De Novo Screening and Mirror Image Isomerization of Linear Peptides Targeting α7 Nicotinic Acetylcholine Receptor.

As ligand-gated ion channels, nicotinic acetylcholine receptors (nAChRs) are widely distributed in the central and peripheral nervous systems and are associated with the pathogenesis of various degenerative neurological diseases. Here, we report the results of phage display-based de novo screening of an 11-residue linear peptide (named LKP1794) that targets the α7 nAChR, which is among the most abundant nAChR subtypes in the brain. Moreover, two d-peptides were generated through mirror image and/or primary sequence inverso isomerization (termed DRKP1794 and DKP1794) and displayed improved inhibitory effects (IC50 = 0.86 and 0.35 µM, respectively) on α7 nAChR compared with the parent l-peptide LKP1794 (IC50 = 2.48 µM), which markedly enhanced serum stability. A peptide-based fluorescence probe was developed using proteolytically resistant DKP1794 to specifically image the α7 nAChR in living cells. This work provides a new peptide tool to achieve inhibitory modulation and specifically image the α7 nAChR.

Structural mechanisms of α7 nicotinic receptor allosteric modulation and activation.

The α7 nicotinic acetylcholine receptor is a pentameric ligand-gated ion channel that plays an important role in cholinergic signaling throughout the nervous system. Its unique physiological characteristics and implications in neurological disorders and inflammation make it a promising but challenging therapeutic target. Positive allosteric modulators overcome limitations of traditional α7 agonists, but their potentiation mechanisms remain unclear. Here, we present high-resolution structures of α7-modulator complexes, revealing partially overlapping binding sites but varying conformational states. Structure-guided functional and computational tests suggest that differences in modulator activity arise from the stable rotation of a channel gating residue out of the pore. We extend the study using a time-resolved cryoelectron microscopy (cryo-EM) approach to reveal asymmetric state transitions for this homomeric channel and also find that a modulator with allosteric agonist activity exploits a distinct channel-gating mechanism. These results define mechanisms of α7 allosteric modulation and activation with implications across the pentameric receptor superfamily.

Real-time tilting and twisting motions of ligand-bound states of α7 nicotinic acetylcholine receptor.

The α7 nicotinic acetylcholine receptor is a member of the nicotinic acetylcholine receptor family and is composed of five α7 subunits arranged symmetrically around a central pore. It is localized in the central nervous system and immune cells and could be a target for treating Alzheimer's disease and schizophrenia. Acetylcholine is a ligand that opens the channel, although prolonged application rapidly decreases the response. Ivermectin was reported as one of the positive allosteric modulators, since the binding of Ivermectin to the channel enhances acetylcholine-evoked α7 currents. One research has suggested that tilting motions of the nicotinic acetylcholine receptor are responsible for channel opening and activation. To verify this hypothesis applies to α7 nicotinic acetylcholine receptor, we utilized a diffracted X-ray tracking method to monitor the stable twisting and tilting motion of nAChR α7 without a ligand, with acetylcholine, with Ivermectin, and with both of them. The results show that the α7 nicotinic acetylcholine receptor twists counterclockwise with the channel transiently opening, transitioning to a desensitized state in the presence of acetylcholine and clockwise without the channel opening in the presence of Ivermectin. We propose that the conformational transition of ACh-bound nAChR α7 may be due to the collective twisting of the five α7 subunits, resulting in the compression and movement, either downward or upward, of one or more subunits, thus manifesting tilting motions. These tilting motions possibly represent the transition from the resting state to channel opening and potentially to the desensitized state.

An original potentiating mechanism revealed by the cryo-EM structures of the human α7 nicotinic receptor in complex with nanobodies.

The human α7 nicotinic receptor is a pentameric channel mediating cellular and neuronal communication. It has attracted considerable interest in designing ligands for the treatment of neurological and psychiatric disorders. To develop a novel class of α7 ligands, we recently generated two nanobodies named E3 and C4, acting as positive allosteric modulator and silent allosteric ligand, respectively. Here, we solved the cryo-electron microscopy structures of the nanobody-receptor complexes. E3 and C4 bind to a common epitope involving two subunits at the apex of the receptor. They form by themselves a symmetric pentameric assembly that extends the extracellular domain. Unlike C4, the binding of E3 drives an agonist-bound conformation of the extracellular domain in the absence of an orthosteric agonist, and mutational analysis shows a key contribution of an N-linked sugar moiety in mediating E3 potentiation. The nanobody E3, by remotely controlling the global allosteric conformation of the receptor, implements an original mechanism of regulation that opens new avenues for drug design.

Recent Advances in the Discovery of Nicotinic Acetylcholine Receptor Allosteric Modulators.

Positive allosteric modulators (PAMs), negative allosteric modulators (NAMs), silent agonists, allosteric activating PAMs and neutral or silent allosteric modulators are compounds capable of modulating the nicotinic receptor by interacting at allosteric modulatory sites distinct from the orthosteric sites. This survey is focused on the compounds that have been shown or have been designed to interact with nicotinic receptors as allosteric modulators of different subtypes, mainly α7 and α4ß2. Minimal chemical changes can cause a different pharmacological profile, which can then lead to the design of selective modulators. Experimental evidence supports the use of allosteric modulators as therapeutic tools for neurological and non-neurological conditions.

Selective α3ß4 Nicotinic Acetylcholine Receptor Ligand as a Potential Tracer for Drug Addiction.

α3ß4 Nicotinic acetylcholine receptor (nAChR) has been recognized as an emerging biomarker for the early detection of drug addiction. Herein, α3ß4 nAChR ligands were designed and synthesized to improve the binding affinity and selectivity of two lead compounds, (S)-QND8 and (S)-T2, for the development of an α3ß4 nAChR tracer. The structural modification was achieved by retaining the key features and expanding the molecular structure with a benzyloxy group to increase the lipophilicity for blood-brain barrier penetration and to extend the ligand-receptor interaction. The preserved key features are a fluorine atom for radiotracer development and a p-hydroxyl motif for ligand-receptor binding affinity. Four (R)- and (S)-quinuclidine-triazole (AK1-AK4) were synthesized and the binding affinity, together with selectivity to α3ß4 nAChR subtype, were determined by competitive radioligand binding assay using [3H]epibatidine as a radioligand. Among all modified compounds, AK3 showed the highest binding affinity and selectivity to α3ß4 nAChR with a Ki value of 3.18 nM, comparable to (S)-QND8 and (S)-T2 and 3069-fold higher affinity to α3ß4 nAChR in comparison to α7 nAChR. The α3ß4 nAChR selectivity of AK3 was considerably higher than those of (S)-QND8 (11.8-fold) and (S)-T2 (294-fold). AK3 was shown to be a promising α3ß4 nAChR tracer for further development as a radiotracer for drug addiction.

Structures of highly flexible intracellular domain of human α7 nicotinic acetylcholine receptor.

The intracellular domain (ICD) of Cys-loop receptors mediates diverse functions. To date, no structure of a full-length ICD is available due to challenges stemming from its dynamic nature. Here, combining nuclear magnetic resonance (NMR) and electron spin resonance experiments with Rosetta computations, we determine full-length ICD structures of the human α7 nicotinic acetylcholine receptor in a resting state. We show that ~57% of the ICD residues are in highly flexible regions, primarily in a large loop (loop L) with the most mobile segment spanning ~50 Å from the central channel axis. Loop L is anchored onto the MA helix and virtually forms two smaller loops, thereby increasing its stability. Previously known motifs for cytoplasmic binding, regulation, and signaling are found in both the helices and disordered flexible regions, supporting the essential role of the ICD conformational plasticity in orchestrating a broad range of biological processes.

Detection of differential selection pressure and functional-specific sites in subunits of vertebrate neuronal nicotinic acetylcholine receptors.

The nicotinic acetylcholine receptors (nAChR) are made of subunits evolved from a common ancestor. Despite the similarity in their sequences and structures, the properties of these subunits vary significantly. Thus, identifying the evolution features and function-related sites specific to each subunit is essential for understanding the characteristics of the subunits and the receptors assembled by them. In this study, we examined the sequence features of the nine neuronal nAChRs subunits from representative vertebrate species. Analysis revealed that all the subunits were subject to strong purifying selection in evolution, and each was under a unique pattern of selection pressures. At the same time, the functional constraints were not uniform within each subunit, with different domains in the molecule being subject to different selection pressures. We also detected potential positive selection events in the subunits or subunit clusters, and identified the sites might be associated with the function specificity of each subunit. Furthermore, positive selection at some domains might contribute to the diversity of subunit function; for example, the ß9 strand might be related to the agonist specificity of α subunit in heteromeric receptor and ß4-ß5 linker could be involved in Ca2+ permeability. Subunits α7, α4 and ß2 subunits possess a strong adaptability in vertebrates. Our results highlighted the importance of tracking functional differentiation in protein sequence underlying functional properties of nAChRs. In summary, our work may provide clues on understanding the diversity and the function specificity of the nAChR subunits, as well as the receptors co-assembled by them.Communicated by Ramaswamy H. Sarma.

New Synthetic Caffeine Analogs as Modulators of the Cholinergic System.

Alzheimer's disease is a multifactorial neurodegenerative disorder. Since cholinergic deficit is a major factor in this disease, two molecular targets for its treatment are the acetylcholinesterase (AChE) and the nicotinic acetylcholine receptors (nAChRs). Given that caffeine is a natural compound that behaves as an AChE inhibitor and as a partial agonist of nAChRs, the aim of this work was to synthetize more potent bifunctional caffeine analogs that modulate these two molecular targets. To this end, a theophylline structure was connected to a pyrrolidine structure through a methylene chain of different lengths (3 to 7 carbon atoms) to give compounds 7-11 All caffeine derivatives inhibited the AChE, of which compound 11 showed the strongest effect. Electrophysiological studies showed that all compounds behave as agonists of the muscle and the neuronal α7 nAChR with greater potency than caffeine. To explore whether the different analogs could affect the nAChR conformational state, the nAChR conformational-sensitive probe crystal violet (CrV) was used. Compounds 9 and 10 conduced the nAChR to a different conformational state comparable with a control nAChR desensitized state. Finally, molecular docking experiments showed that all derivatives interacted with both the catalytic and anionic sites of AChE and with the orthosteric binding site of the nAChR. Thus, the new synthetized compounds can inhibit the AChE and activate muscle and α7 nAChRs with greater potency than caffeine, which suggests that they could be useful leaders for the development of new therapies for the treatment of different neurologic diseases. SIGNIFICANCE STATEMENT: In this work we synthetized caffeine derivatives which can inhibit acetylcholinesterase and activate both muscle and α7 nicotinic acetylcholine receptors (nAChRs) with higher potency than caffeine. These analogs can be divided into two groups: a non-desensitizing and a desensitizing nAChR group. From the nAChR non-desensitizing group, we propose compound 11 as the most interesting analog for further studies since it inhibits acetylcholinesterase with the highest potency and activates the nAChRs in the picomolar range without inducing receptor desensitization.

The Allosteric Activation of α7 nAChR by α-Conotoxin MrIC Is Modified by Mutations at the Vestibular Site.

α-conotoxins are 13-19 amino acid toxin peptides that bind various nicotinic acetylcholine receptor (nAChR) subtypes. α-conotoxin Mr1.7c (MrIC) is a 17 amino acid peptide that targets α7 nAChR. Although MrIC has no activating effect on α7 nAChR when applied by itself, it evokes a large response when co-applied with the type II positive allosteric modulator PNU-120596, which potentiates the α7 nAChR response by recovering it from a desensitized state. A lack of standalone activity, despite activation upon co-application with a positive allosteric modulator, was previously observed for molecules that bind to an extracellular domain allosteric activation (AA) site at the vestibule of the receptor. We hypothesized that MrIC may activate α7 nAChR allosterically through this site. We ran voltage-clamp electrophysiology experiments and in silico peptide docking calculations in order to gather evidence in support of α7 nAChR activation by MrIC through the AA site. The experiments with the wild-type α7 nAChR supported an allosteric mode of action, which was confirmed by the significantly increased MrIC + PNU-120596 responses of three α7 nAChR AA site mutants that were designed in silico to improve MrIC binding. Overall, our results shed light on the allosteric activation of α7 nAChR by MrIC and suggest the involvement of the AA site.

Cholinergic Modulation of Neuroinflammation: Focus on α7 Nicotinic Receptor.

All nervous system pathologies (e.g., neurodegenerative/demyelinating diseases and brain tumours) develop neuroinflammation, a beneficial process during pathological events, aimed at removing damaged cells, toxic agents, and/or pathogens. Unfortunately, excessive inflammation frequently occurs during nervous system disorders, becoming a detrimental event capable of enhancing neurons and myelinating glial cell impairment, rather than improving their survival and activity. Consequently, targeting the neuroinflammation could be relevant for reducing brain injury and rescuing neuronal and glial cell functions. Several studies have highlighted the role of acetylcholine and its receptors in the regulation of central and peripheral inflammation. In particular, α7 nicotinic receptor has been described as one of the main regulators of the "brain cholinergic anti-inflammatory pathway". Its expression in astrocytes and microglial cells and the ability to modulate anti-inflammatory cytokines make this receptor a new interesting therapeutic target for neuroinflammation regulation. In this review, we summarize the distribution and physiological functions of the α7 nicotinic receptor in glial cells (astrocytes and microglia) and its role in the modulation of neuroinflammation. Moreover, we explore how its altered expression and function contribute to the development of different neurological pathologies and exacerbate neuroinflammatory processes.

Different Classes of Antidepressants Inhibit the Rat α7 Nicotinic Acetylcholine Receptor by Interacting within the Ion Channel: A Functional and Structural Study.

Several antidepressants inhibit nicotinic acetylcholine receptors (nAChRs) in a non-competitive and voltage-dependent fashion. Here, we asked whether antidepressants with a different structure and pharmacological profile modulate the rat α7 nAChR through a similar mechanism by interacting within the ion-channel. We applied electrophysiological (recording of the ion current elicited by choline, ICh, which activates α7 nAChRs from rat CA1 hippocampal interneurons) and in silico approaches (homology modeling of the rat α7 nAChR, molecular docking, molecular dynamics simulations, and binding free energy calculations). The antidepressants inhibited ICh with the order: norfluoxetine ~ mirtazapine ~ imipramine < bupropion ~ fluoxetine ~ venlafaxine ~ escitalopram. The constructed homology model of the rat α7 nAChR resulted in the extracellular vestibule and the channel pore is highly negatively charged, which facilitates the permeation of cations and the entrance of the protonated form of antidepressants. Molecular docking and molecular dynamics simulations were carried out within the ion-channel of the α7 nAChR, revealing that the antidepressants adopt poses along the receptor channel, with slightly different binding-free energy values. Furthermore, the inhibition of ICh and free energy values for each antidepressant-receptor complex were highly correlated. Thus, the α7 nAChR is negatively modulated by a variety of antidepressants interacting in the ion-channel.

Structure and gating mechanism of the α7 nicotinic acetylcholine receptor.

The α7 nicotinic acetylcholine receptor plays critical roles in the central nervous system and in the cholinergic inflammatory pathway. This ligand-gated ion channel assembles as a homopentamer, is exceptionally permeable to Ca2+, and desensitizes faster than any other Cys-loop receptor. The α7 receptor has served as a prototype for the Cys-loop superfamily yet has proven refractory to structural analysis. We present cryo-EM structures of the human α7 nicotinic receptor in a lipidic environment in resting, activated, and desensitized states, illuminating the principal steps in the gating cycle. The structures also reveal elements that contribute to its function, including a C-terminal latch that is permissive for channel opening, and an anionic ring in the extracellular vestibule that contributes to its high conductance and calcium permeability. Comparisons among the α7 structures provide a foundation for mapping the gating cycle and reveal divergence in gating mechanisms in the Cys-loop receptor superfamily.

Ligand-induced transition in conformations of vicinal cysteine disulfides in proteins.

Vicinal cysteine disulfides are thought to be associated with specific conformations of cysteine disulfides due to the restricted rotation of single bonds in an eight-membered cyclic disulfide loop. Conformations of vicinal cysteine disulfides are analyzed using χ1 , χ2 , χ3 , χ2 ', χ1 ' torsion angles in the crystal structures of proteins retrieved from Protein Data Bank (PDB). 85% of vicinal disulfides have (+, -)LHStaple conformation with trans configuration of the peptide bond and 9% have (-, -)RHStaple conformation with cis configured peptide bond. Conformational analysis of dipeptide Cys-Cys vicinal disulfide by density functional theory (DFT) further supported (+, -)LHStaple, (-, -)RHStaple, and (+, +)RHStaple as the preferred conformations of vicinal disulfides. Interestingly, the rare conformations of vicinal disulfides are observed in the ligand-bound forms of proteins and have higher disulfide strain energy. Conformations of vicinal disulfides in palmitoyl protein thioesterase 1, AChBP, and α7 nicotinic receptor are changed from preferred (+, -)LHStaple to rare (+, -)AntiLHHook/(+, -)AntiRHHook/(+, +)RHStaple conformation due to binding of ligands. Surprisingly, ligands are proximal to the vicinal disulfides in protein complexes that exhibited rare conformations of vicinal disulfides. The report has identified (+, -) LHStaple/(-, -) RHStaple as the hallmark conformations of vicinal disulfides and unraveled ligand-induced transition in conformations of vicinal cysteine disulfides in proteins.

Enriched environment alleviates post-stroke cognitive impairment through enhancing α7-nAChR expression in rats.

BACKGROUND: Enriched environment (EE) is a simple and effective intervention to improve cognitive function in post-stroke cognitive impairment (PSCI), partly due to the rebalancing of the cholinergic signaling pathway in the hippocampus. α7-nicotinic acetylcholine receptor (α7-nAChR) is a cholinergic receptor whose activation inhibits inflammation and promotes the recovery of neurological function in PSCI patients. However, it is still unclear whether EE can regulate α7-nAChR and activate the cholinergic anti-inflammatory pathway (CAP) in PSCI. OBJECTIVE: To investigate the effects of EE on cognitive impairment, and the role of α7-nAChR in PSCI. METHODS: A PSCI rat model was induced by middle cerebral artery occlusion and reperfusion (MCAO/R) and were reared in standard environment (SE) or EE for 28d, control group with sham surgery. Cognitive function was determined by Morris water maze test. The long-term potentiation (LTP) was assessed by Electrophysiology. Histopathological methods were used to determine infarct volume, α7-nAChR expression and the cytokines and cholinergic proteins expression. RESULTS: Compared with SE group, rats in EE group had better cognitive function, higher expression of α7-nAChR positive neurons in hippocampal CA1 region. In addition, EE attenuated unfavorable changes induced by MCAO/R in cytokines and cholinergic proteins, and also enhanced LTP promoted by nicotine and attenuated by α-BGT; but showed no significantly difference in infarct volume. CONCLUSIONS: EE markedly improves cognitive impairment and enhances neuroplasticity in PSCI rats, which may be closely related to enhancement of α7-nAChR expression.

Enriched environment alleviates post-stroke cognitive impairment through enhancing α7-nAChR expression in rats / Ambiente enriquecido alivia o comprometimento cognitivo pós-AVC em ratos por meio do aumento da expressão de α7-nAChR

ABSTRACT Background: Enriched environment (EE) is a simple and effective intervention to improve cognitive function in post-stroke cognitive impairment (PSCI), partly due to the rebalancing of the cholinergic signaling pathway in the hippocampus. α7-nicotinic acetylcholine receptor (α7-nAChR) is a cholinergic receptor whose activation inhibits inflammation and promotes the recovery of neurological function in PSCI patients. However, it is still unclear whether EE can regulate α7-nAChR and activate the cholinergic anti-inflammatory pathway (CAP) in PSCI. Objective: To investigate the effects of EE on cognitive impairment, and the role of α7-nAChR in PSCI. Methods: A PSCI rat model was induced by middle cerebral artery occlusion and reperfusion (MCAO/R) and were reared in standard environment (SE) or EE for 28d, control group with sham surgery. Cognitive function was determined by Morris water maze test. The long-term potentiation (LTP) was assessed by Electrophysiology. Histopathological methods were used to determine infarct volume, α7-nAChR expression and the cytokines and cholinergic proteins expression. Results: Compared with SE group, rats in EE group had better cognitive function, higher expression of α7-nAChR positive neurons in hippocampal CA1 region. In addition, EE attenuated unfavorable changes induced by MCAO/R in cytokines and cholinergic proteins, and also enhanced LTP promoted by nicotine and attenuated by α-BGT; but showed no significantly difference in infarct volume. Conclusions: EE markedly improves cognitive impairment and enhances neuroplasticity in PSCI rats, which may be closely related to enhancement of α7-nAChR expression.

RESUMO Introdução: O ambiente enriquecido (AE) é uma intervenção simples e eficaz para melhorar a função cognitiva no comprometimento cognitivo pós-AVC, em parte devido ao reequilíbrio da via de sinalização colinérgica no hipocampo. O receptor nicotínico α7 de acetilcolina (α7-nAChR) é um receptor colinérgico cuja ativação inibe inflamação e promove a recuperação da função neurológica em pacientes com comprometimento cognitivo pós-AVC. No entanto, ainda não está claro se o AE pode regular α7-nAChR e ativar a via anti-inflamatória colinérgica (VAC) em comprometimento cognitivo pós-AVC. Objetivo: Investigar os efeitos do AE no comprometimento cognitivo e o papel do α7-nAChR no comprometimento cognitivo pós-AVC. Métodos: Modelo de comprometimento cognitivo pós-AVC foi induzido em ratos por oclusão e reperfusão da artéria cerebral média (MCAO/R), que foram criados em ambiente padrão (AP) ou em AE por 28d; grupo controle com cirurgia simulada. A função cognitiva foi determinada pelo teste do labirinto aquático de Morris. A potenciação de longo prazo (PLP) foi avaliada por eletrofisiologia. Métodos histopatológicos foram usados para determinar o volume do infarto, a expressão de α7-nAChR e a expressão de citocinas e proteínas colinérgicas. Resultados: Em comparação com o grupo AP, os ratos do grupo AE tiveram melhor função cognitiva, com maior expressão de neurônios positivos para α7-nAChR na região CA1 do hipocampo. Além disso, o AE atenuou alterações desfavoráveis induzidas por MCAO/R em citocinas e proteínas colinérgicas, e também aumentou a PLP promovida pela nicotina e atenuada por α-BGT, mas não mostrou nenhuma diferença significativa no volume do infarto. Conclusão: O AE melhora acentuadamente o comprometimento cognitivo e aumenta a neuroplasticidade em ratos com comprometimento cognitivo pós-AVC, o que pode estar intimamente relacionado ao aumento da expressão de α7-nAChR.

In Silico Finding of Key Interaction Mediated α3ß4 and α7 Nicotinic Acetylcholine Receptor Ligand Selectivity of Quinuclidine-Triazole Chemotype.

The selective binding of six (S)-quinuclidine-triazoles and their (R)-enantiomers to nicotinic acetylcholine receptor (nAChR) subtypes α3ß4 and α7, respectively, were analyzed by in silico docking to provide the insight into the molecular basis for the observed stereospecific subtype discrimination. Homology modeling followed by molecular docking and molecular dynamics (MD) simulations revealed that unique amino acid residues in the complementary subunits of the nAChR subtypes are involved in subtype-specific selectivity profiles. In the complementary ß4-subunit of the α3ß4 nAChR binding pocket, non-conserved AspB173 through a salt bridge was found to be the key determinant for the α3ß4 selectivity of the quinuclidine-triazole chemotype, explaining the 47-327-fold affinity of the (S)-enantiomers as compared to their (R)-enantiomer counterparts. Regarding the α7 nAChR subtype, the amino acids promoting a however significantly lower preference for the (R)-enantiomers were the conserved TyrA93, TrpA149 and TrpB55 residues. The non-conserved amino acid residue in the complementary subunit of nAChR subtypes appeared to play a significant role for the nAChR subtype-selective binding, particularly at the heteropentameric subtype, whereas the conserved amino acid residues in both principal and complementary subunits are essential for ligand potency and efficacy.

Efficient screening of ligand-receptor complex formation using fluorescence labeling and size-exclusion chromatography.

Evidence of a complex formation is a crucial step in the structural studies of ligand-receptor interactions. Here we presented a simple and fast approach for qualitative screening of the complex formation between the chimeric extracellular domain of the nicotinic acetylcholine receptor (α7-ECD) and three-finger proteins. Complex formation of snake toxins α-Bgtx and WTX, as well as of recombinant analogs of human proteins Lynx1 and SLURP-1, with α7-ECD was confirmed using fluorescently labeled ligands and size-exclusion chromatography with simultaneous absorbance and fluorescence detection. WTX/α7-ECD complex formation also was confirmed by cryo-EM. The proposed approach could easily be adopted to study the interaction of other receptors with their ligands.

Differing Activity Profiles of the Stereoisomers of 2,3,5,6TMP-TQS, a Putative Silent Allosteric Modulator of α7 nAChR.

Many synthetic compounds to which we attribute specific activities are produced as racemic mixtures of stereoisomers, and it may be that all the desired activity comes from a single enantiomer. We have previously shown this to be the case with the α7 nicotinic acetylcholine receptor positive allosteric modulator (PAM) 3a,4,5,9b-Tetrahydro-4-(1-naphthalenyl)-3H-cyclopentan[c]quinoline-8-sulfonamide (TQS) and the α7 ago-PAM 4BP-TQS. Cis-trans-4-(2,3,5,6-tetramethylphenyl)-3a,4,5,9b-te-trahydro-3H-cyclopenta[c]quinoline-8-sulfonamide (2,3,5,6TMP-TQS), previously published as a "silent allosteric modulator" and an antagonist of α7 allosteric activation, shares the same scaffold with three chiral centers as the aforementioned compounds. We isolated the enantiomers of 2,3,5,6TMP-TQS and determined that the (-) isomer was a significantly better antagonist than the (+) isomer of the allosteric activation of both wild-type α7 and the nonorthosterically activatible C190A α7 mutant by the ago-PAM GAT107 (the active isomer of 4BP-TQS). In contrast, (+)2,3,5,6TMP-TQS proved to be an α7 PAM. (-)2,3,5,6TMP-TQS was shown to antagonize the allosteric activation of α7 by the structurally unrelated ago-PAM B-973B as well as the allosteric activation of the TQS-sensitive α4ß2L15'M mutant. In silico docking of 2,3,5,6TMP-TQS in the putative allosteric activation binding site suggested a specific interaction of the (-) enantiomer with α7T106, and allosteric activation of α7T106 mutants was not inhibited by (-)2,3,5,6TMP-TQS, confirming the importance of this interaction and supporting the model of the allosteric binding site. Comparisons and contrasts between 2,3,5,6TMP-TQS isomers and active and inactive enantiomers of other TQS-related compounds identify the orientation of the cyclopentenyl ring to the plane of the core quinoline to be a crucial determinate of PAM activity. SIGNIFICANCE STATEMENT: Many synthetic ligands are in use as racemic preparations. We show that one enantiomer of the TQS analog Cis-trans-4-(2,3,5,6-tetramethylphenyl)-3a,4,5,9b-te-trahydro-3H-cyclopenta[c]quinoline-8-sulfonamide, originally reported to lack activity when used as a racemic preparation, is an α7 nicotinic acetylcholine receptor positive allosteric modulator (PAM). The other enantiomer is not a PAM, but it is an effective allosteric antagonist. In silico studies and structural comparisons identify essential elements of both the allosteric ligands and receptor binding sites important for these allosteric activities.

Structure-Based Discovery of Dual-Target Hits for Acetylcholinesterase and the α7 Nicotinic Acetylcholine Receptors: In Silico Studies and In Vitro Confirmation.

Despite extensive efforts in the development of drugs for complex neurodegenerative diseases, treatment often remains challenging or ineffective, and hence new treatment strategies are necessary. One approach is the design of multi-target drugs, which can potentially address the complex nature of disorders such as Alzheimer's disease. We report a method for high throughput virtual screening aimed at identifying new dual target hit molecules. One of the identified hits, N,N-dimethyl-1-(4-(3-methyl-[1,2,4]triazolo[4,3-a]pyrimidin-6-yl)phenyl)ethan-1-amine (Ý;mir-2), has dual-activity as an acetylcholinesterase (AChE) inhibitor and as an α7 nicotinic acetylcholine receptor (α7 nAChR) agonist. Using computational chemistry methods, parallel and independent screening of a virtual compound library consisting of 3,848,234 drug-like and commercially available molecules from the ZINC15 database, resulted in an intersecting set of 57 compounds, that potentially possess activity at both of the two protein targets. Based on ligand efficiency as well as scaffold and molecular diversity, 16 of these compounds were purchased for in vitro validation by Ellman's method and two-electrode voltage-clamp electrophysiology. Ý;mir-2 was shown to exhibit the desired activity profile (AChE IC50 = 2.58 ± 0.96 µM; α7 nAChR activation = 7.0 ± 0.9% at 200 µM) making it the first reported compound with this particular profile and providing further evidence of the feasibility of in silico methods for the identification of novel multi-target hit molecules.