Mutagenesis of the Peptide Inhibitor of ASIC3 Channel Introduces Binding to Thumb Domain of ASIC1a but Reduces Analgesic Activity.

Acid-sensing ion channels (ASICs), which act as proton-gating sodium channels, have garnered attention as pharmacological targets. ASIC1a isoform, notably prevalent in the central nervous system, plays an important role in synaptic plasticity, anxiety, neurodegeneration, etc. In the peripheral nervous system, ASIC1a shares prominence with ASIC3, the latter well established for its involvement in pain signaling, mechanical sensitivity, and inflammatory hyperalgesia. However, the precise contributions of ASIC1a in peripheral functions necessitate thorough investigation. To dissect the specific roles of ASICs, peptide ligands capable of modulating these channels serve as indispensable tools. Employing molecular modeling, we designed the peptide targeting ASIC1a channel from the sea anemone peptide Ugr9-1, originally targeting ASIC3. This peptide (A23K) retained an inhibitory effect on ASIC3 (IC50 9.39 µM) and exhibited an additional inhibitory effect on ASIC1a (IC50 6.72 µM) in electrophysiological experiments. A crucial interaction between the Lys23 residue of the A23K peptide and the Asp355 residue in the thumb domain of the ASIC1a channel predicted by molecular modeling was confirmed by site-directed mutagenesis of the channel. However, A23K peptide revealed a significant decrease in or loss of analgesic properties when compared to the wild-type Ugr9-1. In summary, using A23K, we show that negative modulation of the ASIC1a channel in the peripheral nervous system can compromise the efficacy of an analgesic drug. These results provide a compelling illustration of the complex balance required when developing peripheral pain treatments targeting ASICs.

The scorpion toxin BeKm-1 blocks hERG cardiac potassium channels using an indispensable arginine residue.

BeKm-1 is a peptide toxin from scorpion venom that blocks the pore of the potassium channel hERG (Kv11.1) in the human heart. Although individual protein structures have been resolved, the structure of the complex between hERG and BeKm-1 is unknown. Here, we used molecular dynamics and ensemble docking, guided by previous double-mutant cycle analysis data, to obtain an in silico model of the hERG-BeKm-1 complex. Adding to the previous mutagenesis study of BeKm-1, our model uncovers the key role of residue Arg20, which forms three interactions (a salt bridge and hydrogen bonds) with the channel vestibule simultaneously. Replacement of this residue even by lysine weakens the interactions significantly. In accordance, the recombinantly produced BeKm-1R20K mutant exhibited dramatically decreased activity on hERG. Our model may be useful for future drug design attempts.

Potentiating TRPA1 by Sea Anemone Peptide Ms 9a-1 Reduces Pain and Inflammation in a Model of Osteoarthritis.

Progressive articular surface degradation during arthritis causes ongoing pain and hyperalgesia that lead to the development of functional disability. TRPA1 channel significantly contributes to the activation of sensory neurons that initiate neurogenic inflammation and mediates pain signal transduction to the central nervous system. Peptide Ms 9a-1 from the sea anemone Metridium senile is a positive allosteric modulator of TRPA1 and shows significant anti-inflammatory and analgesic activity in different models of pain. We used a model of monosodium iodoacetate (MIA)-induced osteoarthritis to evaluate the anti-inflammatory properties of Ms 9a-1 in comparison with APHC3 (a polypeptide modulator of TRPV1 channel) and non-steroidal anti-inflammatory drugs (NSAIDs) such as meloxicam and ibuprofen. Administration of Ms 9a-1 (0.1 mg/kg, subcutaneously) significantly reversed joint swelling, disability, thermal and mechanical hypersensitivity, and grip strength impairment. The effect of Ms 9a-1 was equal to or better than that of reference drugs. Post-treatment histological analysis revealed that long-term administration of Ms9a-1 could reduce inflammatory changes in joints and prevent the progression of cartilage and bone destruction at the same level as meloxicam. Peptide Ms 9a-1 showed significant analgesic and anti-inflammatory effects in the model of MIA-induced OA, and therefore positive allosteric modulators could be considered for the alleviation of OA symptoms.

Modulation of TRPV1 and TRPA1 Channels Function by Sea Anemones' Peptides Enhances the Viability of SH-SY5Y Cell Model of Parkinson's Disease.

Cellular dysfunction during Parkinson's disease leads to neuroinflammation in various brain regions, inducing neuronal death and contributing to the progression of the disease. Different ion channels may influence the process of neurodegeneration. The peptides Ms 9a-1 and APHC3 can modulate the function of TRPA1 and TRPV1 channels, and we evaluated their cytoprotective effects in differentiated to dopaminergic neuron-like SH-SY5Y cells. We used the stable neuroblastoma cell lines SH-SY5Y, producing wild-type alpha-synuclein and its mutant A53T, which are prone to accumulation of thioflavin-S-positive aggregates. We analyzed the viability of cells, as well as the mRNA expression levels of TRPA1, TRPV1, ASIC1a channels, alpha-synuclein, and tyrosine hydroxylase after differentiation of these cell lines using RT-PCR. Overexpression of alpha-synuclein showed a neuroprotective effect and was accompanied by a reduction of tyrosine hydroxylase expression. A mutant alpha-synuclein A53T significantly increased the expression of the pro-apoptotic protein BAX and made cells more susceptible to apoptosis. Generally, overexpression of alpha-synuclein could be a model for the early stages of PD, while expression of mutant alpha-synuclein A53T mimics a genetic variant of PD. The peptides Ms 9a-1 and APHC3 significantly reduced the susceptibility to apoptosis of all cell lines but differentially influenced the expression of the genes of interest. Therefore, these modulators of TRPA1 and TRPV1 have the potential for the development of new therapeutic agents for neurodegenerative disease treatment.

Analysis of Structural Determinants of Peptide MS 9a-1 Essential for Potentiating of TRPA1 Channel.

The TRPA1 channel is involved in a variety of physiological processes and its activation leads to pain perception and the development of inflammation. Peptide Ms 9a-1 from sea anemone Metridium senile is a positive modulator of TRPA1 and causes significant analgesic and anti-inflammatory effects by desensitization of TRPA1-expressing sensory neurons. For structural and functional analysis of Ms 9a-1, we produced four peptides-Ms 9a-1 without C-terminal domain (abbreviated as N-Ms), short C-terminal domain Ms 9a-1 alone (C-Ms), and two homologous peptides (Ms 9a-2 and Ms 9a-3). All tested peptides possessed a reduced potentiating effect on TRPA1 compared to Ms 9a-1 in vitro. None of the peptides reproduced analgesic and anti-inflammatory properties of Ms 9a-1 in vivo. Peptides N-Ms and C-Ms were able to reduce pain induced by AITC (selective TRPA1 agonist) but did not decrease AITC-induced paw edema development. Fragments of Ms 9a-1 did not effectively reverse CFA-induced thermal hyperalgesia and paw edema. Ms 9a-2 and Ms 9a-3 possessed significant effects and anti-inflammatory properties in some doses, but their unexpected efficacy and bell-shape dose-responses support the hypothesis of other targets involved in their effects in vivo. Therefore, activity comparison of Ms 9a-1 fragments and homologues peptides revealed structural determinants important for TRPA1 modulation, as well as analgesic and anti-inflammatory properties of Ms9a-1.

Peptides from the Sea Anemone Metridium senile with Modified Inhibitor Cystine Knot (ICK) Fold Inhibit Nicotinic Acetylcholine Receptors.

Nicotinic acetylcholine receptors (nAChRs) play an important role in the functioning of the central and peripheral nervous systems, and other organs of living creatures. There are several subtypes of nAChRs, and almost all of them are considered as pharmacological targets in different pathological states. The crude venom of the sea anemone Metridium senile showed the ability to interact with nAChRs. Four novel peptides (Ms11a-1-Ms11a-4) with nAChR binding activity were isolated. These peptides stabilized by three disulfide bridges have no noticeable homology with any known peptides. Ms11a-1-Ms11a-4 showed different binding activity towards the muscle-type nAChR from the Torpedo californica ray. The study of functional activity and selectivity for the most potent peptide (Ms11a-3) revealed the highest antagonism towards the heterologous rat α9α10 nAChR compared to the muscle and α7 receptors. Structural NMR analysis of two toxins (Ms11a-2 and Ms11a-3) showed that they belong to a new variant of the inhibitor cystine knot (ICK) fold but have a prolonged loop between the fifth and sixth cysteine residues. Peptides Ms11a-1-Ms11a-4 could represent new pharmacological tools since they have structures different from other known nAChRs inhibitors.

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.

ω-Tbo-IT1-New Inhibitor of Insect Calcium Channels Isolated from Spider Venom.

Novel disulfide-containing polypeptide toxin was discovered in the venom of the Tibellus oblongus spider. We report on isolation, spatial structure determination and electrophysiological characterization of this 41-residue toxin, called ω-Tbo-IT1. It has an insect-toxic effect with LD50 19 µg/g in experiments on house fly Musca domestica larvae and with LD50 20 µg/g on juvenile Gromphadorhina portentosa cockroaches. Electrophysiological experiments revealed a reversible inhibition of evoked excitatory postsynaptic currents in blow fly Calliphora vicina neuromuscular junctions, while parameters of spontaneous ones were not affected. The inhibition was concentration dependent, with IC50 value 40 ± 10 nM and Hill coefficient 3.4 ± 0.3. The toxin did not affect frog neuromuscular junctions or glutamatergic and GABAergic transmission in rat brains. Ca(2+) currents in Calliphora vicina muscle were not inhibited, whereas in Periplaneta americana cockroach neurons at least one type of voltage gated Ca(2+) current was inhibited by ω-Tbo-IT1. Thus, the toxin apparently acts as an inhibitor of presynaptic insect Ca(2+) channels. Spatial structure analysis of the recombinant ω-Tbo-IT1 by NMR spectroscopy in aqueous solution revealed that the toxin comprises the conventional ICK fold containing an extended ß-hairpin loop and short ß-hairpin loop which are capable of making "scissors-like mutual motions".

Novel class of spider toxin: active principle from the yellow sac spider Cheiracanthium punctorium venom is a unique two-domain polypeptide.

Venom of the yellow sac spider Cheiracanthium punctorium (Miturgidae) was found unique in terms of molecular composition. Its principal toxic component CpTx 1 (15.1 kDa) was purified, and its full amino acid sequence (134 residues) was established by protein chemistry and mass spectrometry techniques. CpTx 1 represents a novel class of spider toxin with modular architecture. It consists of two different yet homologous domains (modules) each containing a putative inhibitor cystine knot motif, characteristic of the widespread single domain spider neurotoxins. Venom gland cDNA sequencing provided precursor protein (prepropeptide) structures of three CpTx 1 isoforms (a-c) that differ by single residue substitutions. The toxin possesses potent insecticidal (paralytic and lethal), cytotoxic, and membrane-damaging activities. In both fly and frog neuromuscular preparations, it causes stable and irreversible depolarization of muscle fibers leading to contracture. This effect appears to be receptor-independent and is inhibited by high concentrations of divalent cations. CpTx 1 lyses cell membranes, as visualized by confocal microscopy, and destabilizes artificial membranes in a manner reminiscent of other membrane-active peptides by causing numerous defects of variable conductance and leading to bilayer rupture. The newly discovered class of modular polypeptides enhances our knowledge of the toxin universe.