4-(Azolyl)-Benzamidines as a Novel Chemotype for ASIC1a Inhibitors.

Acid-sensing ion channels (ASICs) play a key role in the perception and response to extracellular acidification changes. These proton-gated cation channels are critical for neuronal functions, like learning and memory, fear, mechanosensation and internal adjustments like synaptic plasticity. Moreover, they play a key role in neuronal degeneration, ischemic neuronal injury, seizure termination, pain-sensing, etc. Functional ASICs are homo or heterotrimers formed with (ASIC1-ASIC3) homologous subunits. ASIC1a, a major ASIC isoform in the central nervous system (CNS), possesses an acidic pocket in the extracellular region, which is a key regulator of channel gating. Growing data suggest that ASIC1a channels are a potential therapeutic target for treating a variety of neurological disorders, including stroke, epilepsy and pain. Many studies were aimed at identifying allosteric modulators of ASIC channels. However, the regulation of ASICs remains poorly understood. Using all available crystal structures, which correspond to different functional states of ASIC1, and a molecular dynamics simulation (MD) protocol, we analyzed the process of channel inactivation. Then we applied a molecular docking procedure to predict the protein conformation suitable for the amiloride binding. To confirm the effect of its sole active blocker against the ASIC1 state transition route we studied the complex with another MD simulation run. Further experiments evaluated various compounds in the Enamine library that emerge with a detectable ASIC inhibitory activity. We performed a detailed analysis of the structural basis of ASIC1a inhibition by amiloride, using a combination of in silico approaches to visualize its interaction with the ion pore in the open state. An artificial activation (otherwise, expansion of the central pore) causes a complex modification of the channel structure, namely its transmembrane domain. The output protein conformations were used as a set of docking models, suitable for a high-throughput virtual screening of the Enamine chemical library. The outcome of the virtual screening was confirmed by electrophysiological assays with the best results shown for three hit compounds.

Integrated workflow for the identification of new GABAA R positive allosteric modulators based on the in†silico screening with further in†vitro validation. Case study using Enamine's stock chemical space.

Numerous studies reported an association between GABAA R subunit genes and epilepsy, eating disorders, autism spectrum disorders, neurodevelopmental disorders, and bipolar disorders. This study was aimed to find some potential positive allosteric modulators and was performed by combining the in silico approach with further in vitro evaluation of its real activity. We started from the GABAA R-diazepam complexes and assembled a lipid embedded protein ensemble to refine it via molecular dynamics (MD) simulation. Then we focused on the interaction of α1ß2γ2 with some Z-drugs (non-benzodiazepine compounds) using an Induced Fit Docking (IFD) into the relaxed binding site to generate a pharmacophore model. The pharmacophore model was validated with a reference set and applied to decrease the pre-filtered Enamine database before the main docking procedure. Finally, we succeeded in identifying a set of compounds, which met all features of the docking model. The aqueous solubility and stability of these compounds in mouse plasma were assessed. Then they were tested for the biological activity using the rat Purkinje neurons and CHO cells with heterologously expressed human α1ß2γ2 GABAA receptors. Whole-cell patch clamp recordings were used to reveal the GABA induced currents. Our study represents a convenient and tunable model for the discovery of novel positive allosteric modulators of GABAA receptors. A High-throughput virtual screening of the largest available database of chemical compounds resulted in the selection of 23 compounds. Further electrophysiological tests allowed us to determine a set of 3 the most outstanding active compounds. Considering the structural features of leader compounds, the study can develop into the MedChem project soon.

omega-Lsp-IA, a novel modulator of P-type Ca2+ channels.

A novel polypeptide, designated omega-Lsp-IA, which modulates P-type Ca(2+) channels, was purified from the venom of the spider Geolycosa sp. omega-Lsp-IA contains 47 amino acid residues and 4 intramolecular disulfide bridges. It belongs to a group of spider toxins affecting Ca(2+) channels and presumably forms the inhibitor cystine knot (ICK) fold. Peculiar structural features (a cluster of positively charged residues in the C-terminal loop of the peptide and a regular distribution of hydrophobic residues) that may play a decisive role in the omega-Lsp-IA mechanism of action were located. Recombinant omega-Lsp-IA was produced in prokaryotic expression system and was shown to be structurally and functionally identical to the native toxin. At saturating concentration (10nM), the peptide clearly slows down the activation kinetics and partially inhibits the amplitude of P-current in rat cerebellar Purkinje neurons. Prominent deceleration of the activation kinetics is manifested as the appearance of a five-fold slower component of the current activation. The specificity of action of omega-Lsp-IA on different Ca(2+) channel types was studied in isolated hippocampal neurons of rat. omega-Agatoxin IVA completely removed the effect of omega-Lsp-IA on the whole-cell Ca(2+) current. Therefore, omega-Lsp-IA appears to act specifically on P-type Ca(2+) channels.

G-protein-independent modulation of P-type calcium channels by mu-opioids in Purkinje neurons of rat.

P-type calcium channels play a key role in the synaptic transmission between mammalian central neurons since a major part of calcium entering pre-synaptic terminals is delivered via these channels. Using conventional whole-cell patch clamp techniques we have studied the effect of mu-opioids on P-type calcium channels in acutely isolated Purkinje neurons from rat cerebellum. The selective mu-opioid agonist DAMGO (10nM) produced a small, but consistent facilitation of current through P-type calcium channels (10+/-1%, n=27, p<0.001). The effect of DAMGO was rapid (less than 10s) and fully reversible. This effect was both concentration and voltage-dependent. The EC(50) for the effect of DAMGO was 1.3+/-0.4nM and the saturating concentration was 100nM. The endogenous selective agonist of mu-opioid receptors, endomorphin-1 demonstrated similar action. Intracellular perfusion of Purkinje neurons with GTPgammaS (0.5mM) or GDPbetaS (0.5mM), as well as strong depolarizing pre-pulses (+50mV), did not eliminate facilitatory action of DAMGO on P-channels indicating that this effect is not mediated by G-proteins. Furthermore, the effect of DAMGO was preserved in the presence of a non-specific inhibitor of PKA and PKC (H7, 10microM) inside the cell. DAMGO-induced facilitation of P-current was almost completely abolished by the selective mu-opioid antagonist CTOP (100nM). These observations indicate that mu-type opioid receptors modulate P-type calcium channels in Purkinje neurons via G-protein-independent mechanism.