Curare alkaloids from Matis Dart Poison: Comparison with d-tubocurarine in interactions with nicotinic, 5-HT3 serotonin and GABAA receptors.

Several novel bisbenzylisoquinoline alkaloids (BBIQAs) have recently been isolated from a Matis tribe arrow poison and shown by two-electrode voltage-clamp to inhibit mouse muscle nicotinic acetylcholine receptors (nAChR). Here, using radioligand assay with Aplysia californica AChBP and radioiodinated α-bungarotoxin ([125I]-αBgt), we show that BBIQA1, BBIQA2, and d-tubocurarine (d-TC) have similar affinities to nAChR orthosteric site. However, a competition with [125I]-αBgt for binding to the Torpedo californica muscle-type nAChR revealed that BBIQAs1, 2, and 3 are less potent (IC50s = 26.3, 8.75, and 17.0 μM) than d-TC (IC50 = 0.39 μM), while with α7 nAChR in GH4C1 cells, BBIQA1 was less potent that d-TC (IC50s = 162 μM and 7.77 μM, respectively), but BBIQA2 was similar (IC50 = 5.52 μM). In inhibiting the Ca2+ responses induced by acetylcholine in Neuro2a cells expressing the mouse adult α1β1εδ nAChR or human α7 nAChR, BBIQAs1 and 2 had similar potencies to d-TC (IC50s in the range 0.75-3.08 μM). Our data suggest that BBIQA1 and BBIQA2 can inhibit adult muscle α1β1εδ nAChR by both competitive and noncompetitive mechanisms. Further experiments on neuronal α3β2, α4β2, and α9α10 nAChRs, expressed in Xenopus laevis oocytes, showed that similar potencies for BBIQAs1, 2, and d-TC. With α3β2γ2 GABAAR currents were almost completely inhibited by d-TC at a high (100 μM) concentration, but BBIQAs1 and 2 were less potent (only 40-50% inhibition), whereas in competition with Alexa Fluor 546-α-cobratoxin for binding to α1β3γ2 GABAAR in Neuro2a cells, d-TC and these analogs had comparable affinities. Especially interesting effects of BBIQAs1 and 2 in comparison with d-TC were observed for 5-HT3AR: BBIQA1 and BBIQA2 were 5- and 87-fold less potent than d-TC (IC50 = 22.63 nM). Thus, our results reveal that these BBIQAs differ from d-TC in their potencies towards certain Cys-loop receptors, and we suggest that understanding the reasons behind this might be useful for future drug design.The work of ENS, IAI, DSK, IVS, AIG, LVS, and VIT was supported by the Russian Science Foundation Grant 16-14-00215 (http://rscf. ru/en). The work of IEK was supported by the Russian Foundation for Basic Research Grant 18- 04-01366 (http://www.rfbr.ru/rffi/eng). The work of SCRL was supported by a Medical Research Council Grant MR L021676 (https://mrc.ukri.org/)

Novel long-chain neurotoxins from Bungarus candidus distinguish the two binding sites in muscle-type nicotinic acetylcholine receptors

αδ-Bungarotoxins, a novel group of long-chain α-neurotoxins, manifest different affinity to two agonist/competitive antagonist binding sites of muscle-type nicotinic acetylcholine receptors (nAChRs), being more active at the interface of α–δ subunits. Three isoforms (αδ-BgTx-1–3) were identified in Malayan Krait (Bungarus candidus) from Thailand by genomic DNA analysis; two of them (αδ-BgTx-1 and 2) were isolated from its venom. The toxins comprise 73 amino acid residues and 5 disulfide bridges, being homologous to α-bungarotoxin (α-BgTx), a classical blocker of muscle-type and neuronal α7, α8, and α9α10 nAChRs. The toxicity of αδ-BgTx-1 (LD50 = 0.17–0.28 µg/g mouse, i.p. injection) is essentially as high as that of α-BgTx. In the chick biventer cervicis nerve–muscle preparation, αδ-BgTx-1 completely abolished acetylcholine response, but in contrast with the block by α-BgTx, acetylcholine response was fully reversible by washing. αδ-BgTxs, similar to α-BgTx, bind with high affinity to α7 and muscle-type nAChRs. However, the major difference of αδ-BgTxs from α-BgTx and other naturally occurring α-neurotoxins is that αδ-BgTxs discriminate the two binding sites in the Torpedo californica and mouse muscle nAChRs showing up to two orders of magnitude higher affinity for the α–δ site as compared with α–ε or α–γ binding site interfaces. Molecular modeling and analysis of the literature provided possible explanations for these differences in binding mode; one of the probable reasons being the lower content of positively charged residues in αδ-BgTxs. Thus, αδ-BgTxs are new tools for studies on nAChRs

Design of New α-Conotoxins: From Computer Modeling to Synthesis of Potent Cholinergic Compounds

Abstract: A series of 14 new analogs of α-conotoxin PnIA Conus pennaceus was synthesized and tested for binding to the human α7 nicotinic acetylcholine receptor (nAChR) and acetylcholine-binding proteins (AChBP) Lymnaea stagnalis and Aplysia californica. Based on computer modeling and the X-ray structure of the A. californica AChBP complex with the PnIA[A10L, D14K] analog [1], single and multiple amino acid substitutions were introduced in α-conotoxin PnIA aimed at compounds of higher affinity and selectivity. Three analogs, PnIA[L5H], PnIA[A10L, D14K] and PnIA[L5R, A10L, D14R], have high affinities for AChBPs or α7 nAChR, as found in competition with radioiodinated α-bungarotoxin. That is why we prepared radioiodinated derivatives of these α-conotoxins, demonstrated their specific binding and found that among the tested synthetic analogs, most had almost 10-fold higher affinity in competition with radioactive α-conotoxins as compared to competition with radioactive α-bungarotoxin. Thus, radioiodinated α-conotoxins are a more sensitive tool for checking the activity of novel α-conotoxins and other compounds quickly dissociating from the receptor complexes

From crystal structure of α-conotoxin GIC in complex with Ac-AChBP to molecular determinants of its high selectivity for α3β2 nAChR

Acetylcholine binding proteins (AChBPs) are unique spatial homologs of the ligand-binding domains of nicotinic acetylcholine receptors (nAChRs), and they reproduce some pharmacological properties of nAChRs. X-ray crystal structures of AСhBP in complex with α-conotoxins provide important insights into the interactions of α-conotoxins with distinct nAChR subtypes. Although considerable efforts have been made to understand why α-conotoxin GIC is strongly selective for α3β2 nAChR, this question has not yet been solved. Here we present the structure of α-conotoxin GIC in complex with Aplysia californica AChBP (Ac-AChBP) at a resolution of 2.1 Å. Based on this co-crystal structure complemented with molecular docking data, we suggest the key residues of GIC in determining its high affinity and selectivity for human α3β2 vs α3β4 nAChRs. These suggestions were checked by radioligand and electrophysiology experiments, which confirmed the functional role of detected contacts for GIC interactions with Ac-AChBP and α3β2 nAChR subtypes. While GIC elements responsible for its high affinity binding with Ac-AChBP and α3β2 nAChR were identified, our study also showed the limitations of computer modelling in extending the data from the X-ray structures of the AChBP complexes to all nAChR subtypes

Interaction of three-finger proteins from snake venoms and from mammalian brain with the cys-loop receptors and their models

International audienceWith the use of surface plasmon resonance (SPR) it was shown that ws-Lynx1, a water-soluble analog of the three-finger membrane-bound protein Lynx1, that modulates the activity of brain nicotinic acetylcholine receptors (nAChRs), interacts with the acetylcholine-binding protein (AChBP) with high affinity, K D = 62 nM. This result agrees with the earlier demonstrated competition of ws-Lynx1 with radioiodinated α-bungarotoxin for binding to AChBP. For the first time it was shown that ws-Lynx1 binds to GLIC, prokaryotic Cys-loop receptor (K D = 1.3 μM). On the contrary, SPR revealed that α-cobratoxin, a three-finger protein from cobra venom, does not bind to GLIC. Obtained results indicate that SPR is a promising method for analysis of topography of ws-Lynx1 binding sites using its mutants and those of AChBP and GLIC

6-Bromohypaphorine from Marine Nudibranch Mollusk Hermissenda crassicornis is an Agonist of Human α7 Nicotinic Acetylcholine Receptor

6-Bromohypaphorine (6-BHP) has been isolated from the marine sponges Pachymatisma johnstoni, Aplysina sp., and the tunicate Aplidium conicum, but data on its biological activity were not available. For the nudibranch mollusk Hermissenda crassicornis no endogenous compounds were known, and here we describe the isolation of 6-BHP from this mollusk and its effects on different nicotinic acetylcholine receptors (nAChR). Two-electrode voltage-clamp experiments on the chimeric α7 nAChR (built of chicken α7 ligand-binding and glycine receptor transmembrane domains) or on rat α4β2 nAChR expressed in Xenopus oocytes revealed no action of 6-BHP. However, in radioligand analysis, 6-BHP competed with radioiodinated α-bungarotoxin for binding to human α7 nAChR expressed in GH4C1 cells (IC50 23 ± 1 μM), but showed no competition on muscle-type nAChR from Torpedo californica. In Ca2+-imaging experiments on the human α7 nAChR expressed in the Neuro2a cells, 6-BHP in the presence of PNU120596 behaved as an agonist (EC50 ~80 μM). To the best of our knowledge, 6-BHP is the first low-molecular weight compound from marine source which is an agonist of the nAChR subtype. This may have physiological importance because H. crassicornis, with its simple and tractable nervous system, is a convenient model system for studying the learning and memory processes