Mutational analysis of G-protein coupled receptor – FFA2

FFA2 (GPR43) is a receptor for short-chain fatty acids (SCFAs), acetate, and propionate. FFA2 is predominantly expressed in islets, a subset of immune cells, adipocytes, and the gastrointestinal tract which suggest a possible role in inflammatory and metabolic conditions. We have previously described the identification and characterization of novel phenylacetamides as allosteric agonists of FFA2. In the current study, we have investigated the molecular determinants contributing to receptor activation with the endogenous and synthetic ligands as well as allosteric interactions between these two sites. The mutational analysis revealed previously unidentified sites that may allosterically regulate orthosteric ligand’s function as well as residues potentially important for the interactions between orthosteric and allosteric binding sites.     

Allosteric small molecules unveil a role of an extracellular E2/transmembrane helix 7 junction for G protein-coupled receptor activation

G protein-coupled receptors represent the largest superfamily of cell membrane-spanning receptors. We used allosteric small molecules as a novel approach to better understand conformational changes underlying the inactive-to-active switch in native receptors. Allosteric molecules bind outside the orthosteric area for the endogenous receptor activator. The human muscarinic M(2) acetylcholine receptor is prototypal for the study of allosteric interactions. We measured receptor-mediated G protein activation, applied a series of structurally diverse muscarinic allosteric agents, and analyzed their cooperative effects with orthosteric receptor agonists. A strong negative cooperativity of receptor binding was observed with acetylcholine and other full agonists, whereas a pronounced negative cooperativity of receptor activation was observed with the partial agonist pilocarpine. Applying a newly synthesized allosteric tool, point mutated receptors, radioligand binding, and a three-dimensional receptor model, we found that the deviating allosteric/orthosteric interactions are mediated through the core region of the allosteric site. A key epitope is M(2)Trp(422) in position 7.35 that is located at the extracellular top of transmembrane helix 7 and that contacts, in the inactive receptor, the extracellular loop E2. Trp 7.35 is critically involved in the divergent allosteric/orthosteric cooperativities with acetylcholine and pilocarpine, respectively. In the absence of allosteric agents, Trp 7.35 is essential for receptor binding of the full agonist and for receptor activation by the partial agonist. This study provides first evidence for a role of an allosteric E2/transmembrane helix 7 contact region for muscarinic receptor activation by orthosteric agonists.     

Negative allosteric modulators of cannabinoid receptor 2: protein modeling,binding site identification and molecular dynamics simulations in the presence of an orthosteric agonist

Abstract

Selective activation of the cannabinoid receptor subtype 2 (CB2) shows promise for treating pain, inflammation, multiple sclerosis, cancer, ischemic/reperfusion injury and osteoporosis. Target selectivity and off-target side effects are two major limiting factors for orthosteric ligands, and therefore, the search for allosteric modulators (AMs) is a widely used drug discovery approach. To date, only a limited number of negative CB2 AMs have been identified, possessing only micromolar activity at best, and the CB2 receptor’s allosteric site(s) are not well characterized. Herein, we used computational approaches including receptor modeling, site mapping, docking, molecular dynamics (MD) simulations and binding free energy calculations to predict, characterize and validate allosteric sites within the complex of the CB2 receptor with bound orthosteric agonist CP55,940. After docking of known negative CB2 allosteric modulators (NAMs), dihydro-gambogic acid (DHGA) and trans-β-caryophyllene (TBC) (note that TBC also shows agonist activity), at the predicted allosteric sites, the best total complex with CB2, CP55,940 and NAM was embedded into a hydrated lipid bilayer and subjected to a 200 ns MD simulation. The presence of an AM affected the CB2–CP55,940 complex, altering the relative positioning of the toggle switch residues and promoting a strong π–π interaction between Phe117^3.36 and Trp258^6.48. Binding of either TBC or DHGA to a putative allosteric pocket directly adjacent to the orthosteric ligand reduced the binding free energy of CP55,940, which is consistent with the expected effect of a negative AM. The identified allosteric sites present immense scope for the discovery of novel classes of CB2 AMs.     

Identification of an allosteric binding site for Zn2+ on the beta2 adrenergic receptor

The activity of G protein-coupled receptors (GPCRs) can be modulated by a diverse spectrum of drugs ranging from full agonists to partial agonists, antagonists, and inverse agonists. The vast majority of these ligands compete with native ligands for binding to orthosteric binding sites. Allosteric ligands have also been described for a number of GPCRs. However, little is known about the mechanism by which these ligands modulate the affinity of receptors for orthosteric ligands. We have previously reported that Zn(II) acts as a positive allosteric modulator of the beta(2)-adrenergic receptor (beta(2)AR). To identify the Zn(2+) binding site responsible for the enhancement of agonist affinity in the beta(2)AR, we mutated histidines located in hydrophilic sequences bridging the seven transmembrane domains. Mutation of His-269 abolished the effect of Zn(2+) on agonist affinity. Mutations of other histidines had no effect on agonist affinity. Further mutagenesis of residues adjacent to His-269 demonstrated that Cys-265 and Glu-225 are also required to achieve the full allosteric effect of Zn(2+) on agonist binding. Our results suggest that bridging of the cytoplasmic extensions of TM5 and TM6 by Zn(2+) facilitates agonist binding. These results are in agreement with recent biophysical studies demonstrating that agonist binding leads to movement of TM6 relative to TM5.     

Interdependent allosteric free fatty acid receptor 2 modulators synergistically induce functional selective activation and desensitization in neutrophils

The non-activating allosteric modulator AZ1729, specific for free fatty acid receptor 2 (FFAR2), transfers the orthosteric FFAR2 agonists propionate and the P2Y₂R specific agonist ATP into activating ligands that trigger an assembly of the neutrophil superoxide generating NADPH-oxidase. The homologous priming effect on the propionate response and the heterologous receptor cross-talk sensitized ATP response mediated by AZ1729 are functional characteristics shared with Cmp58, another non-activating allosteric FFAR2 modulator. In addition, AZ1729 also turned Cmp58 into a potent activator of the superoxide generating neutrophil NADPH-oxidase, and in agreement with the allosteric modulation concept, the effect was reciprocal in that Cmp58 turned AZ1729 into a potent activating allosteric agonist. The activation signals down-stream of FFAR2 when stimulated by the two interdependent allosteric modulators were biased in that, unlike for orthosteric agonists, the two complementary modulators together triggered an activation of the NADPH-oxidase, but not any transient rise in the cytosolic concentration of free calcium ions (Ca²⁺). Furthermore, following AZ1729/Cmp58 activation, the signaling by the desensitized FFAR2s was functionally selective in that the orthosteric agonist propionate could still induce a transient rise in intracellular Ca²⁺. The novel neutrophil activation and receptor down-stream signaling pattern mediated by the two cross-sensitizing allosteric FFAR2 modulators represent a new regulatory mechanism that controls receptor signaling.     

Multiple allosteric sites on muscarinic receptors

Proteins and small molecules are capable of regulating the agonist binding and function of G-protein coupled receptors by multiple allosteric mechanisms. In the case of muscarinic receptors, there is the well-characterised allosteric site that binds, for example, gallamine and brucine. The protein kinase inhibitor, KT5720, has now been shown to bind to a second allosteric site and to regulate agonist and antagonist binding. The binding of brucine and gallamine does not affect KT5720 binding nor its effects on the dissociation of [3H]-N-methylscopolamine from M1 receptors. Therefore it is possible to have a muscarinic receptor with three small ligands bound simultaneously. A model of the M1 receptor, based on the recently determined structure of rhodopsin, has the residues that have been shown to be important for gallamine binding clustered within and to one side of a cleft in the extracellular face of the receptor. This cleft may represent the access route of acetylcholine to its binding site.     

A sphingosine 1-phosphate receptor 2 selective allosteric agonist

Molecular probe tool compounds for the Sphingosine 1-phosphate receptor 2 (S1PR2) are important for investigating the multiple biological processes in which the S1PR2 receptor has been implicated. Amongst these are NF-κB-mediated tumor cell survival and fibroblast chemotaxis to fibronectin. Here we report our efforts to identify selective chemical probes for S1PR2 and their characterization. We employed high throughput screening to identify two compounds which activate the S1PR2 receptor. SAR optimization led to compounds with high nanomolar potency. These compounds, XAX-162 and CYM-5520, are highly selective and do not activate other S1P receptors. Binding of CYM-5520 is not competitive with the antagonist JTE-013. Mutation of receptor residues responsible for binding to the zwitterionic headgroup of sphingosine 1-phosphate (S1P) abolishes S1P activation of the receptor, but not activation by CYM-5520. Competitive binding experiments with radiolabeled S1P demonstrate that CYM-5520 is an allosteric agonist and does not displace the native ligand. Computational modeling suggests that CYM-5520 binds lower in the orthosteric binding pocket, and that co-binding with S1P is energetically well tolerated. In summary, we have identified an allosteric S1PR2 selective agonist compound.     

Chemotype-selective Modes of Action of κ-Opioid Receptor Agonists

The crystal structures of opioid receptors provide a novel platform for inquiry into opioid receptor function. The molecular determinants for activation of the κ-opioid receptor (KOR) were studied using a combination of agonist docking, functional assays, and site-directed mutagenesis. Eighteen positions in the putative agonist binding site of KOR were selected and evaluated for their effects on receptor binding and activation by ligands representing four distinct chemotypes: the peptide dynorphin A(1–17), the arylacetamide U-69593, and the non-charged ligands salvinorin A and the octahydroisoquinolinone carboxamide 1xx. Minimally biased docking of the tested ligands into the antagonist-bound KOR structure generated distinct binding modes, which were then evaluated biochemically and pharmacologically. Our analysis identified two types of mutations: those that affect receptor function primarily via ligand binding and those that primarily affect function. The shared and differential mechanisms of agonist binding and activation in KOR are further discussed. Usually, mutations affecting function more than binding were located at the periphery of the binding site and did not interact strongly with the various ligands. Analysis of the crystal structure along with the present results provide fundamental insights into the activation mechanism of the KOR and suggest that “functional” residues, along with water molecules detected in the crystal structure, may be directly involved in transduction of the agonist binding event into structural changes at the conserved rotamer switches, thus leading to receptor activation.     

Allosteric interactions between GB1 and GB2 subunits are required for optimal GABA(B) receptor function

Recent studies on G-protein-coupled receptors revealed that they can dimerize. However, the role of each subunit in the activation process remains unclear. The gamma-amino-n-butyric acid type B (GABA(B)) receptor is comprised of two subunits: GB1 and GB2. Both consist of an extracellular domain (ECD) and a heptahelical domain composed of seven transmembrane alpha-helices, loops and the C-terminus (HD). Whereas GB1 ECD plays a critical role in ligand binding, GB2 is required not only to target GB1 subunit to the cell surface but also for receptor activation. Here, by analysing chimeric GB subunits, we show that only GB2 HD contains the determinants required for G-protein signalling. However, the HD of GB1 improves coupling efficacy. Conversely, although GB1 ECD is sufficient to bind GABA(B) ligands, the ECD of GB2 increases the agonist affinity on GB1, and is necessary for agonist activation of the receptor. These data indicate that multiple allosteric interactions between the two subunits are required for wild-type functioning of the GABA(B) receptor and highlight further the importance of the dimerization process in GPCR activation.     

A Key Agonist-induced Conformational Change in the Cannabinoid Receptor CB1 Is Blocked by the Allosteric Ligand Org 27569

Allosteric ligands that modulate how G protein-coupled receptors respond to traditional orthosteric drugs are an exciting and rapidly expanding field of pharmacology. An allosteric ligand for the cannabinoid receptor CB1, Org 27569, exhibits an intriguing effect; it increases agonist binding, yet blocks agonist-induced CB1 signaling. Here we explored the mechanism behind this behavior, using a site-directed fluorescence labeling approach. Our results show that Org 27569 blocks conformational changes in CB1 that accompany G protein binding and/or activation, and thus inhibit formation of a fully active CB1 structure. The underlying mechanism behind this behavior is that simultaneous binding of Org 27569 produces a unique agonist-bound conformation, one that may resemble an intermediate structure formed on the pathway to full receptor activation.     

The Activity of GAT107, an Allosteric Activator and Positive Modulator of α7 Nicotinic Acetylcholine Receptors (nAChR), Is Regulated by Aromatic Amino Acids That Span the Subunit Interface

GAT107, the (+)-enantiomer of racemic 4-(4-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide, is a strong positive allosteric modulator (PAM) of α7 nicotinic acetylcholine receptor (nAChR) activation by orthosteric agonists with intrinsic allosteric agonist activities. The direct activation produced by GAT107 in electrophysiological studies is observed only as long as GAT107 is freely diffusible in solution, although the potentiating activity primed by GAT107 can persist for over 30 min after drug washout. Direct activation is sensitive to α7 nAChR antagonist methyllycaconitine, although the primed potentiation is not. The data are consistent with GAT107 activity arising from two different sites. We show that the coupling between PAMs and the binding of orthosteric ligands requires tryptophan 55 (Trp-55), which is located at the subunit interface on the complementary surface of the orthosteric binding site. Mutations of Trp-55 increase the direct activation produced by GAT107 and reduce or prevent the synergy between allosteric and orthosteric binding sites, so that these mutants can also be directly activated by other PAMs such as PNU-120596 and TQS, which do not activate wild-type α7 in the absence of orthosteric agonists. We identify Tyr-93 as an essential element for orthosteric activation, because Y93C mutants are insensitive to orthosteric agonists but respond to GAT107. Our data show that both orthosteric and allosteric activation of α7 nAChR require cooperative activity at the interface between the subunits in the extracellular domain. These cooperative effects rely on key aromatic residues, and although mutations of Trp-55 reduce the restraints placed on the requirement for orthosteric agonists, Tyr-93 can conduct both orthosteric activation and desensitization among the subunits.     

Communication over the Network of Binary Switches Regulates the Activation of A2A Adenosine Receptor

Dynamics and functions of G-protein coupled receptors (GPCRs) are accurately regulated by the type of ligands that bind to the orthosteric or allosteric binding sites. To glean the structural and dynamical origin of ligand-dependent modulation of GPCR activity, we performed total ~ 5 μsec molecular dynamics simulations of A_2A adenosine receptor (A_2AAR) in its apo, antagonist-bound, and agonist-bound forms in an explicit water and membrane environment, and examined the corresponding dynamics and correlation between the 10 key structural motifs that serve as the allosteric hotspots in intramolecular signaling network. We dubbed these 10 structural motifs “binary switches” as they display molecular interactions that switch between two distinct states. By projecting the receptor dynamics on these binary switches that yield 2¹⁰ microstates, we show that (i) the receptors in apo, antagonist-bound, and agonist-bound states explore vastly different conformational space; (ii) among the three receptor states the apo state explores the broadest range of microstates; (iii) in the presence of the agonist, the active conformation is maintained through coherent couplings among the binary switches; and (iv) to be most specific, our analysis shows that W246, located deep inside the binding cleft, can serve as both an agonist sensor and actuator of ensuing intramolecular signaling for the receptor activation. Finally, our analysis of multiple trajectories generated by inserting an agonist to the apo state underscores that the transition of the receptor from inactive to active form requires the disruption of ionic-lock in the DRY motif.     

Contrasting Properties of α7-Selective Orthosteric and Allosteric Agonists Examined on Native Nicotinic Acetylcholine Receptors

Subtype-selective ligands are important tools for the pharmacological characterisation of neurotransmitter receptors. This is particularly the case for nicotinic acetylcholine receptors (nAChRs), given the heterogeneity of their subunit composition. In addition to agonists and antagonists that interact with the extracellular orthosteric nAChR binding site, a series of nAChR allosteric modulators have been identified that interact with a distinct transmembrane site. Here we report studies conducted with three pharmacologically distinct nicotinic ligands, an orthosteric agonist (compound B), a positive allosteric modulator (TQS) and an allosteric agonist (4BP-TQS). The primary focus of the work described in this study is to examine the suitability of these compounds for the characterisation of native neuronal receptors (both rat and human). However, initial experiments were conducted on recombinant nAChRs demonstrating the selectivity of these three compounds for α7 nAChRs. In patch-clamp recordings on rat primary hippocampal neurons we found that all these compounds displayed pharmacological properties that mimicked closely those observed on recombinant α7 nAChRs. However, it was not possible to detect functional responses with compound B, an orthosteric agonist, using a fluorescent intracellular calcium assay on either rat hippocampal neurons or with human induced pluripotent stem cell-derived neurons (iCell neurons). This is, presumably, due to the rapid desensitisation of α7 nAChR that is induced by orthosteric agonists. In contrast, clear agonist-evoked responses were observed in fluorescence-based assays with the non-desensitising allosteric agonist 4BP-TQS and also when compound B was co-applied with the non-desensitising positive allosteric modulator TQS. In summary, we have demonstrated the suitability of subtype-selective orthosteric and allosteric ligands for the pharmacological identification and characterisation of native nAChRs and the usefulness of ligands that minimise receptor desensitisation for the characterisation of α7 nAChRs in fluorescence-based assays.     

Carboxypeptidase M Is a Positive Allosteric Modulator of the Kinin B1 Receptor

Ligand binding to extracellular domains of G protein-coupled receptors can result in novel and nuanced allosteric effects on receptor signaling. We previously showed that the protein-protein interaction of carboxypeptidase M (CPM) and kinin B1 receptor (B1R) enhances B1R signaling in two ways; 1) kinin binding to CPM causes a conformational activation of the B1R, and 2) CPM-generated des-Arg-kinin agonist is efficiently delivered to the B1R. Here, we show CPM is also a positive allosteric modulator of B1R signaling to its agonist, des-Arg¹⁰-kallidin (DAKD). In HEK cells stably transfected with B1R, co-expression of CPM enhanced DAKD-stimulated increases in intracellular Ca²⁺ or phosphoinositide turnover by a leftward shift of the dose-response curve without changing the maximum. CPM increased B1R affinity for DAKD by ∼5-fold but had no effect on basal B1R-dependent phosphoinositide turnover. Soluble, recombinant CPM bound to HEK cells expressing B1Rs without stimulating receptor signaling. CPM positive allosteric action was independent of enzyme activity but depended on interaction of its C-terminal domain with the B1R extracellular loop 2. Disruption of the CPM/B1R interaction or knockdown of CPM in cytokine-treated primary human endothelial cells inhibited the allosteric enhancement of CPM on B1R DAKD binding or ERK1/2 activation. CPM also enhanced the DAKD-induced B1R conformational change as detected by increased intramolecular fluorescence or bioluminescence resonance energy transfer. Thus, CPM binding to extracellular loop 2 of the B1R results in positive allosteric modulation of B1R signaling, and disruption of this interaction could provide a novel therapeutic approach to reduce pathological B1R signaling.     

Positive versus negative modulation of different endogenous chemokines for CC-chemokine receptor 1 by small molecule agonists through allosteric versus orthosteric binding

7 transmembrane-spanning (7TM) chemokine receptors having multiple endogenous ligands offer special opportunities to understand the molecular basis for allosteric mechanisms. Thus, CC-chemokine receptor 1 (CCR1) binds CC-chemokine 3 and 5 (CCL3 and CCL5) with K(d) values of 7.3 and 0.16 nm, respectively, as determined in homologous competition binding assays. However, CCL5 appears to have a >10,000-fold lower affinity in competition against (125)I-CCL3. Mutational mapping revealed that CCL3 and CCL5 both are strongly affected by systematic truncations of the N-terminal extension, whereas only CCL5 and not CCL3 activation is affected by substitutions in the main ligand binding pocket including the conserved GluVII:06 anchor point. A series of metal ion chelator complexes were found to act as full agonists on CCR1 and to be critically affected by the same substitutions in the main ligand binding pocket as CCL5 but not by mutations in the extracellular domain. In agreement with the overlapping binding sites, the small non-peptide agonists displaced radiolabeled CCL5 with high affinity. Interestingly, the same compounds acted as allosteric enhancers of the binding of CCL3, with which they did not overlap in binding site, leading to an increased B(max) and affinity of this chemokine mainly due to an increased association rate. It is concluded that a small molecule agonist through binding deep in the main ligand binding pocket can act as an allosteric enhancer for one endogenous chemokine and at the same time as a competitive blocker of the binding of another endogenous chemokine.     

Structural Determinants of Allosteric Agonism and Modulation at the M4 Muscarinic Acetylcholine Receptor: IDENTIFICATION OF LIGAND-SPECIFIC AND GLOBAL ACTIVATION MECHANISMS*

The recently identified small molecule, 3-amino-5-chloro-6-methoxy-4-methylthieno[2,3-b]pyridine-2-carboxylic acid cyclopropylamide (LY2033298), is the first selective allosteric modulator of the muscarinic acetylcholine receptors (mAChRs) that mediates both receptor activation and positive modulation of the endogenous agonist, acetylcholine (ACh), via the same allosteric site on the M₄ mAChR. We thus utilized this novel chemical tool, as well as ACh, the bitopic (orthosteric/allosteric) agonist, McN-A-343, and the clinically efficacious M₁/M₄ mAChR-preferring agonist, xanomeline, in conjunction with site-directed mutagenesis of four different regions of the M₄ mAChR (extracellular loops 1, 2, and 3, and transmembrane domain 7), to identify regions that govern ligand-specific modes of binding, signaling, and allosteric modulation. In the first extracellular loop (E1), we identified Ile⁹³ and Lys⁹⁵ as key residues that specifically govern the signaling efficacy of LY2033298 and its binding cooperativity with ACh, whereas Phe¹⁸⁶ in the E2 loop was identified as a key contributor to the binding affinity of the modulator for the allosteric site, and Asp⁴³² in the E3 loop appears to be involved in the functional (activation) cooperativity between the modulator and the endogenous agonist. In contrast, the highly conserved transmembrane domain 7 residues, Tyr⁴³⁹ and Tyr⁴⁴³, were identified as contributing to a key activation switch utilized by all classes of agonists. These results provide new insights into the existence of multiple activation switches in G protein-coupled receptors (GPCRs), some of which can be selectively exploited by allosteric agonists, whereas others represent global activation mechanisms for all classes of ligand.     

A novel mechanism of G protein-coupled receptor functional selectivity. Muscarinic partial agonist McN-A-343 as a bitopic orthosteric/allosteric ligand

Many G protein-coupled receptors (GPCRs) possess allosteric binding sites distinct from the orthosteric site utilized by their cognate ligands, but most GPCR allosteric modulators reported to date lack signaling efficacy in their own right. McN-A-343 (4-(N-(3-chlorophenyl)carbamoyloxy)-2-butynyltrimethylammonium chloride) is a functionally selective muscarinic acetylcholine receptor (mAChR) partial agonist that can also interact allosterically at the M(2) mAChR. We hypothesized that this molecule simultaneously utilizes both an allosteric and the orthosteric site on the M(2) mAChR to mediate these effects. By synthesizing progressively truncated McN-A-343 derivatives, we identified two, which minimally contain 3-chlorophenylcarbamate, as pure allosteric modulators. These compounds were positive modulators of the orthosteric antagonist N-[(3)H]methylscopolamine, but in functional assays of M(2) mAChR-mediated ERK1/2 phosphorylation and guanosine 5'-3-O-([(35)S]thio)triphosphate binding, they were negative modulators of agonist efficacy. This negative allosteric effect was diminished upon mutation of Y177A in the second extracellular (E2) loop of the M(2) mAChR that is known to reduce prototypical allosteric modulator potency. Our results are consistent with McN-A-343 being a bitopic orthosteric/allosteric ligand with the allosteric moiety engendering partial agonism and functional selectivity. This finding suggests a novel and largely unappreciated mechanism of "directed efficacy" whereby functional selectivity may be engendered in a GPCR by utilizing an allosteric ligand to direct the signaling of an orthosteric ligand encoded within the same molecule.     

Further exploration of M1 allosteric agonists: Subtle structural changes abolish M1 allosteric agonism and result in pan-mAChR orthosteric antagonism

This letter describes the further exploration of two series of M₁ allosteric agonists, TBPB and VU0357017, previously reported from our lab. Within the TPBP scaffold, either electronic or steric perturbations to the central piperidine ring led to a loss of selective M₁ allosteric agonism and afforded pan-mAChR antagonism, which was demonstrated to be mediated via the orthosteric site. Additional SAR around a related M₁ allosteric agonist family (VU0357017) identified similar, subtle ‘molecular switches’ that modulated modes of pharmacology from allosteric agonism to pan-mAChR orthosteric antagonism. Therefore, all of these ligands are best classified as bi-topic ligands that possess high affinity binding at an allosteric site to engender selective M₁ activation, but all bind, at higher concentrations, to the orthosteric ACh site, leading to non-selective orthosteric site binding and mAChR antagonism.     

Synthesis and evaluation of a conditionally-silent agonist for the α7 nicotinic acetylcholine receptor

We introduce the term ‘silent agonists’ to describe ligands that can place the α7 nicotinic acetylcholine receptor (nAChR) into a desensitized state with little or no apparent activation of the ion channel, forming a complex that can subsequently generate currents when treated with an allosteric modulator. KC-1 (5′-phenylanabaseine) was synthesized and identified as a new silent agonist for the α7 nAChR; it binds to the receptor but does not activate α7 nAChR channel opening when applied alone, and its agonism is revealed by co-application with the type II positive allosteric modulator PNU-120596 in the Xenopus oocyte system. The concise synthesis was accomplished in three steps with the C–C bonds formed via Pd-catalyzed mono-arylation and organolithium coupling with N-Boc piperidinone. Comparative structural analyses indicate that a positive charge, an H-bond acceptor, and an aryl ring in a proper arrangement are needed to constitute one class of silent agonist for the α7 nAChR. Because silent agonists may act on signaling pathways not involving ion channel opening, this class of α7 nAChR ligands may constitute a new alternative for the development of α7 nAChR therapeutics.