Design, Synthesis, and Structure-Activity Relationships of Novel Peptide Derivatives of the Severe Acute Respiratory Syndrome-Coronavirus-2 Spike-Protein that Potently Inhibit Nicotinic Acetylcholine Receptors.

The spike-protein of SARS-CoV-2 has a distinctive amino-acid sequence (682RRARS686) that forms a cleavage site for the enzyme furin. Strikingly, the structure of the spike-protein loop containing the furin cleavage site bears substantial similarity to neurotoxin peptides found in the venoms of certain snakes and marine cone snails. Leveraging this relationship, we designed and synthesized disulfide-constrained peptides with amino-acid sequences corresponding to the furin cleavage-sites of wild-type (B.1 variant) SARS-CoV-2 or the Alpha, Delta, and Omicron variants. Remarkably, some of these peptides potently inhibited α7 and α9α10 nicotinic acetylcholine receptors (nAChR) with nM affinity and showed SARS-CoV-2 variant and nAChR subtype-dependent potencies. Nuclear magnetic resonance spectroscopy and molecular dynamics were used to rationalize structure-activity relationships between peptides and their cognate receptors. These findings delineate nAChR subtypes that can serve as high-affinity spike-protein targets in tissues central to COVID-19 pathophysiology and identify ligands and target receptors to inform the development of novel SARS-CoV-2 therapeutics.

Synthesis of Aristoquinoline Enantiomers and Their Evaluation at the α3ß4 Nicotinic Acetylcholine Receptor.

The first synthesis of aristoquinoline (1), a naturally occurring nicotinic acetylcholine receptor (nAChR) antagonist, was accomplished using two different approaches. Comparison of the synthetic material's spectroscopic data to that of the isolated alkaloid identified a previously misassigned stereogenic center. An evaluation of each enantiomer's activity at the α3ß4 nAChR revealed that (+)-1 is significantly more potent than (-)-1. This unexpected finding suggests that naturally occurring 1 possesses the opposite absolute configuration from indole-containing Aristotelia alkaloids.

Selective Penicillamine Substitution Enables Development of a Potent Analgesic Peptide that Acts through a Non-Opioid-Based Mechanism.

Venom-derived compounds are of broad interest in neuropharmacology and drug development. α-Conotoxins are small disulfide-containing peptides from Conus snails that target nicotinic acetylcholine receptors (nAChRs) and are in clinical development for non-opioid-based treatment of intractable pain. Although refined by evolution for interaction with target prey receptors, enhancements of pharmacological properties are needed for use in mammalian systems. Therefore, we synthesized analogues of α-conotoxin RgIA using a combination of selective penicillamine substitutions together with natural and non-natural amino acid replacements. This approach resulted in a peptide with 9000-fold increased potency on the human α9α10 nAChR and improved resistance to disulfide shuffling compared to the native peptide. The lead analogue, RgIA-5474, potently blocked α9α10 nAChRs, but not opioid- or other pain-related targets. In addition, RgIA-5474 effectively reversed chemotherapy-induced neuropathic pain.

Synthesis and evaluation of disulfide-rich cyclic α-conotoxin [S9A]TxID analogues as novel α3ß4 nAChR antagonists.

Head-to-tail cyclization is an effective strategy to improve the biological stability of peptides. The α-conotoxin [S9A]TxID is a peptide that inhibits α3ß4 nAChR with high activity and selectivity. Herein, we established a method for cyclizing and oxidative folding of [S9A]TxID, and six cyclic analogues of [S9A]TxID were chemically synthesized with various linker lengths. We used the electrophysiology assay to measure activity values of these cyclic analogues, and obtained the most potent analogue c[S9A]TxID-6, which was more stable than native [S9A]TxID against proteinase K.

Cysteine [2,4] Disulfide Bond as a New Modifiable Site of α-Conotoxin TxIB.

α-Conotoxin TxIB, a selective antagonist of α6/α3ß2ß3 nicotinic acetylcholine receptor, could be a potential therapeutic agent for addiction and Parkinson's disease. As a peptide with a complex pharmacophoric conformation, it is important and difficult to find a modifiable site which can be modified effectively and efficiently without activity loss. In this study, three xylene scaffolds were individually reacted with one pair of the cysteine residues ([1,3] or [2,4]), and iodine oxidation was used to form a disulfide bond between the other pair. Overall, six analogs were synthesized with moderate isolated yields from 55% to 65%, which is four times higher than the traditional two-step oxidation with orthogonal protection on cysteines. The cysteine [2,4] modified analogs, with higher stability in human serum than native TxIB, showed obvious inhibitory effect and selectivity on α6/α3ß2ß3 nicotinic acetylcholine receptors (nAChRs), which was 100 times more than the cysteine [1,3] modified ones. This result demonstrated that the cysteine [2,4] disulfide bond is a new modifiable site of TxIB, and further modification can be a simple and feasible strategy for the exploitation and utilization of α-Conotoxin TxIB in drug discovery.

Computational and Functional Mapping of Human and Rat α6ß4 Nicotinic Acetylcholine Receptors Reveals Species-Specific Ligand-Binding Motifs.

Nicotinic acetylcholine receptors (nAChRs) are pharmacological targets for the treatment of neuropathic pain, and the α6ß4 subtype has been identified as particularly promising. Rat α6ß4 nAChRs are less sensitive to some ligands than the human homologue potentially complicating the use of rodent α6ß4 receptors for screening therapeutic compounds. We used molecular dynamics simulations coupled with functional assays to study the interaction between α-conotoxin PeIA and α6ß4 nAChRs and to identify key ligand-receptor interactions that contribute to species differences in α-conotoxin potency. Our results show that human and rat α6ß4 nAChRs have distinct ligand-binding motifs and show markedly different sensitivities to α-conotoxins. These studies facilitated the creation of PeIA-5667, a peptide that shows 270-fold higher potency for rat α6ß4 nAChRs over native PeIA and similar potency for the human homologue. Our results may inform the design of therapeutic ligands that target α6ß4 nAChRs for the treatment of neuropathic pain.

α-Conotoxin TxID and [S9K]TxID, α3ß4 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 α3ß4 nicotinic acetylcholine receptor (nAChR) is strongly associated with nicotine reward and withdrawal symptom. α-Conotoxin TxID, cloned from Conus textile, is a strong α3ß4 nAChR antagonist, which has weak inhibition activity of α6/α3ß4 nAChR. Meanwhile, its analogue [S9K]TxID only inhibits α3ß4 nAChR (IC50 = 6.9 nM), and has no inhibitory activity to other nAChRs. The present experiment investigates the effect of α3ß4 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 α3ß4 nAChR may be a potential target for anti-nicotine addiction treatment. [S9K]TxID, α3ß4 nAChR antagonist, exhibit a superior effect for anti-nicotine addiction, which is promising to develop a novel smoking cessation drug.

Effect of 4-Fluoro-N-(4-Sulfamoylbenzyl) Benzene Sulfonamide on Acquisition and Expression of Nicotine-Induced Behavioral Sensitization and Striatal Adenosine Levels.

INTRODUCTION: Behavioral sensitization is a phenomenon that develops from intermittent exposure to nicotine and other psychostimulants, which often leads to heightened locomotor activity and then relapse. Sulfonamides that act as carbonic anhydrase inhibitors have a documented role in enhancing dopaminergic tone and normalizing neuroplasticity by stabilizing glutamate release. OBJECTIVE: The aim of the current study was to explore synthetic sulfonamides derivative 4-fluoro-N-(4-sulfamoylbenzyl) benzene-sulfonamide (4-FBS) (with documented carbonic anhydrase inhibitory activity) on acquisition and expression of nicotine-induced behavioral sensitization. METHODS: In the acquisition phase, selected 5 groups of mice were exposed to saline or nicotine 0.5mg/kg intraperitoneal (i.p) for 7 consecutive days. Selected 3 groups were administered with 4-FBS 20, 40, and 60 mg/kg p.o. along with nicotine. After 3 days of the drug-free period, ie, day 11, a challenge dose of nicotine was injected to all groups except saline and locomotor activity was recorded for 30 minutes. In the expression phase, mice were exposed to saline and nicotine only 0.5 mg/kg i.p for 7 consecutive days. After 3 days of the drug-free period, ie, day 11, 4-FBS at 20, 40, and 60 mg/kg were administered to the selected groups, one hour after drug a nicotine challenge dose was administered, and locomotion was recorded. At the end of behavioral experiments, all animals were decapitated and the striatum was excised and screened for changes in adenosine levels, using HPLC-UV. RESULTS: Taken together, our findings showed that 4-FBS in all 3 doses, in both sets of experiments significantly attenuated nicotine-induced behavioral sensitization in mice. Additionally, 4-FBS at 60mg/kg significantly lowered the adenosine level in the striatum. CONCLUSION: The behavioral and adenosine modulation is promising, and more receptors level studies are warranted to explore the exact mechanism of action of 4-FBS.

Design, synthesis and biological activities of piperidine-spirooxadiazole derivatives as α7 nicotinic receptor antagonists.

α: 7 nicotinic acetylcholine receptors (nAChRs) expressed in the nervous and immune systems have been suggested to play important roles in the control of inflammation. However, the lack of antagonist tools specifically inhibiting α7 nAChR impedes the validation of the channel as therapeutic target. To discover a selective α7 antagonist, we started a pharmacophore-based virtual screening and identified a piperidine-spirooxadiazole derivative T761-0184 that acts as a α7 antagonist. A series of novel piperidine-spirooxadiazole derivatives were subsequently synthesized and evaluated using two-electrode voltage clamp (TEVC) assay in Xenopus oocytes. Lead compounds from two series inhibited α7 with their IC50 values ranging from 3.3 µM to 13.7 µM. Compound B10 exhibited α7 selectivity over other α4ß2 and α3ß4 nAChR subtypes. The analysis of structure-activity relationship (SAR) provides valuable insights for further development of selective α7 nAChR antagonists.

3-Quinuclidinyl-α-methoxydiphenylacetate: A multi-targeted ligand with antimuscarinic and antinicotinic effects designed for the treatment of anticholinesterase poisoning.

Racemic 3-quinuclidinyl-α-methoxydiphenylacetate (MB266) was synthesised. Its activity at muscarinic acetylcholine receptors (mAChRs), and muscle and neuronal nicotinic acetylcholine receptors (nAChRs), was compared to that of atropine and racemic 3-quinucidinyl benzilate (QNB) using a functional assay based on agonist-induced elevation of intracellular calcium ion concentration in CN21, Chinese Hamster Ovary (CHO) and SHSY5Y human cell lines. MB266 acted as an antagonist at acetylcholine receptors, displaying 18-fold selectivity for mAChR versus nAChR (compared to the 15,200-fold selectivity observed for QNB). Thus O-methylation of QNB reduced the affinity for mAChR antagonism and increased the relative potency at both muscle and neuronal nAChRs. Despite MB266 having a pharmacological profile potentially useful for the treatment of anticholinesterase poisoning, its administration did not improve the neuromuscular function in a soman-poisoned guinea-pig diaphragm preparation pretreated with the organophosphorus nerve agent soman. Consideration should be given to exploring the potential of MB266 for possible anticonvulsant action in vitro as part of a multi-targeted ligand approach.

Philanthotoxin Analogues That Selectively Inhibit Ganglionic Nicotinic Acetylcholine Receptors with Exceptional Potency.

Philanthotoxin-433 (PhTX-433) is an active component of the venom from the Egyptian digger wasp, Philanthus triangulum. PhTX-433 nonselectively inhibits several excitatory ligand-gated ion channels, and we recently showed that its synthetic analogue, PhTX-343, exhibits strong selectivity for neuronal over muscle-type nicotinic acetylcholine receptors (nAChRs). Here, we examined the action of 17 analogues of PhTX-343 against ganglionic (α3ß4) and brain (α4ß2) nAChRs expressed in Xenopus oocytes by using a two-electrode voltage clamp at -100 mV. IC50 values for PhTX-343 inhibition of α3ß4 and α4ß2 receptors were 7.7 and 80 nM, respectively. All the studied analogues had significantly higher potency at α3ß4 nAChRs with IC50 values as low as 0.16 nM and with up to 91-fold selectivity for α3ß4 over α4ß2 receptors. We conclude that PhTX-343 analogues displaying both a saturated ring and an aromatic moiety in the hydrophobic headgroup of the molecule demonstrate exceptional potency and selectivity for α3ß4 nAChRs.

PeIA-5466: A Novel Peptide Antagonist Containing Non-natural Amino Acids That Selectively Targets α3ß2 Nicotinic Acetylcholine Receptors.

Pharmacologically distinguishing α3ß2 nicotinic acetylcholine receptors (nAChRs) from closely related subtypes, particularly α6ß2, has been challenging due to the lack of subtype-selective ligands. We created analogs of α-conotoxin (α-Ctx) PeIA to identify ligand-receptor interactions that could be exploited to selectively increase potency and selectivity for α3ß2 nAChRs. A series of PeIA analogs were synthesized by replacing amino acid residues in the second disulfide loop with standard or nonstandard residues and assessing their activity on α3ß2 and α6/α3ß2ß3 nAChRs heterologously expressed in Xenopus laevis oocytes. Asparagine11 was found to occupy a pivotal position, and when replaced with negatively charged amino acids, selectivity for α3ß2 over α6/α3ß2ß3 nAChRs was substantially increased. Second generation peptides were then designed to further improve both potency and selectivity. One peptide, PeIA-5466, was ∼300-fold more potent on α3ß2 than α6/α3ß2ß3 and is the most α3ß2-selective antagonist heretofore reported.

Synthetic Pinnatoxins A and G Reversibly Block Mouse Skeletal Neuromuscular Transmission In Vivo and In Vitro.

Pinnatoxins (PnTXs) A-H constitute an emerging family belonging to the cyclic imine group of phycotoxins. Interest has been focused on these fast-acting and highly-potent toxins because they are widely found in contaminated shellfish. Despite their highly complex molecular structure, PnTXs have been chemically synthetized and demonstrated to act on various nicotinic acetylcholine receptor (nAChR) subtypes. In the present work, PnTX-A, PnTX-G and analogue, obtained by chemical synthesis with a high degree of purity (>98%), have been studied in vivo and in vitro on adult mouse and isolated nerve-muscle preparations expressing the mature muscle-type (α1)2ß1δε nAChR. The results show that PnTX-A and G acted on the neuromuscular system of anesthetized mice and blocked the compound muscle action potential (CMAP) in a dose- and time-dependent manner, using a minimally invasive electrophysiological method. The CMAP block produced by both toxins in vivo was reversible within 6-8 h. PnTX-A and G, applied to isolated extensor digitorum longus nerve-muscle preparations, blocked reversibly isometric twitches evoked by nerve stimulation. The action of PnTX-A was reversed by 3,4-diaminopyridine. Both toxins exerted no direct action on muscle fibers, as revealed by direct muscle stimulation. PnTX-A and G blocked synaptic transmission at mouse neuromuscular junctions and PnTX-A amino ketone analogue (containing an open form of the imine ring) had no effect on neuromuscular transmission. These results indicate the importance of the cyclic imine for interacting with the adult mammalian muscle-type nAChR. Modeling and docking studies revealed molecular determinants responsible for the interaction of PnTXs with the muscle-type nAChR.

New Rigid Nicotine Analogues, Carrying a Norbornane Moiety, Are Potent Agonists of α7 and α3* Nicotinic Receptors.

A three-dimensional database search has been applied to design a series of endo- and exo-3-(pyridin-3-yl)bicyclo[2.2.1]heptan-2-amines as nicotinic receptor ligands. The synthesized compounds were tested in radioligand binding assay on rat cortex against [3H]-cytisine and [3H]-methyllycaconitine to measure their affinity for α4ß2* and α7* nicotinic receptors. The new derivatives showed some preference for the α4ß2* over the α7* subtype, with their affinity being dependent on the endo/exo isomerism and on the methylation degree of the basic nitrogen. The endo primary amines displayed the lowest Ki values on both receptor subtypes. Selected compounds (1a, 2a, 3a, and 6a) were tested on heterologously expressed α4ß2, α7, and α3ß2 receptors and on SHSY-5Y cells. Compounds 1a and 2a showed α4ß2 antagonistic properties while behaved as full agonists on recombinant α7 and on SHSY5Y cells. On the α3ß2 subtype, only the chloro derivative 2a showed full agonist activity and submicromolar potency (EC50 = 0.43 µM). The primary amines described here represent new chemotypes for the α7 and α3* receptor subtypes.

Di- and heptavalent nicotinic analogues to interfere with α7 nicotinic acetylcholine receptors.

In the field of nicotinic acetylcholine receptors (nAChRs), recognized as important therapeutic targets, much effort has been dedicated to the development of nicotinic analogues to agonize or antagonize distinct homo- and heteropentamers nAChR subtypes, selectively. In this work we developed di- and heptavalent nicotinic derivatives based on ethylene glycol (EG) and cyclodextrin cores, respectively. The compounds showed a concentration dependent inhibition of acetylcholine-induced currents on α7 nAChR expressed by Xenopus oocytes. Interesting features were observed with the divalent nicotinic derivatives, acting as antagonists with varied inhibitory concentrations (IC50) in function of the spacer arm length. The best divalent compounds showed a 16-fold lowered IC50 compared to the monovalent reference (12 vs 195 µM). Docking investigations provide guidelines to rationalize these experimental findings.

Structure-Activity Studies Reveal the Molecular Basis for GABAB-Receptor Mediated Inhibition of High Voltage-Activated Calcium Channels by α-Conotoxin Vc1.1.

α-Conotoxins are disulfide-bonded peptides from cone snail venoms and are characterized by their affinity for nicotinic acetylcholine receptors (nAChR). Several α-conotoxins with distinct selectivity for nAChR subtypes have been identified as potent analgesics in animal models of chronic pain. However, a number of α-conotoxins have been shown to inhibit N-type calcium channel currents in rodent dissociated dorsal root ganglion (DRG) neurons via activation of G protein-coupled GABAB receptors (GABABR). Therefore, it is unclear whether activation of GABABR or inhibition of α9α10 nAChRs is the analgesic mechanism. To investigate the mechanisms by which α-conotoxins provide analgesia, we synthesized a suite of Vc1.1 analogues where all residues, except the conserved cysteines, in Vc1.1 were individually replaced by alanine (A), lysine (K), and aspartic acid (D). Our results show that the amino acids in the first loop play an important role in binding of the peptide to the receptor, whereas those in the second loop play an important role for the selectivity of the peptide for the GABABR over α9α10 nAChRs. We designed a cVc1.1 analogue that is >8000-fold selective for GABABR-mediated inhibition of high voltage-activated (HVA) calcium channels over α9α10 nAChRs and show that it is analgesic in a mouse model of chronic visceral hypersensitivity (CVH). cVc1.1[D11A,E14A] caused dose-dependent inhibition of colonic nociceptors with greater efficacy in ex vivo CVH colonic nociceptors relative to healthy colonic nociceptors. These findings suggest that selectively targeting GABABR-mediated HVA calcium channel inhibition by α-conotoxins could be effective for the treatment of chronic visceral pain.

Designing selective modulators for the nicotinic receptor subtypes: challenges and opportunities.

Nicotinic receptors are membrane proteins involved in several physiological processes. They are considered suitable drug targets for various CNS disorders or conditions, as shown by the large number of compounds which have entered clinical trials. In recent years, nonconventional agonists have been discovered: positive allosteric modulators, allosteric agonists, site-specific agonists and silent desensitizers are compounds able to modulate the receptor interacting at sites different from the orthodox one, or to desensitize the receptor without prior opening. While these new findings can further complicate the pharmacology of these proteins and the design and optimization of ligands, they undoubtedly offer new opportunities to find drugs for the many therapeutic indications involving nicotinic receptors.

Effects of serum, enzyme, thiol, and forced degradation on the stabilities of αO-Conotoxin GeXIVA[1,2] and GeXIVA [1,4].

αO-conotoxin GeXIVA, which is a potent antagonist of α9α10 nicotinic acetylcholine receptor (nAChR), is of great interest as a potential analgesic for chronic neuropathic pain. It has three isomers, of which both GeXIVA[1,2] and GeXIVA[1,4] showed similar low nanomolar IC50 s in potent blocking rat α9α10 nAChRs. Here, we first reported stabilities of GeXIVA[1,2] and GeXIVA[1,4] in various biochemical circumstances, including human serum, enzymatic degradation, and thiol, which would be the key factors to affect stabilities of the two isomers in vivo. Simultaneously, forced degradation was carried out to evaluate stabilities of the two isomers. GeXIVA[1,2] and GeXIVA[1,4] were unstable when they were incubated in serum and digestive enzymes at 37°C. Their disulfide bond frameworks were easy to be scrambled in GSH and HSA. For different stress conditions, their stabilities were impacted greatly by oxidation, temperature, and alkaline conditions. The results may provide a foundation for storage conditions, structural modification, and pharmaceutical preparation of GeXIVA[1,2] and GeXIVA[1,4].

Design, synthesis, and biological activity of 5'-phenyl-1,2,5,6-tetrahydro-3,3'-bipyridine analogues as potential antagonists of nicotinic acetylcholine receptors.

Starting from a known non-specific agonist (1) of nicotinic acetylcholine receptors (nAChRs), rationally guided structural-based design resulted in the discovery of a small series of 5'-phenyl-1,2,5,6-tetrahydro-3,3'-bipyridines (3a-3e) incorporating a phenyl ring off the pyridine core of 1. The compounds were synthesized via successive Suzuki couplings on a suitably functionalized pyridine starting monomer 4 to append phenyl and pyridyl substituents off the 3- and 5-positions, respectively, and then subsequent modifications were made on the flanking pyridyl ring to provide target compounds. Compound 3a is a novel antagonist, which is highly selective for α3ß4 nAChR (Ki=123nM) over the α4ß2 and α7 receptors.

Synthesis, Nicotinic Acetylcholine Receptor Binding, and in Vitro and in Vivo Pharmacological Properties of 2'-Fluoro-(substituted thiophenyl)deschloroepibatidine Analogues.

The synthesis, nAChR in vitro and in vivo pharmacological properties of 2'-fluoro-3'-(substituted thiophenyl)deschloroepibatidine analogues (5a-f, 6a-d, and 7a-c) are presented herein. All had subnanomolar affinity at α4ß2*-nAChRs. Contrary to lead structure epibatidine, a potent nAChR agonist, all were potent α4ß2- and α3ß4-AChR antagonists in an in vitro functional assay. In vivo, the compounds were also nAChR antagonists with various degrees of agonist activity. Compounds 5e, 5f, 6a, 6c, 6d, and 7c had no agonist effects in the tail-flick, hot-plate, hypothermia, or spontaneous activity tests, whereas 5a-d, 7a and 7b did not have agonist activity in the tail-flick and hot-plate tests but, like varenicline, were agonists in the hypothermia and spontaneous activity tests. Compound 6b had agonist activity in all four in vivo tests. All the compounds were antagonists of nicotine-induced antinociception in the tail-flick test, and all except 5c, 5d, 5f, and 6b were antagonists of nicotine-induced antinociception in the hot-plate test. Compound 7c, which had a Ki = 0.86 nM in the binding assay similar potency at α4ß2/α3ß4 with selectivity relative to α7 nAChRs, had an AD50 value of 0.001 µg/kg in the tail-flick test with no agonist activity in the in vitro or in vivo test had one of the more interesting profiles.