocked the delayed rectifier potassium channel.

Martentoxin, a novel K+-channel-blocking peptide: purification, cDNA and genomic cloning, and electrophysiological and pharmacological characterization.

Martentoxin, a novel K+-channel-specific peptide has been purified and characterized from the venom of the East-Asian scorpion (Buthus martensi Karsch). The whole cDNA precursor sequence suggested that martentoxin was composed of 37 residues with a unique sequence compared with other scorpion neurotoxins. The genomic DNA of martentoxin showed an additional intron situated unexpectedly in the 5' UTR region, besides one located close to the C-terminal of the signal peptide. The patch-clamp recording found that martentoxin at the applied dose of 100 nm could strongly block large-conductance Ca2+-activated K+ (BKCa) currents in adrenal medulla chromaffin cells, and BKCa currents blocked by martentoxin could be fully recovered within 30 seconds after washing, which is at least 10 times faster than recovery after charybdotoxin. Meanwhile, a biosensor binding assay showed a fast association rate and a slow dissociation rate of martentoxin binding on rat brain synaptosomes. The binding of martentoxin on rat brain synaptosomes could be inhibited regularly by charybdotoxin, and gradually by toosendanin in a concentration-dependent manner, but not by either apamin or P03 from Buthus martensi. The results thus indicate that martentoxin is a new member in the family of K+-channel-blocking ligands.