Nicotinic acetylcholine receptors: Therapeutic targets for novel ligands to treat pain and inflammation.

Nicotinic acetylcholine receptors (nAChRs) have been historically defined as ligand-gated ion channels and function as such in the central and peripheral nervous systems. Recently, however, non-ionic signaling mechanisms via nAChRs have been demonstrated in immune cells. Furthermore, the signaling pathways where nAChRs are expressed can be activated by endogenous ligands other than the canonical agonists acetylcholine and choline. In this review, we discuss the involvement of a subset of nAChRs containing α7, α9, and/or α10 subunits in the modulation of pain and inflammation via the cholinergic anti-inflammatory pathway. Additionally, we review the most recent advances in the development of novel ligands and their potential as therapeutics.

Conus regius-Derived Conotoxins: Novel Therapeutic Opportunities from a Marine Organism.

Conus regius is a marine venomous mollusk of the Conus genus that captures its prey by injecting a rich cocktail of bioactive disulfide bond rich peptides called conotoxins. These peptides selectively target a broad range of ion channels, membrane receptors, transporters, and enzymes, making them valuable pharmacological tools and potential drug leads. C. regius-derived conotoxins are particularly attractive due to their marked potency and selectivity against specific nicotinic acetylcholine receptor subtypes, whose signalling is involved in pain, cognitive disorders, drug addiction, and cancer. However, the species-specific differences in sensitivity and the low stability and bioavailability of these conotoxins limit their clinical development as novel therapeutic agents for these disorders. Here, we give an overview of the main pharmacological features of the C. regius-derived conotoxins described so far, focusing on the molecular mechanisms underlying their potential therapeutic effects. Additionally, we describe adoptable chemical engineering solutions to improve their pharmacological properties for future potential clinical translation.

From Crystal Structures of RgIA4 in Complex with Ac-AChBP to Molecular Determinants of Its High Potency of α9α10 nAChR.

α9-containing nicotinic acetylcholine receptors (nAChRs) have been shown to play critical roles in neuropathic pain. The α-conotoxin (α-CTx) RgIA and its analog RgIA4 were identified as the most selective inhibitor of α9α10 nAChR. However, the mechanism of their selectivity toward α9α10 nAChR remains elusive. Here, we reported the co-crystal structure of RgIA and RgIA4 in complex with Aplysia californica acetylcholine binding protein (Ac-AChBP) at resolution of 2.6 Å, respectively. Based on the structure of the complexes, together with molecular dynamic simulation (MD-simulation), we suggested the key residues of α9α10 nAChR in determining its high affinity for RgIA/RgIA4. This is the first time the complex between pain-related conotoxins and Ac-AChBP was reported and the complementary side of α9 subunit in binding of the antagonists shown. These results provide realistic template for the design of new therapeutic in neuropathic pain.

On-resin strategy to label α-conotoxins: Cy5-RgIA, a potent α9α10 nicotinic acetylcholine receptor imaging probe.

In-solution conjugation is the most commonly used strategy to label peptides and proteins with fluorophores. However, lack of site-specific control and high costs of fluorophores are recognised limitations of this approach. Here, we established facile access to grams of Cy5-COOH via a two-step synthetic route, demonstrated that Cy5 is stable to HF treatment and therefore compatible with Boc-SPPS, and coupled Cy5 to the N-terminus of α-conotoxin RgIA while still attached to the resin. Folding of the two-disulfide containing Cy5-RgIA benefitted from the hydrophobic nature of Cy5 resulting in only the globular disulfide bond isomer. In contrast, wild-type α-RgIA folded into the inactive ribbon and bioactive globular isomer under the same conditions. Labelled α-RgIA retained its ability to inhibit acetylcholine(100 µM)-evoked current reversibly with an IC50 of 5.0 nM (Hill coefficient = 1.7) for α-RgIA and an IC50 of 1.6 (Hill coefficient = 1.2) for Cy5-RgIA at the α9α10 nicotinic acetylcholine receptors (nAChRs) heterologeously expressed in Xenopus oocytes. Cy5-RgIA was then used to successfully visualise nAChRs in RAW264.7 mouse macrophage cell line. This work introduced not only a new and valuable nAChR probe, but also a new versatile synthetic strategy that facilitates production of milligram to gram quantities of fluorophore-labelled peptides at low cost, which is often required for in vivo experiments. The strategy is compatible with Boc- and Fmoc-chemistry, allows for site-specific labelling of free amines anywhere in the peptide sequence, and can also be used for the introduction of Cy3/Cy5 FRET pairs.

d-Amino Acid Substitution of α-Conotoxin RgIA Identifies its Critical Residues and Improves the Enzymatic Stability.

α-Conotoxin RgIA is a selective and potent competitive antagonist of rat α9α10 nicotinic acetylcholine receptors (nAChR), but it is much less potent towards human α9α10 nAChR. Furthermore, RgIA is susceptible to proteolytic degradation due to containing four arginine residues. These disadvantages greatly limit its use for clinical applications. The purpose of this research was to identify critical stereocenters of RgIA and discover more stable analogues, enhancing its bioavailability by using the d-amino acid scan method. The activity of each variant was investigated against rat and human α9α10 nAChRs, which were expressed in Xenopus oocytes. Experimental assays showed that 14 out of 15 analogues had a substantial reduction in potency towards rat α9α10 nAChR. Noticeably, analogue 13 retained full biological activity compared with RgIA. Meanwhile, two other analogues, 14 and 15, of which l-Args were substituted with d-Args, exhibited a significantly increased potency towards human α9α10 nAChR, although these analogues showed decreased activities against rat α9α10 nAChR. Additionally, these three analogues exhibited a high resistance against enzymatic degradation in human serum and simulated intestinal fluid (SIF). Collectively, our findings suggest that a d-amino acid scan is a useful strategy for investigating how the side-chain chirality of amino acids affects the structure and function of peptides and may facilitate the development of more stable analogues to increase therapeutic potential.

Corrigendum: Crystal structure of the monomeric extracellular domain of α9 nicotinic receptor subunit in complex with α-conotoxin RgIA: Molecular dynamics insights into RgIA binding to α9α10 nicotinic receptors.

[This corrects the article DOI: 10.3389/fphar.2019.00474.].

α-Conotoxin RgIA and oligoarginine R8 in the mice model alleviate long-term oxaliplatin induced neuropathy.

Оligoarginines were recently discovered (Lebedev et al., 2019 Nov) as a novel class of nicotinic acetylcholine receptors (nAChRs) inhibitors, octaoligoarginine R8 showing a relatively high affinity (44 nM) for the α9/α10 nAChR. Since the inhibition of α9/α10 nAChR by α-conotoxin RgIA and its analogs is a possible way to drugs against neuropathic pain, here in a mice model we compared R8 with α-conotoxin RgIA in the effects on the chemotherapy-induced peripheral neuropathy (CIPN), namely on the long-term oxaliplatin induced neuropathy. Tests of cold allodynia, hot plate, Von Frey and grip strength analysis revealed for R8 and α-conotoxin RgIA similar positive effects, expressed most prominently after two weeks of administration. Histological analysis of the dorsal root ganglia sections showed for R8 and RgIA a similar partial correction of changes in the nuclear morphology of neurons. Since α9/α10 nAChR might be not the only drug target for R8, we analyzed the R8 action on rat TRPV1 and TRPA1, well-known nociceptive receptors. Against rTRPV1 at 25 µM there was no inhibition, while for rTRPA1 IC50 was about 20 µM. Thus, involvement of rTRPA1 cannot be excluded, but in view of the R8 much higher affinity for α9/α10 nAChR the latter seems to be the main target and the easily synthesized R8 can be considered as a potential candidate for a drug design.

Alkaloid ligands enable function of homomeric human α10 nicotinic acetylcholine receptors.

In the nervous system, nicotinic acetylcholine receptors (nAChRs) rapidly transduce a chemical signal into one that is electrical via ligand-gated ion flux through the central channel of the receptor. However, some nAChR subunits are expressed by non-excitable cells where signal transduction apparently occurs through non-ionic mechanisms. One such nAChR subunit, α10, is present in a discreet subset of immune cells and has been implicated in pathologies including cancer, neuropathic pain, and chronic inflammation. Longstanding convention holds that human α10 subunits require co-assembly with α9 subunits for function. Here we assessed whether cholinergic ligands can enable or uncover ionic functions from homomeric α10 nAChRs. Xenopus laevis oocytes expressing human α10 subunits were exposed to a panel of ligands and examined for receptor activation using voltage-clamp electrophysiology. Functional expression of human α10 nAChRs was achieved by exposing the oocytes to the alkaloids strychnine, brucine, or methyllycaconitine. Furthermore, acute exposure to the alkaloid ligands significantly enhanced ionic responses. Acetylcholine-gated currents mediated by α10 nAChRs were potently inhibited by the snake toxins α-bungarotoxin and α-cobratoxin but not by α-conotoxins that target α9 and α9α10 nAChRs. Our findings indicate that human α10 homomers are expressed in oocytes and exposure to certain ligands can enable ionic functions. To our knowledge, this is the first demonstration that human α10 subunits can assemble as functional homomeric nAChRs. These findings have potential implications for receptor regulatory-mechanisms and will enable structural, functional, and further pharmacological characterization of human α10 nAChRs.

The α9α10 nicotinic acetylcholine receptors antagonist α-conotoxin RgIA reverses colitis signs in murine dextran sodium sulfate model.

Nicotinic acetylcholine receptors can regulate inflammation primarily through the vagus nerve via the cholinergic anti-inflammatory pathway. α9α10 nicotinic receptors (nAChRs) are a new promising target for chronic pain and inflammation. Recently, α9α10 selective α-conotoxin antagonists were shown to have antinociception effect in neuropathic and tonic inflammatory pain animal models. However, limited data available on the role of α9α10 nAChRs in experimental colitis. In this study, we report for the first time, the role of α9α10 nAChRs in the dextran sodium sulfate (DSS) experimental animal colitis model. We determined the effect of the α9α10 nAChRs antagonist, α-conotoxin RgIA (α-RgIA) in DSS-induced colitis model in adult male and female C57BL/6 J mice. DSS solution was freely given in the drinking water for seven consecutive days, and tap water was given on the 8th day. We then sacrificed mice on day 8 to examine the entire colon. Disease severity, colon tissue histology, and tumor necrosis factor-α (TNF-α) were evaluated. The lower doses (0.02 and 0.1 nmol/mouse, s.c.) of α-RgIA treatment in DSS-treated mice were inactive, whereas the higher dose (0.2 nmol/mouse, s.c.) reversed the disease activity index (DAI) score, loss of body weight, total histological damage score, as well as the colonic level of TNF-α compared to the DSS-control group. Moreover, the highest dose of α-RgIA (0.2 nmol/mouse, s.c.) significantly rescued the colon length shortening in DSS-treated mice compared to the DSS-control mice. The availability of α9*-selective conotoxins has opened new avenues in pharmacology research and potential targets in inflammatory disorders.

Crystal Structure of the Monomeric Extracellular Domain of α9 Nicotinic Receptor Subunit in Complex With α-Conotoxin RgIA: Molecular Dynamics Insights Into RgIA Binding to α9α10 Nicotinic Receptors.

The α9 subunit of nicotinic acetylcholine receptors (nAChRs) exists mainly in heteropentameric assemblies with α10. Accumulating data indicate the presence of three different binding sites in α9α10 nAChRs: the α9(+)/α9(-), the α9(+)/α10(-), and the α10(+)/α9(-). The major role of the principal (+) side of the extracellular domain (ECD) of α9 subunit in binding of the antagonists methyllylcaconitine and α-bungarotoxin was shown previously by the crystal structures of the monomeric α9-ECD with these molecules. Here we present the 2.26-Å resolution crystal structure of α9-ECD in complex with α-conotoxin (α-Ctx) RgIA, a potential drug for chronic pain, the first structure reported for a complex between an nAChR domain and an α-Ctx. Superposition of this structure with those of other α-Ctxs bound to the homologous pentameric acetylcholine binding proteins revealed significant similarities in the orientation of bound conotoxins, despite the monomeric state of the α9-ECD. In addition, ligand-binding studies calculated a binding affinity of RgIA to the α9-ECD at the low micromolar range. Given the high identity between α9 and α10 ECDs, particularly at their (+) sides, the presented structure was used as template for molecular dynamics simulations of the ECDs of the human α9α10 nAChR in pentameric assemblies. Our results support a favorable binding of RgIA at α9(+)/α9(-) or α10(+)/α9(-) rather than the α9(+)/α10(-) interface, in accordance with previous mutational and functional data.

Interaction of rat α9α10 nicotinic acetylcholine receptor with α-conotoxin RgIA and Vc1.1: Insights from docking, molecular dynamics and binding free energy contributions.

The α9α10 nicotinic acetylcholine receptor (nAChR) is an effective therapeutic target for neuropathic pain. α-Conotoxin RgIA and Vc1.1 are two well-known peptides blocking α9α10 nAChR potently and selectively, which have been extensively investigated as drug candidates. Several key residues were established in previous experimental research. However, the mechanism of the specific interaction still needs to be elucidated in more detail. In this work, we explored the interaction mechanism between RgIA/Vc1.1 and rat α9α10 nAChR using docking and molecular dynamics (MD) simulations. Energy and network analysis programs were used to reveal key residues responsible for their interaction. Our results indicated that the most critical residues were in accord with previous studies. Importantly, several novel residues, including Tyr95, Trp151 in α9 (+)α10 (-) interface as well as Tyr196, Arg59in α10 (+)α9 (-) interface, were found in our models. Furthermore, we analyzed noncovalent interaction energies between RgIA/Vc1.1 and rat α9α10 nAChR. The results showed that three negatively charged residues (Glu197 in α10 subunit, Asp168 in α9 subunit and Asp205 in α10 subunit) were involved in the interaction between RgIA and rat α9α10 nAChR. In contrast, the interaction between Vc1.1 and rat α9α10 nAChR was mediated by the positively charged residues Arg59, Arg81 in α9 (-) subunit. These findings provided further insights into the molecular mechanisms of interaction between RgIA and Vc1.1 and rat α9α10 nAChR.

Nicotinic acetylcholine receptors in neuropathic and inflammatory pain.

Nicotinic acetylcholine receptors (nAChRs) are actively being investigated as therapeutic targets for the treatment of pain and inflammation, but despite more than 30 years of research, there are currently no FDA-approved analgesics that are specific for these receptors. Much of the initial research effort focused on the α4ß2 nAChR subtype, but more recently, additional subtypes have been identified as promising new leads and include α6ß4, α7, and α9-containing nAChRs. This Review will focus on the distribution of these nAChRs in the cell types involved in neuropathic pain and inflammation and the activity of currently available nicotinic ligands.

The α9α10 nicotinic receptor antagonist α-conotoxin RgIA prevents neuropathic pain induced by oxaliplatin treatment.

Oxaliplatin, a third-generation diaminocyclohexane platinum drug, is widely used alone or in combination with 5-fluorouracil and leucovorin to treat metastatic colorectal, ovarian, and pancreatic cancers. Oxaliplatin long-term treatment is associated with the development of a dose-limiting painful neuropathy that dramatically impairs the patient's quality of life and therapy possibility. To study novel strategies to treat oxaliplatin-induced neuropathy, we evaluated α-conotoxin RgIA, a peptide that potently blocks the α9α10 nicotinic acetylcholine receptor (nAChR) subtype in a rat model of oxaliplatin-dependent neurotoxicity (2.4mgkg(-1) oxaliplatin intraperitoneally daily for 21days). The administration of RgIA (2 and 10nmol injected intramuscularly once a day concomitantly with oxaliplatin treatment), reduced the oxaliplatin-dependent hypersensitivity to mechanical and thermal noxious and non-noxious stimuli. Moreover, morphological modifications of L4-L5 dorsal root ganglia were significantly prevented. In the spinal cord the numerical increase of astrocyte cell density present in oxaliplatin-treated rats is partially prevented by RgIA treatment. Nevertheless, the administration of the α-conotoxin is able per se to elicit a numerical increase and a morphological activation of microglia and astrocytes in specific brain areas.

α-conotoxin RgIA protects against the development of nerve injury-induced chronic pain and prevents both neuronal and glial derangement.

Neuropathic pain affects millions of people worldwide, causing substantial disability and greatly impairing quality of life. Commonly used analgesics or antihyperalgesic compounds are generally characterized by limited therapeutic outcomes. Thus, there is a compelling need for novel therapeutic strategies able to prevent nervous tissue alterations responsible for chronic pain. The α9α10 nicotinic acetylcholine receptor antagonist α-conotoxin RgIA (RgIA), a peptide isolated from the venom of a carnivorous cone snail, induces relief in both acute and chronic pain models. To evaluate potential disease-modifying effects of RgIA, the compound was given to rats following chronic constriction injury (CCI) of the sciatic nerve. Two or 10 nmol RgIA injected intramuscularly once a day for 14 days reduced the painful response to suprathreshold stimulation, increased pain threshold to nonnoxious stimuli, and normalized alterations in hind limb weight bearing. Histological analysis of the sciatic nerve revealed that RgIA prevented CCI-induced decreases of axonal compactness and diameter, loss of myelin sheath, and decreases in the fiber number. Moreover, RgIA significantly reduced edema and inflammatory infiltrate, including a decrease of CD86(+) macrophages. In L4-L5 dorsal root ganglia, RgIA prevented morphometric changes and reduced the inflammatory infiltrate consistent with a disease-modifying effect. In the dorsal horn of the spinal cord, RgIA prevented CCI-induced activation of microglia and astrocytes. These data suggest that RgIA-like compounds may represent a novel class of therapeutics for neuropathic pain that protects peripheral nervous tissues as well as prevents central maladaptive plasticity by inhibiting glial cell activation.

Presence of multiple binding sites on α9α10 nAChR receptors alludes to stoichiometric-dependent action of the α-conotoxin, Vc1.1.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels involved in fast synaptic transmission. nAChRs are pentameric receptors formed from a combination of different or similar subunits to produce heteromeric or homomeric channels. The heteromeric, α9α10 nAChR subtype is well-known for its role in the auditory system, being expressed in cochlear hair cells. These nAChRs have also been shown to be involved in immune-modulation. Antagonists of α9α10 nAChRs, like the α-conotoxin Vc1.1, have analgesic effects in neuropathic pain. Unlike other nAChR subtypes there is no evidence that functional receptor stoichiometries of α9α10 exist. By using 2-electrode voltage clamp methods and maintaining a constant intracellular Ca(2+) concentration, we observed a biphasic activation curve for ACh that is dependent on receptor stoichiometry. Vc1.1, but not the α9α10 antagonists RgIA or atropine, inhibits ACh-evoked currents in a biphasic manner. Characteristics of the ACh and Vc1.1 activation and inhibition curves can be altered by varying the ratio of α9 and α10 mRNA injected into oocytes, changing the curves from biphasic to monophasic when an excess of α10 mRNA is used. These results highlight the difference in the pharmacological profiles of at least two different α9α10 nAChR stoichiometries, possibly (α9)3(α10)2 and (α9)2(α10)3. As a result, we infer that there is an additional binding site for ACh and Vc1.1 at the α9-α9 interface on the hypothesized (α9)3(α10)2 nAChR, in addition to the α10-α9 and or α9-α10 interfaces that are common to both stoichiometries. This study provides further evidence that receptor stoichiometry contributes another layer of complexity in understanding Cys-loop receptors.