Opioid Analgesic as a Positive Allosteric Modulator of Acid-Sensing Ion Channels.

Tafalgin (Taf) is a tetrapeptide opioid used in clinical practice in Russia as an analgesic drug for subcutaneous administration as a solution (4 mg/mL; concentration of 9 mM). We found that the acid-sensing ion channels (ASICs) are another molecular target for this molecule. ASICs are proton-gated sodium channels that mediate nociception in the peripheral nervous system and contribute to fear and learning in the central nervous system. Using electrophysiological methods, we demonstrated that Taf could increase the integral current through heterologically expressed ASIC with half-maximal effective concentration values of 0.09 mM and 0.3 mM for rat and human ASIC3, respectively, and 1 mM for ASIC1a. The molecular mechanism of Taf action was shown to be binding to the channel in the resting state and slowing down the rate of desensitization. Taf did not compete for binding sites with both protons and ASIC3 antagonists, such as APETx2 and amiloride (Ami). Moreover, Taf and Ami together caused an unusual synergistic effect, which was manifested itself as the development of a pronounced second desensitizing component. Thus, the ability of Taf to act as a positive allosteric modulator of these channels could potentially cause promiscuous effects in clinical practice. This fact must be considered in patients' treatment.

Dual Modulator of ASIC Channels and GABAA Receptors from Thyme Alters Fear-Related Hippocampal Activity.

Acid-sensing ion channels (ASICs) are proton-gated ion channels that mediate nociception in the peripheral nervous system and contribute to fear and learning in the central nervous system. Sevanol was reported previously as a naturally-occurring ASIC inhibitor from thyme with favorable analgesic and anti-inflammatory activity. Using electrophysiological methods, we found that in the high micromolar range, the compound effectively inhibited homomeric ASIC1a and, in sub- and low-micromolar ranges, positively modulated the currents of α1ß2γ2 GABAA receptors. Next, we tested the compound in anxiety-related behavior models using a targeted delivery into the hippocampus with parallel electroencephalographic measurements. In the open field, 6 µM sevanol reduced both locomotor and θ-rhythmic activity similar to GABA, suggesting a primary action on the GABAergic system. At 300 µM, sevanol markedly suppressed passive avoidance behavior, implying alterations in conditioned fear memory. The observed effects could be linked to distinct mechanisms involving GABAAR and ASIC1a. These results elaborate the preclinical profile of sevanol as a candidate for drug development and support the role of ASIC channels in fear-related functions of the hippocampus.

Anxiolytic, Analgesic and Anti-Inflammatory Effects of Peptides Hmg 1b-2 and Hmg 1b-4 from the Sea Anemone Heteractis magnifica.

Acid-sensing ion channels (ASICs) have been known as sensors of a local pH change within both physiological and pathological conditions. ASIC-targeting peptide toxins could be potent molecular tools for ASIC-manipulating in vitro, and for pathology treatment in animal test studies. Two sea anemone toxins, native Hmg 1b-2 and recombinant Hmg 1b-4, both related to APETx-like peptides, inhibited the transient current component of human ASIC3-Δ20 expressed in Xenopus laevis oocytes, but only Hmg 1b-2 inhibited the rat ASIC3 transient current. The Hmg 1b-4 action on rASIC3 as a potentiator was confirmed once again. Both peptides are non-toxic molecules for rodents. In open field and elevated plus maze tests, Hmg 1b-2 had more of an excitatory effect and Hmg 1b-4 had more of an anxiolytic effect on mouse behavior. The analgesic activity of peptides was similar and comparable to diclofenac activity in an acid-induced muscle pain model. In models of acute local inflammation induced by λ-carrageenan or complete Freund's adjuvant, Hmg 1b-4 had more pronounced and statistically significant anti-inflammatory effects than Hmg 1b-2. It exceeded the effect of diclofenac and, at a dose of 0.1 mg/kg, reduced the volume of the paw almost to the initial volume. Our data highlight the importance of a comprehensive study of novel ASIC-targeting ligands, and in particular, peptide toxins, and present the slightly different biological activity of the two similar toxins.

Nocistatin and Products of Its Proteolysis Are Dual Modulators of Type 3 Acid-Sensing Ion Channels (ASIC3) with Algesic and Analgesic Properties.

The neuropeptide nocistatin (NS) is expressed by the nervous system cells and neutrophils as a part of a precursor protein and can undergo stepwise limited proteolysis. Previously, it was shown that rat NS (rNS) is able to activate acid-sensing ion channels (ASICs) and that this effect correlates with the acidic nature of NS. Here, we investigated changes in the properties of rNS in the course of its proteolytic degradation by comparing the effects of the full-size rNS and its two cleavage fragments on the rat isoform 3 ASICs (ASIC3) expressed in X. laevis oocytes and pain perception in mice. The rNS acted as both positive and negative modulator by lowering the steady-state desensitization of ASIC3 at pH 6.8-7.0 and reducing the channel's response to stimuli at pH 6.0-6.9, respectively. The truncated rNSΔ21 peptide lacking 21 amino acid residues from the N-terminus retained the positive modulatory activity, while the C-terminal pentapeptide (rNSΔ30) acted only as a negative ASIC3 modulator. The effects of the studied peptides were confirmed in animal tests: rNS and rNSΔ21 induced a pain-related behavior, whereas rNSΔ30 showed the analgesic effect. Therefore, we have shown that the mode of rNS action changes during its stepwise degradation, from an algesic molecule through a pain enhancer to a pain reliever (rNSΔ30 pentapeptide), which can be considered as a promising drug candidate.

Lignans as Pharmacological Agents in Disorders Related to Oxidative Stress and Inflammation: Chemical Synthesis Approaches and Biological Activities.

Plant lignans exhibit a wide range of biological activities, which makes them the research objects of potential use as therapeutic agents. They provide diverse naturally-occurring pharmacophores and are available for production by chemical synthesis. A large amount of accumulated data indicates that lignans of different structural groups are apt to demonstrate both anti-inflammatory and antioxidant effects, in many cases, simultaneously. In this review, we summarize the comprehensive knowledge about lignan use as a bioactive agent in disorders associated with oxidative stress and inflammation, pharmacological effects in vitro and in vivo, molecular mechanisms underlying these effects, and chemical synthesis approaches. This article provides an up-to-date overview of the current data in this area, available in PubMed, Scopus, and Web of Science databases, screened from 2000 to 2022.

Retinoic Acid-Differentiated Neuroblastoma SH-SY5Y Is an Accessible In Vitro Model to Study Native Human Acid-Sensing Ion Channels 1a (ASIC1a).

Human neuroblastoma SH-SY5Y is a prominent neurobiological tool used for studying neuropathophysiological processes. We investigated acid-sensing (ASIC) and transient receptor potential vanilloid-1 (TRPV1) and ankyrin-1 (TRPA1) ion channels present in untreated and differentiated neuroblastoma SH-SY5Y to propose a new means for their study in neuronal-like cells. Using a quantitative real-time PCR and a whole-cell patch-clamp technique, ion channel expression profiles, functionality, and the pharmacological actions of their ligands were characterized. A low-level expression of ASIC1a and ASIC2 was detected in untreated cells. The treatment with 10 µM of retinoic acid (RA) for 6 days resulted in neuronal differentiation that was accompanied by a remarkable increase in ASIC1a expression, while ASIC2 expression remained almost unaltered. In response to acid stimuli, differentiated cells showed prominent ASIC-like currents. Detailed kinetic and pharmacological characterization suggests that homomeric ASIC1a is a dominant isoform among the present ASIC channels. RA-treatment also reduced the expression of TRPV1 and TRPA1, and minor electrophysiological responses to their agonists were found in untreated cells. Neuroblastoma SH-SY5Y treated with RA can serve as a model system to study the effects of different ligands on native human ASIC1a in neuronal-like cells. This approach can improve the characterization of modulators for the development of new neuroprotective and analgesic drugs.

Animal, Herb, and Microbial Toxins for Structural and Pharmacological Study of Acid-Sensing Ion Channels.

Acid-sensing ion channels (ASICs) are of the most sensitive molecular sensors of extracellular pH change in mammals. Six isoforms of these channels are widely represented in membranes of neuronal and non-neuronal cells, where these molecules are involved in different important regulatory functions, such as synaptic plasticity, learning, memory, and nociception, as well as in various pathological states. Structural and functional studies of both wild-type and mutant ASICs are essential for human care and medicine for the efficient treatment of socially significant diseases and ensure a comfortable standard of life. Ligands of ASICs serve as indispensable tools for these studies. Such bioactive compounds can be synthesized artificially. However, to date, the search for such molecules has been most effective amongst natural sources, such as animal venoms or plants and microbial extracts. In this review, we provide a detailed and comprehensive structural and functional description of natural compounds acting on ASICs, as well as the latest information on structural aspects of their interaction with the channels. Many of the examples provided in the review demonstrate the undoubted fundamental and practical successes of using natural toxins. Without toxins, it would not be possible to obtain data on the mechanisms of ASICs' functioning, provide detailed study of their pharmacological properties, or assess the contribution of the channels to development of different pathologies. The selectivity to different isoforms and variety in the channel modulation mode allow for the appraisal of prospective candidates for the development of new drugs.

Sevanol and Its Analogues: Chemical Synthesis, Biological Effects and Molecular Docking.

Among acid-sensing ion channels (ASICs), ASIC1a and ASIC3 subunits are the most widespread and prevalent in physiological and pathophysiological conditions. They participate in synaptic plasticity, learning and memory, as well as the perception of inflammatory and neurological pain, making these channels attractive pharmacological targets. Sevanol, a natural lignan isolated from Thymus armeniacus, inhibits the activity of ASIC1a and ASIC3 isoforms, and has a significant analgesic and anti-inflammatory effect. In this work, we described the efficient chemical synthesis scheme of sevanol and its analogues, which allows us to analyze the structure-activity relationships of the different parts of this molecule. We found that the inhibitory activity of sevanol and its analogues on ASIC1a and ASIC3 channels depends on the number and availability of the carboxyl groups of the molecule. At the structural level, we predicted the presence of a sevanol binding site based on the presence of molecular docking in the central vestibule of the ASIC1a channel. We predicted that this site could also be occupied in part by the FRRF-amide peptide, and the competition assay of sevanol with this peptide confirmed this prediction. The intravenous (i.v.), intranasal (i.n.) and, especially, oral (p.o.) administration of synthetic sevanol in animal models produced significant analgesic and anti-inflammatory effects. Both non-invasive methods of sevanol administration (i.n. and p.o.) showed greater efficacy than the invasive (i.v.) method, thus opening new horizons for medicinal uses of sevanol.

Alkaloid Lindoldhamine Inhibits Acid-Sensing Ion Channel 1a and Reveals Anti-Inflammatory Properties.

Acid-sensing ion channels (ASICs), which are present in almost all types of neurons, play an important role in physiological and pathological processes. The ASIC1a subtype is the most sensitive channel to the medium's acidification, and it plays an important role in the excitation of neurons in the central nervous system. Ligands of the ASIC1a channel are of great interest, both fundamentally and pharmaceutically. Using a two-electrode voltage-clamp electrophysiological approach, we characterized lindoldhamine (a bisbenzylisoquinoline alkaloid extracted from the leaves of Laurus nobilis L.) as a novel inhibitor of the ASIC1a channel. Lindoldhamine significantly inhibited the ASIC1a channel's response to physiologically-relevant stimuli of pH 6.5-6.85 with IC50 range 150-9 µM, but produced only partial inhibition of that response to more acidic stimuli. In mice, the intravenous administration of lindoldhamine at a dose of 1 mg/kg significantly reversed complete Freund's adjuvant-induced thermal hyperalgesia and inflammation; however, this administration did not affect the pain response to an intraperitoneal injection of acetic acid (which correlated well with the function of ASIC1a in the peripheral nervous system). Thus, we describe lindoldhamine as a novel antagonist of the ASIC1a channel that could provide new approaches to drug design and structural studies regarding the determinants of ASIC1a activation.

Multiple Modulation of Acid-Sensing Ion Channel 1a by the Alkaloid Daurisoline.

Acid-sensing ion channels (ASICs) are proton-gated sodium-selective channels that are expressed in the peripheral and central nervous systems. ASIC1a is one of the most intensively studied isoforms due to its importance and wide representation in organisms, but it is still largely unexplored as a target for therapy. In this study, we demonstrated response of the ASIC1a to acidification in the presence of the daurisoline (DAU) ligand. DAU alone did not activate the channel, but in combination with protons, it produced the second peak component of the ASIC1a current. This second peak differs from the sustained component (which is induced by RF-amide peptides), as the second (DAU-induced) peak is completely desensitized, with the same kinetics as the main peak. The co-application of DAU and mambalgin-2 indicated that their binding sites do not overlap. Additionally, we found an asymmetry in the pH activation curve of the channel, which was well-described by a mathematical model based on the multiplied probabilities of protons binding with a pool of high-cooperative sites and a single proton binding with a non-cooperative site. In this model, DAU targeted the pool of high-cooperative sites and, when applied with protons, acted as an inhibitor of ASIC1a activation. Moreover, DAU's occupation of the same binding site most probably reverses the channel from steady-state desensitization in the pH 6.9-7.3 range. DAU features disclose new opportunities in studies of ASIC structure and function.

Endogenous Neuropeptide Nocistatin Is a Direct Agonist of Acid-Sensing Ion Channels (ASIC1, ASIC2 and ASIC3).

Acid-sensing ion channel (ASIC) channels belong to the family of ligand-gated ion channels known as acid-sensing (proton-gated) ion channels. Only a few activators of ASICs are known. These are exogenous and endogenous molecules that cause a persistent, slowly desensitized current, different from an acid-induced current. Here we describe a novel endogenous agonist of ASICs-peptide nocistatin produced by neuronal cells and neutrophils as a part of prepronociceptin precursor protein. The rat nocistatin evoked currents in X. laevis oocytes expressing rat ASIC1a, ASIC1b, ASIC2a, and ASIC3 that were very similar in kinetic parameters to the proton-gated response. Detailed characterization of nocistatin action on rASIC1a revealed a proton-like dose-dependence of activation, which was accompanied by a dose-dependent decrease in the sensitivity of the channel to the protons. The toxin mambalgin-2, antagonist of ASIC1a, inhibited nocistatin-induced current, therefore the close similarity of mechanisms for ASIC1a activation by peptide and protons could be suggested. Thus, nocistatin is the first endogenous direct agonist of ASICs. This data could give a key to understanding ASICs activation regulation in the nervous system and also could be used to develop new drugs to treat pathological processes associated with ASICs activation, such as neurodegeneration, inflammation, and pain.

Analgesic Activity of Acid-Sensing Ion Channel 3 (ASIС3) Inhibitors: Sea Anemones Peptides Ugr9-1 and APETx2 versus Low Molecular Weight Compounds.

Acid-sensing ion channel 3 (ASIC3) makes an important contribution to the development and maintenance of inflammatory and acid-induced pain. We compared different ASIC3 inhibitors (peptides from sea anemones (APETx2 and Ugr9-1) and nonpeptide molecules (sevanol and diclofenac)) in anti-inflammatory action and analgesic effects. All tested compounds had distinct effects on pH-induced ASIC3 current. APETx2 inhibited only transient current, whereas Ugr9-1 and sevanol decreased transient and sustained components of the current. The effect on mice was evaluated after administering an intramuscular injection in the acetic acid writhing pain model and the complete Freund's adjuvant-induced thermal hyperalgesia/inflammation test. The bell-shaped dependence of the analgesic effect was observed for APETx2 in the acetic acid-induced writhing test, as well as for sevanol and peptide Ugr9-1 in the thermal hyperalgesia test. This dependence could be evidence of the nonspecific action of compounds in high doses. Compounds reducing both components of ASIC3 current produced more significant pain relief than APETx2, which is an effective inhibitor of a transient current only. Therefore, the comparison of the efficacy of ASIC3 inhibitors revealed the importance of ASIC3-sustained currents' inhibition for promotion of acidosis-related pain relief.

Proton-independent activation of acid-sensing ion channel 3 by an alkaloid, lindoldhamine, from Laurus nobilis.

BACKGROUND AND PURPOSE: Acid-sensing ion channels (ASICs) play an important role in synaptic plasticity and learning, as well as in nociception and mechanosensation. ASICs are involved in pain and in neurological and psychiatric diseases, but their therapeutic potential is limited by the lack of ligands activating them at physiological pH. EXPERIMENTAL APPROACH: We extracted, purified and determined the structure of a bisbenzylisoquinoline alkaloid, lindoldhamine, (LIN) from laurel leaves. Its effect on ASIC3 channels were characterized, using two-electrode voltage-clamp electrophysiological recordings from Xenopus laevis oocytes. KEY RESULTS: At pH 7.4 or higher, LIN activated a sustained, proton-independent, current through rat and human ASIC3 channels, but not rat ASIC1a or ASIC2a channels. LIN also potentiated proton-induced transient currents and promoted recovery from desensitization in human, but not rat, ASIC3 channels. CONCLUSIONS AND IMPLICATIONS: We describe a novel ASIC subtype-specific agonist LIN, which induced proton-independent activation of human and rat ASIC3 channels at physiological pH. LIN also acts as a positive allosteric modulator of human, but not rat, ASIC3 channels. This unique, species-selective, ligand of ASIC3, opens new avenues in studies of ASIC structure and function, as well as providing new approaches to drug design.

Endogenous Isoquinoline Alkaloids Agonists of Acid-Sensing Ion Channel Type 3.

Acid-sensing ion channels (ASICs) ASIC3 expressed mainly in peripheral sensory neurons play an important role in pain perception and inflammation development. In response to acidic stimuli, they can generate a unique biphasic current. At physiological pH 7.4, human ASIC3 isoform (hASIC3) is desensitized and able to generate only a sustained current. We found endogenous isoquinoline alkaloids (EIAs), which restore hASIC3 from desensitization and recover the transient component of the current. Similarly, rat ASIC3 isoform (rASIC3) can also be restored from desensitization (at pH < 7.0) by EIAs with the same potency. At physiological pH and above, EIAs at high concentrations were able to effectively activate hASIC3 and rASIC3. Thus, we found first endogenous agonists of ASIC3 channels that could both activate and prevent or reverse desensitization of the channel. The decrease of EIA levels could be suggested as a novel therapeutic strategy for treatment of pain and inflammation.

Conversed mutagenesis of an inactive peptide to ASIC3 inhibitor for active sites determination.

Peptide Ugr9-1 from the venom of sea anemone Urticina grebelnyi selectively inhibits the ASIC3 channel and significantly reverses inflammatory and acid-induced pain in vivo. A close homolog peptide Ugr 9-2 does not have these features. To find the pharmacophore residues and explore structure-activity relationships of Ugr 9-1, we performed site-directed mutagenesis of Ugr 9-2 and replaced several positions by the corresponding residues from Ugr 9-1. Mutant peptides Ugr 9-2 T9F and Ugr 9-2 Y12H were able to inhibit currents of the ASIC3 channels 2.2 times and 1.3 times weaker than Ugr 9-1, respectively. Detailed analysis of the spatial models of Ugr 9-1, Ugr 9-2 and both mutant peptides revealed the presence of the basic-aromatic clusters on opposite sides of the molecule, each of which is responsible for the activity. Additionally, Ugr9-1 mutant with truncated N- and C-termini retained similar with the Ugr9-1 action in vitro and was equally potent in vivo model of thermal hypersensitivity. All together, these results are important for studying the structure-activity relationships of ligand-receptor interaction and for the future development of peptide drugs from animal toxins.

Sea anemone peptide with uncommon ß-hairpin structure inhibits acid-sensing ion channel 3 (ASIC3) and reveals analgesic activity.

Three novel peptides were isolated from the venom of the sea anemone Urticina grebelnyi. All of them are 29 amino acid peptides cross-linked by two disulfide bridges, with a primary structure similar to other sea anemone peptides belonging to structural group 9a. The structure of the gene encoding the shared precursor protein of the identified peptides was determined. One peptide, π-AnmTX Ugr 9a-1 (short name Ugr 9-1), produced a reversible inhibition effect on both the transient and the sustained current of human ASIC3 channels expressed in Xenopus laevis oocytes. It completely blocked the transient component (IC50 10 ± 0.6 µM) and partially (48 ± 2%) inhibited the amplitude of the sustained component (IC50 1.44 ± 0.19 µM). Using in vivo tests in mice, Ugr 9-1 significantly reversed inflammatory and acid-induced pain. The other two novel peptides, AnmTX Ugr 9a-2 (Ugr 9-2) and AnmTX Ugr 9a-3 (Ugr 9-3), did not inhibit the ASIC3 current. NMR spectroscopy revealed that Ugr 9-1 has an uncommon spatial structure, stabilized by two S-S bridges, with three classical ß-turns and twisted ß-hairpin without interstrand disulfide bonds. This is a novel peptide spatial structure that we propose to name boundless ß-hairpin.

Lignan from thyme possesses inhibitory effect on ASIC3 channel current.

A novel compound was identified in the acidic extract of Thymus armeniacus collected in the Lake Sevan region of Armenia. This compound, named "sevanol," to our knowledge is the first low molecular weight natural molecule that has a reversible inhibition effect on both the transient and the sustained current of human ASIC3 channels expressed in Xenopus laevis oocytes. Sevanol completely blocked the transient component (IC(50) 353 ± 23 µM) and partially (∼45%) inhibited the amplitude of the sustained component (IC(50) of 234 ± 53 µM). Other types of acid-sensing ion channel (ASIC) channels were intact to sevanol application, except ASIC1a, which showed more than six times less affinity to it as compared with the inhibitory action on the ASIC3 channel. To elucidate the structure of sevanol, the set of NMR spectra in two solvents (d(6)-DMSO and D(2)O) was collected, and the complete chemical structure was confirmed by liquid chromatography-mass spectrometry with electrospray ionization (LC-ESI(+)-MS) fragmentation. This compound is a new lignan built up of epiphyllic acid and two isocitryl esters in positions 9 and 10. In vivo administration of sevanol (1-10 mg/kg) significantly reversed thermal hyperalgesia induced by complete Freund's adjuvant injection and reduced response to acid in a writhing test. Thus, we assume the probable considerable role of sevanol in known analgesic and anti-inflammatory properties of thyme.