Interactions of Globular and Ribbon [γ4E]GID with α4β2 Neuronal Nicotinic Acetylcholine Receptor

The α4β2 nAChR is implicated in a range of diseases and disorders including nicotine addiction, epilepsy and Parkinson’s and Alzheimer’s diseases. Designing α4β2 nAChR selective inhibitors could help define the role of the α4β2 nAChR in such disease states. In this study, we aimed to modify globular and ribbon α-conotoxin GID to selectively target the α4β2 nAChR through competitive inhibition of the α4(+)β2(−) or α4(+)α4(−) interfaces. The binding modes of the globular α-conotoxin [γ4E]GID with rat α3β2, α4β2 and α7 nAChRs were deduced using computational methods and were validated using published experimental data. The binding mode of globular [γ4E]GID at α4β2 nAChR can explain the experimental mutagenesis data, suggesting that it could be used to design GID variants. The predicted mutational energy results showed that globular [γ4E]GID is optimal for binding to α4β2 nAChR and its activity could not likely be further improved through amino-acid substitutions. The binding mode of ribbon GID with the (α4)₃(β2)₂ nAChR was deduced using the information from the cryo-electron structure of (α4)₃(β2)₂ nAChR and the binding mode of ribbon AuIB. The program FoldX predicted the mutational energies of ribbon [γ4E]GID at the α4(+)α4(−) interface, and several ribbon[γ4E]GID mutants were suggested to have desirable properties to inhibit (α4)₃(β2)₂ nAChR.     

Design of new α-conotoxins: from computer modeling to synthesis of potent cholinergic compounds

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, 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.     

ω-Conotoxins GVIA,MVIIA and CVID: SAR and Clinical Potential

Highly selective N-type voltage-gated calcium (Ca_V) channel inhibitors from cone snail venom (the ω-conotoxins) have emerged as a new class of therapeutics for the treatment of chronic and neuropathic pain. Earlier in 2005, Prialt (Elan) or synthetic ω-conotoxin MVIIA, was the first ω-conotoxin to be approved by Food and Drug Administration for human use. This review compares the action of three ω-conotoxins, GVIA, MVIIA and CVID, describing their structure-activity relationships and potential as leads for the design of improved N-type therapeutics that are more useful in the treatment of chronic pain.     

Recombinant Expression and Characterization of α-Conotoxin LvIA in Escherichia coli

α-Conotoxin LvIA is derived from Conus lividus, native to Hainan, and is the most selective inhibitor of α3β2 nicotinic acetylcholine receptors (nAChRs) known to date. In this study, an efficient approach for the production of recombinant α-Conotoxin LvIA is described. Tandem repeats of a LvIA gene fragment were constructed and fused with a KSI gene and a His₆ tag in a Escherichia coli (E. coli) expression vector pET-31b(+). The recombinant plasmids were transformed into E. coli and were found to express well. The KSI-(LvIA)_n-His₆ fusion protein was purified by metal affinity chromatography and then cleaved with CNBr to release recombinant LvIA (rLvIA). High yields of fusion protein ranging from 100 to 500 mg/L culture were obtained. The pharmacological profile of rLvIA was determined by two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes expressing rat nAChR subtypes. The rLvIA antagonized the α3β2 nAChR subtype selectively with a nano-molar IC₅₀. The rLvIA was analgesic in a mouse hot-plate test model of pain. Overall, this study provides an effective method to synthesize α-conotoxin LvIA in an E. coli recombinant expression system, and this approach could be useful to obtain active conopeptides in large quantity and at low cost.     

Brevetoxin and Conotoxin Interactions with Single-Domain Voltage-Gated Sodium Channels from a Diatom and Coccolithophore

The recently characterized single-domain voltage-gated ion channels from eukaryotic protists (EukCats) provide an array of novel channel proteins upon which to test the pharmacology of both clinically and environmentally relevant marine toxins. Here, we examined the effects of the hydrophilic µ-CTx PIIIA and the lipophilic brevetoxins PbTx-2 and PbTx-3 on heterologously expressed EukCat ion channels from a marine diatom and coccolithophore. Surprisingly, none of the toxins inhibited the peak currents evoked by the two EukCats tested. The lack of homology in the outer pore elements of the channel may disrupt the binding of µ-CTx PIIIA, while major structural differences between mammalian sodium channels and the C-terminal domains of the EukCats may diminish interactions with the brevetoxins. However, all three toxins produced significant negative shifts in the voltage dependence of activation and steady state inactivation, suggesting alternative and state-dependent binding conformations that potentially lead to changes in the excitability of the phytoplankton themselves.     

α-Conotoxins and α-Cobratoxin Promote,while Lipoxygenase and Cyclooxygenase Inhibitors Suppress the Proliferation of Glioma C6 Cells

Among the brain tumors, glioma is the most common. In general, different biochemical mechanisms, involving nicotinic acetylcholine receptors (nAChRs) and the arachidonic acid cascade are involved in oncogenesis. Although the engagement of the latter in survival and proliferation of rat C6 glioma has been shown, there are practically no data about the presence and the role of nAChRs in C6 cells. In this work we studied the effects of nAChR antagonists, marine snail α-conotoxins and snake α-cobratoxin, on the survival and proliferation of C6 glioma cells. The effects of the lipoxygenase and cyclooxygenase inhibitors either alone or together with α-conotoxins and α-cobratoxin were studied in parallel. It was found that α-conotoxins and α-cobratoxin promoted the proliferation of C6 glioma cells, while nicotine had practically no effect at concentrations below 1 µL/mL. Nordihydroguaiaretic acid, a nonspecific lipoxygenase inhibitor, and baicalein, a 12-lipoxygenase inhibitor, exerted antiproliferative and cytotoxic effects on C6 cells. nAChR inhibitors weaken this effect after 24 h cultivation but produced no effects at longer times. Quantitative real-time polymerase chain reaction showed that mRNA for α4, α7, β2 and β4 subunits of nAChR were expressed in C6 glioma cells. This is the first indication for involvement of nAChRs in mechanisms of glioma cell proliferation.     

Optimal Cleavage and Oxidative Folding of α-Conotoxin TxIB as a Therapeutic Candidate Peptide

Alpha6beta2 nicotinic acetylcholine receptors (nAChRs) are potential therapeutic targets for the treatment of several neuropsychiatric diseases, including addiction and Parkinson’s disease. Alpha-conotoxin (α-CTx) TxIB is a uniquely selective ligand, which blocks α6/α3β2β3 nAChRs only, but does not block the other subtypes. Therefore, α-CTx TxIB is a valuable therapeutic candidate peptide. Synthesizing enough α-CTx TxIB with high yield production is required for conducting wide-range testing of its potential medicinal applications. The current study optimized the cleavage of synthesized α-CTx TxIB resin-bounded peptide and folding of the cleaved linear peptide. Key parameters influencing cleavage and oxidative folding of α-CTx TxIB were examined, such as buffer, redox agents, pH, salt, co-solvent and temperature. Twelve conditions were used for cleavage optimization. Fifty-four kinds of one-step oxidative solution were used to assess their effects on each α-CTx TxIB isomers’ yield. The result indicated that co-solvent choices were particularly important. Completely oxidative folding of globular isomer was achieved when the NH₄HCO₃ or Tris-HCl folding buffer at 4 °C contained 40% of co-solvent DMSO, and GSH:GSSG (2:1) or GSH only with pH 8~8.7.     

α-Conotoxin TxID and [S9K]TxID, α3β4 nAChR Antagonists,Attenuate Expression and Reinstatement of Nicotine-Induced Conditioned Place Preference in Mice

Tobacco smoking has become a prominent health problem faced around the world. The α3β4 nicotinic acetylcholine receptor (nAChR) is strongly associated with nicotine reward and withdrawal symptom. α-Conotoxin TxID, cloned from Conus textile, is a strong α3β4 nAChR antagonist, which has weak inhibition activity of α6/α3β4 nAChR. Meanwhile, its analogue [S9K]TxID only inhibits α3β4 nAChR (IC₅₀ = 6.9 nM), and has no inhibitory activity to other nAChRs. The present experiment investigates the effect of α3β4 nAChR antagonists (TxID and [S9K]TxID) on the expression and reinstatement of nicotine-induced conditioned place preference (CPP) and explores the behaviors of acute nicotine in mice. The animal experimental results showed that TxID and [S9K] TxID could inhibit the expression and reinstatement of CPP, respectively. Moreover, both had no effect in acute nicotine experiment and the locomotor activity in mice. Therefore, these findings reveal that the α3β4 nAChR may be a potential target for anti-nicotine addiction treatment. [S9K]TxID, α3β4 nAChR antagonist, exhibit a superior effect for anti-nicotine addiction, which is promising to develop a novel smoking cessation drug.     

Structural and Functional Characterization of a Novel α-Conotoxin Mr1.7 from Conus marmoreus Targeting Neuronal nAChR α3β2, α9α10 and α6/α3β2β3 Subtypes

In the present study, we synthesized and, structurally and functionally characterized a novel α4/7-conotoxin Mr1.7 (PECCTHPACHVSHPELC-NH₂), which was previously identified by cDNA libraries from Conus marmoreus in our lab. The NMR solution structure showed that Mr1.7 contained a 3₁₀-helix from residues Pro⁷ to His¹⁰ and a type I β-turn from residues Pro¹⁴ to Cys¹⁷. Electrophysiological results showed that Mr1.7 selectively inhibited the α3β2, α9α10 and α6/α3β2β3 neuronal nicotinic acetylcholine receptors (nAChRs) with an IC₅₀ of 53.1 nM, 185.7 nM and 284.2 nM, respectively, but showed no inhibitory activity on other nAChR subtypes. Further structure-activity studies of Mr1.7 demonstrated that the PE residues at the N-terminal sequence of Mr1.7 were important for modulating its selectivity, and the replacement of Glu² by Ala resulted in a significant increase in potency and selectivity to the α3β2 nAChR. Furthermore, the substitution of Ser¹² with Asn in the loop2 significantly increased the binding of Mr1.7 to α3β2, α3β4, α2β4 and α7 nAChR subtypes. Taken together, this work expanded our knowledge of selectivity and provided a new way to improve the potency and selectivity of inhibitors for nAChR subtypes.