Radioligand Binding Data Research Articles

Screening 800 putative endocrine disrupting chemicals in a representative mammal, bird, and fish using a neurochemical cell-free testing platform

There is global demand for novel ecotoxicity testing tools that are based on alternative to animal models, have high throughput potential, and may be applicable to a wide diversity of taxa. Here we scaled up a microplate-based cell-free neurochemical testing platform to screen 800 putative endocrine disrupting chemicals from the U.S. Environmental Protection Agency's ToxCast e1k library against the glutamate (NMDA), muscarinic acetylcholine (mACh), and dopamine (D2) receptors. Each assay was tested in cellular membranes isolated from brain tissues from a representative bird (zebra finch = Taeniopygia castanotis), mammal (mink = Neogale vison), and fish (rainbow trout = Oncorhynchus mykiss). The primary objective of this short communication was to make the results database accessible, while also summarising key attributes of assay performance and presenting some initial observations. In total, 7200 species-chemical-assay combinations were tested, of which 453 combinations were classified as a hit (radioligand binding changed by at least 3 standard deviations). There were some differences across species, and most hits were found for the D2 and NMDA receptors. The most active chemical was C.I. Solvent Yellow 14 followed by Diphenhydramine hydrochloride, Gentian Violet, SR271425, and Zamifenacin. Nine chemicals were tested across multiple plates with a mean relative standard deviation of the specific radioligand binding data being 24.6%. The results demonstrate that cell-free assays may serve as screening tools for large chemical libraries especially for ecological species not easily studied using traditional methods.

Shedding Light on the D1 -Like Receptors: A Fluorescence-Based Toolbox for Visualization of the D1 and D5 Receptors.

Dopamine D1 -like receptors are the most abundant type of dopamine receptors in the central nervous system and, even after decades of discovery, still highly interesting for the study of neurological diseases. We herein describe the synthesis of a new set of fluorescent ligands, structurally derived from D1 R antagonist SCH-23390 and labeled with two different fluorescent dyes, as tool compounds for the visualization of D1 -like receptors. Pharmacological characterization in radioligand binding studies identified UR-NR435 (25) as a high-affinity ligand for D1 -like receptors (pKi (D1 R)=8.34, pKi (D5 R)=7.62) with excellent selectivity towards D2 -like receptors. Compound 25 proved to be a neutral antagonist at the D1 R and D5 R in a Gs heterotrimer dissociation assay, an important feature to avoid receptor internalization and degradation when working with whole cells. The neutral antagonist 25 displayed rapid association and complete dissociation to the D1 R in kinetic binding studies using confocal microscopy verifying its applicability for fluorescence microscopy. Moreover, molecular brightness studies determined a single-digit nanomolar binding affinity of the ligand, which was in good agreement with radioligand binding data. For this reason, this fluorescent ligand is a useful tool for a sophisticated characterization of native D1 receptors in a variety of experimental setups.

Fluorescent Tools for the Imaging of Dopamine D2 -Like Receptors.

The family of dopamine D2 -like receptors represents an interesting target for a variety of neurological diseases, e. g. Parkinson's disease (PD), addiction, or schizophrenia. In this study we describe the synthesis of a new set of fluorescent ligands as tools for visualization of dopamine D2 -like receptors. Pharmacological characterization in radioligand binding studies identified UR-MN212 (20) as a high-affinity ligand for D2 -like receptors (pKi (D2long R)=8.24, pKi (D3 R)=8.58, pKi (D4 R)=7.78) with decent selectivity towards D1 -like receptors. Compound 20 is a neutral antagonist in a Go1 activation assay at the D2long R, D3 R, and D4 R, which is an important feature for studies using whole cells. The neutral antagonist 20, equipped with a 5-TAMRA dye, displayed rapid association to the D2long R in binding studies using confocal microscopy demonstrating its suitability for fluorescence microscopy. Furthermore, in molecular brightness studies, the ligand's binding affinity could be determined in a single-digit nanomolar range that was in good agreement with radioligand binding data. Therefore, the fluorescent compound can be used for quantitative characterization of native D2 -like receptors in a broad variety of experimental setups.

The molecular basis of drug selectivity for α5 subunit-containing GABAA receptors

α5 subunit-containing γ-aminobutyric acid type A (GABAA) receptors represent a promising drug target for neurological and neuropsychiatric disorders. Altered expression and function contributes to neurodevelopmental disorders such as Dup15q and Angelman syndromes, developmental epilepsy and autism. Effective drug action without side effects is dependent on both α5-subtype selectivity and the strength of the positive or negative allosteric modulation (PAM or NAM). Here we solve structures of drugs bound to the α5 subunit. These define the molecular basis of binding and α5 selectivity of the β-carboline, methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM), type II benzodiazepine NAMs, and a series of isoxazole NAMs and PAMs. For the isoxazole series, each molecule appears as an ‘upper’ and ‘lower’ moiety in the pocket. Structural data and radioligand binding data reveal a positional displacement of the upper moiety containing the isoxazole between the NAMs and PAMs. Using a hybrid molecule we directly measure the functional contribution of the upper moiety to NAM versus PAM activity. Overall, these structures provide a framework by which to understand distinct modulator binding modes and their basis of α5-subtype selectivity, appreciate structure–activity relationships, and empower future structure-based drug design campaigns.

Quipazine: Classical hallucinogen? Novel psychedelic?

Quipazine, first identified in the 1960s, has been the topic of >1000 published papers. On the basis of available 5-HT2 serotonin receptor radioligand binding data and various preclinical studies, it might be thought that quipazine bears the hallmarks of a classical hallucinogen or psychedelic agent – agents currently being examined for their potential use in treating certain neuropsychiatric disorders. Nevertheless, by definition, such agents require the availability of human data, which, in the case of quipazine, are lacking. Because quipazine is also a 5-HT3 receptor agonist, future human studies with this agent might prove problematic because 5-HT3 agonists are known to produce emesis. Nevertheless, continued investigation of novel quipazine analogs with modified pharmacological profiles might prove worthwhile.

Rationalizing the binding and α subtype selectivity of synthesized imidazodiazepines and benzodiazepines at GABAA receptors by using molecular docking studies

The pharmacological actions exerted by benzodiazepines are dependent on the discrete α protein subunits of the γ-aminobutyric acid type A receptor (GABAA R). Recent developments via a cryo-EM structure of the α1β3γ2L GABAA R ion channel provide crucial insights into ligand efficacy and binding affinity at this subtype. We investigated the molecular interactions of diazepam and alprazolam bound GABAA R structures (6HUP and 6HUO) to determine key binding interaction domains. A halogen bond between the chlorine atoms of diazepam and alprazolam with the group on the backbone of the α1 histidine amino acid 102 is important to the positive allosteric modulatory actions of diazepam and alprazolam in the α1β3γ2L GABAA R ion channel. In order to gain insight into α subtype selectivity we designed and synthesized close structural analogs of diazepam and alprazolam. These compounds were then docked into the recently publish cryo-EM structures of GABAA Rs (6HUP and 6HUO). This modeling along with radio-ligand binding data resulted in the conclusion that the non-classical bioisosteric replacement of the chlorine atom at C7 with an ethinyl group (compound 5) resulted in an 11-fold gain in α5 binding selectivity over the α1 subtype. Moreover, the potency of compound 5 resulted in a ligand with less sedation than diazepam, while still maintaining the same anxiolytic potency. These modeling data extend our understanding of the structural requirements for α-subtype-selective compounds that can be utilized to achieve improved medical treatments. It is clear that the ethinyl group in place of a halogen atom decreases the affinity and efficacy of benzodiazepines and imidazodiazepines at α1 subtypes, which results in less sedation and ataxia.

Enhancement of Muscimol Binding and Gating by Allosteric Modulators of the GABAAReceptor: Relating Occupancy to State Functions

Muscimol is a psychoactive isoxazole derived from the mushroom <i>Amanita muscaria</i> and a potent orthosteric agonist of the GABA_A receptor. The binding of [³H]muscimol has been used to evaluate the distribution of GABA_A receptors in the brain, and studies of modulation of [³H]muscimol binding by allosteric GABAergic modulators such as barbiturates and steroid anesthetics have provided insight into the modes of action of these drugs on the GABA_A receptor. It has, however, not been feasible to directly apply interaction parameters derived from functional studies to describe the binding of muscimol to the receptor. Here, we employed the Monod-Wyman-Changeux concerted transition model to analyze muscimol binding isotherms. We show that the binding isotherms from recombinant <i>α</i>1<i>β</i>3 GABA_A receptors can be qualitatively predicted using electrophysiological data pertaining to properties of receptor activation and desensitization in the presence of muscimol. The model predicts enhancement of [³H]muscimol binding in the presence of the steroids allopregnanolone and pregnenolone sulfate, although the steroids interact with distinct sites and either enhance (allopregnanolone) or reduce (pregnenolone sulfate) receptor function. We infer that the concerted transition model can be used to link radioligand binding and electrophysiological data. <h3>Significance Statement</h3> The study employs a three-state resting-active-desensitized model to link radioligand binding and electrophysiological data. We show that the binding isotherms can be qualitatively predicted using parameters estimated in electrophysiological experiments and that the model accurately predicts the enhancement of [³H]muscimol binding in the presence of the potentiating steroid allopregnanolone and the inhibitory steroid pregnenolone sulfate.

NanoBRET binding assay for histamine H2 receptor ligands using live recombinant HEK293T cells

Fluorescence/luminescence-based techniques play an increasingly important role in the development of test systems for the characterization of future drug candidates, especially in terms of receptor binding in the field of G protein-coupled receptors (GPCRs). In this article, we present the establishment of a homogeneous live cell-based BRET binding assay for the histamine H2 receptor with different fluorescently labeled squaramide-type compounds synthesized in the course of this study. Py-1-labeled ligand 8 (UR-KAT478) was found to be most suitable in BRET saturation binding experiments with respect to receptor affinity (pKd = 7.35) and signal intensity. Real-time kinetic experiments showed a full association of 8 within approximately 30 min and a slow dissociation of the ligand from the receptor. Investigation of reference compounds in BRET-based competition binding with 8 yielded pKi values in agreement with radioligand binding data. This study shows that the BRET binding assay is a versatile test system for the characterization of putative new ligands at the histamine H2 receptor and represents a valuable fluorescence-based alternative to canonical binding assays.

Enhancement of Muscimol Binding and Gating by Allosteric Modulators of the GABAA Receptor: Relating Occupancy to State Functions.

Muscimol is a psychoactive isoxazole derived from the mushroom Amanita muscaria and a potent orthosteric agonist of the GABAA receptor. The binding of [3H]muscimol has been used to evaluate the distribution of GABAA receptors in the brain, and studies of modulation of [3H]muscimol binding by allosteric GABAergic modulators such as barbiturates and steroid anesthetics have provided insight into the modes of action of these drugs on the GABAA receptor. It has, however, not been feasible to directly apply interaction parameters derived from functional studies to describe the binding of muscimol to the receptor. Here, we employed the Monod-Wyman-Changeux concerted transition model to analyze muscimol binding isotherms. We show that the binding isotherms from recombinant α1β3 GABAA receptors can be qualitatively predicted using electrophysiological data pertaining to properties of receptor activation and desensitization in the presence of muscimol. The model predicts enhancement of [3H]muscimol binding in the presence of the steroids allopregnanolone and pregnenolone sulfate, although the steroids interact with distinct sites and either enhance (allopregnanolone) or reduce (pregnenolone sulfate) receptor function. We infer that the concerted transition model can be used to link radioligand binding and electrophysiological data. SIGNIFICANCE STATEMENT: The study employs a three-state resting-active-desensitized model to link radioligand binding and electrophysiological data. We show that the binding isotherms can be qualitatively predicted using parameters estimated in electrophysiological experiments and that the model accurately predicts the enhancement of [3H]muscimol binding in the presence of the potentiating steroid allopregnanolone and the inhibitory steroid pregnenolone sulfate.

The A2aR C‐terminus provides improved total and active expression yields for adenosine receptor chimeras

Heterologous expression of many G‐protein coupled receptors (GPCRs) is a major bottleneck in drug discovery efforts for therapeutic development of the receptor. The goal of this study was to utilize domains from a well‐trafficked GPCR to aid in improving the trafficking of a related receptor. The adenosine A2a receptor (A2aR) shows exceptional expression and trafficking to the plasma membrane in yeast; however, this is not the case for other adenosine receptors. A2aR has a longer C‐terminus than the other adenosine receptors, which may contribute to its exceptional trafficking to the plasma membrane. To test the possibility to improve trafficking of the adenosine A1 receptor (A1R), chimeric receptors containing the seven transmembrane domains of A1R and the full‐length or truncated A2aR C‐terminus were constructed. The chimeric receptor showed improved localization to the plasma membrane and was capable of binding radioligand with native A1R affinity. Functionally active A1R receptor variants were produced at a theoretical yield of 95 pmol/mg total membrane protein, estimated using radioligand binding data, which are greater than three‐fold higher than previously reported yields from other heterologous expression systems, and should facilitate biophysical characterization and drug discovery efforts. © 2018 American Institute of Chemical Engineers AIChE J, 64: 4297–4307, 2018

Species-specific susceptibility to cannabis-induced convulsions.

Background and PurposeNumerous claims are made for cannabis' therapeutic utility upon human seizures, but concerns persist about risks. A potential confounder is the presence of both Δ9‐tetrahydrocannabinol (THC), variously reported to be pro‐ and anticonvulsant, and cannabidiol (CBD), widely confirmed as anticonvulsant. Therefore, we investigated effects of prolonged exposure to different THC/CBD cannabis extracts on seizure activity and associated measures of endocannabinoid (eCB) system signalling.Experimental ApproachCannabis extract effects on in vivo neurological and behavioural responses, and on bioanalyte levels, were measured in rats and dogs. Extract effects on seizure activity were measured using electroencephalography telemetry in rats. eCB signalling was also investigated using radioligand binding in cannabis extract‐treated rats and treatment‐naïve rat, mouse, chicken, dog and human tissue.Key ResultsProlonged exposure to cannabis extracts caused spontaneous, generalized seizures, subserved by epileptiform discharges in rats, but not dogs, and produced higher THC, but lower 11‐hydroxy‐THC (11‐OH‐THC) and CBD, plasma concentrations in rats versus dogs. In the same rats, prolonged exposure to cannabis also impaired cannabinoid type 1 receptor (CB1 receptor)‐mediated signalling. Profiling CB1 receptor expression, basal activity, extent of activation and sensitivity to THC suggested interspecies differences in eCB signalling, being more pronounced in a species that exhibited cannabis extract‐induced seizures (rat) than one that did not (dog).Conclusions and ImplicationsSustained cannabis extract treatment caused differential seizure, behavioural and bioanalyte levels between rats and dogs. Supporting radioligand binding data suggest species differences in eCB signalling. Interspecies variations may have important implications for predicting cannabis‐induced convulsions from animal models.Linked ArticlesThis article is part of a themed section on 8th European Workshop on Cannabinoid Research. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.10/issuetoc

Discovery of benzothiazolylquinoline conjugates as novel human A3 receptor antagonists: biological evaluations and molecular docking studies.

Adenosine is known as an endogenous purine nucleoside and it modulates a wide variety of physiological responses by interacting with adenosine receptors. Among the four adenosine receptor subtypes, the A3 receptor is of major interest in this study as it is overexpressed in some cancer cell lines. Herein, we have highlighted the strategy of designing the hA3 receptor targeted novel benzothiazolylquinoline scaffolds. The radioligand binding data of the reported compounds are rationalized with the molecular docking results. Compound 6a showed best potency and selectivity at hA3 among other adenosine receptors.

Functional and radioligand binding characterization of the α1L-adrenoceptor subtype of the human vas deferens.

Alpha1 -adrenoceptor antagonists can cause ejaculatory dysfunction as an adverse effect. Contractions of the human vas deferens are mediated via α1A -adrenoceptors, and this study investigated whether the low affinity state of this receptor (α1L -adrenoceptor) is involved in mediating contractions of this tissue. The potency of agonists and the affinity of receptor subtype selective antagonists were determined in functional experiments and in [(3) H]tamsulosin binding experiments to identify the α1 -adrenoceptor subtype population present in the human vas deferens. The α1A -adrenoceptor selective agonist A61603 was a full agonist and was 250-fold more potent than noradrenaline. Prazosin antagonized contractile responses to phenylephrine with a low affinity (pKd = 8.6). Only high concentrations of RS17053 antagonized responses to phenylephrine and yielded a relatively low affinity estimate of 7.0. BMY7378 (α1D -adrenoceptor selective) gave a low affinity estimate (pKd = 6.7), whilst tamsulosin (α1A - and α1D -adrenoceptor selective) had a high affinity (pKd = 9.9). [(3) H]Tamsulosin bound to human vas deferens membranes with a high affinity (pKd = 10.0). Prazosin, RS17053 and BMY7378 competed with [(3) H]tamsulosin with low affinities for a single population of binding sites (pKd values of 8.5, 7.2 and 6.3, respectively). These functional and radioligand binding data indicate that the human vas deferens possesses a homogeneous population of α1 -adrenoceptors which have the pharmacological properties of the putative α1L -adrenoceptor, the same functional receptor previously identified in the human prostate.

Are receptor concentrations correlated across tissues within individuals? A case study examining glucocorticoid and mineralocorticoid receptor binding.

Hormone receptors are a necessary (although not sufficient) part of the process through which hormones like corticosterone create physiological responses. However, it is currently unknown to what extent receptor concentrations across different target tissues may be correlated within individual animals. In this study, we examined this question using a large dataset of radioligand binding data for glucocorticoid receptors (GRs) and mineralocorticoid receptors (MRs) in 13 different tissues in the house sparrow (Passer domesticus) (n=72). Our data revealed that individual house sparrows tended to exhibit higher or lower receptor binding across all tissues, which could be part of what creates the physiological and behavioral syndromes associated with different hormonal profiles. However, although statistically significant, the correlations between tissues were very weak. Thus, when each tissue was independently regressed on receptor concentrations in the other tissues, multivariate analysis revealed significant relationships only for sc fat (for GR) and whole brain, hippocampus, kidney, omental fat, and sc fat (for MR). We also found significant pairwise correlations only between receptor concentrations in brain and hippocampus, and brain and kidney (both for MR). This research reveals that although there are generalized individual consistencies in GR and MR concentrations, possibly due to such factors as hormonal regulation and genetic effects, the ability of 2 different tissues to respond to the same hormonal signal appears to be affected by additional factors that remain to be identified.

The dopamine uptake inhibitor, WIN 35,428, has sigma2 receptor agonist effects that contribute to its self‐administration (848.10)

Sigma receptors (σRs) are intracellular chaperone proteins implicated in several diseases, as well as psychiatric disorders and drug abuse. Several in vivo studies show blockade with σR antagonists of several cocaine effects, though not its self administration (SA). The present study assessed whether that insensitivity was specific to cocaine SA using the dopamine uptake inhibitors (WIN35,428, methylphenidate, nomifensine) in rats trained with cocaine SA. As with cocaine, σR antagonists (BD 1008, BD 1047, BD 1063, SN 79, SN 167, (±)SM 21, CM 304, CM 398) were ineffective against methylphenidate and nomifensine SA across a range of doses that antagonized SA of σR agonists (DTG, PRE 084, (+)pentazocine). In contrast, all antagonists tested, except CM 304 produced dose dependent insurmountable antagonism of WIN35,428 SA. CM 304 was equipotent in blocking the SA of DTG, (+)‐pentazocine, and PRE‐084, but only marginally potent in blocking SA of WIN35,428. The antagonists SN 79, SN 167, CM 398, and (±)SM 21 were more potent in blocking DTG than either PRE 084 or (+)pentazocine SA. These antagonists also blocked WIN35,428 SA, though three fold less potently than against DTG. The results are consistent with σR radioligand binding data for WIN35,428, the nonselective σ1/2R agonist effects of DTG, selective σ1R antagonist effects of CM 304, and that WIN 35,428 has σ2R agonist effects that contribute to its self administration. Char 1219

L-DOPA disrupts adenosine A2A–cannabinoid CB1–dopamine D2 receptor heteromer cross-talk in the striatum of hemiparkinsonian rats: Biochemical and behavioral studies

Long-term therapy with l-3,4-dihydroxyphenylalanine (l-DOPA), still the most effective treatment in Parkinson's disease (PD), is associated with severe motor complications such as dyskinesia. Experimental and clinical data have indicated that adenosine A2A receptor antagonists can provide symptomatic improvement by potentiating l-DOPA efficacy and minimizing its side effects. It is known that the G-protein-coupled adenosine A2A, cannabinoid CB1 and dopamine D2 receptors may interact and form functional A2A-CB1–D2 receptor heteromers in co-transfected cells as well as in rat striatum. These data suggest that treatment with a combination of drugs or a single compound selectively acting on A2A–CB1–D2 heteromers may represent an alternative therapeutic treatment of PD. We investigated the expression of A2A–CB1–D2 receptor heteromers in the striatum of both naïve and hemiparkinsonian rats (HPD-rats) bearing a unilateral 6-hydroxydopamine (6-OHDA) lesion, and assessed how receptor heteromer expression and biochemical properties were affected by l-DOPA treatment. Radioligand binding data showed that A2A–CB1–D2 receptor heteromers are present in the striatum of both naïve and HPD-rats. However, behavioral results indicated that the combined administration of A2A (MSX-3 or SCH58261) and CB1 (rimonabant) receptor antagonists, in the presence of l-DOPA does not produce a response different from administration of the A2A receptor antagonist alone. These behavioral results prompted identification of heteromers in l-DOPA-treated animals. Interestingly, the radioligand binding results in samples from lesioned animals suggest that the heteromer is lost following acute or chronic treatment with l-DOPA.

Synthesis and Functional Characterization of Imbutamine Analogs as Histamine H3 and H4 Receptor Ligands

Imbutamine (4-(1H-imidazol-4-yl)butanamine) is a potent histamine H3 (H3R) and H4 receptor (H4R) agonist (EC50 values: 3 and 66 nM, respectively). Aiming at improved selectivity for the H4R, the imidazole ring in imbutamine was methyl-substituted or replaced by various differently substituted heterocycles (1,2,3-triazoles, 1,2,4-triazoles, pyridines, pyrimidines) as potential bioisosteres. Investigations in [(35)S]GTPγS binding assays using membranes of Sf9 insect cells expressing the respective human histamine receptor subtype revealed only very weak activity of most of the synthesized hetarylalkylamines at both receptors. By contrast, the introduction of substituents at the 4-imidazolyl ring was most effective regarding H4R selectivity. This holds for methyl substitution in position 2 and, especially, in position 5. 5-Methylimbutamine (H4R: EC50 = 59 nM, α = 0.8) was equipotent with imbutamine at the hH4R, but revealed about 16-fold selectivity for the hH4R compared to the hH3R (EC50 980 nM, α = 0.36), whereas imbutamine preferred the hH3R. The functional activities were in agreement with radioligand binding data. The results support the hypothesis that, by analogy with histamine, methyl substitution in histamine homologs offers a way to shift the selectivity in favor of the H4R.

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Preface

Clozapine, atypical antipsychotics, and the benefits of fast-off D2 dopamine receptor antagonism

Drug-receptor interactions are traditionally quantified in terms of affinity and efficacy, but there is increasing awareness that the drug-on-receptor residence time also affects clinical performance. While most interest has hitherto been focused on slow-dissociating drugs, D(2) dopamine receptor antagonists show less extrapyramidal side effects but still have excellent antipsychotic activity when they dissociate swiftly. Fast dissociation of clozapine, the prototype of the "atypical antipsychotics", has been evidenced by distinct radioligand binding approaches both on cell membranes and intact cells. The surmountable nature of clozapine in functional assays with fast-emerging responses like calcium transients is confirmatory. Potential advantages and pitfalls of the hitherto used techniques are discussed, and recommendations are given to obtain more precise dissociation rates for such drugs. Surmountable antagonism is necessary to allow sufficient D(2) receptor stimulation by endogenous dopamine in the striatum. Simulations are presented to find out whether this can be achieved during sub-second bursts in dopamine concentration or rather during much slower, activity-related increases thereof. While the antagonist's dissociation rate is important to distinguish between both mechanisms, this becomes much less so when contemplating time intervals between successive drug intakes, i.e., when pharmacokinetic considerations prevail. Attention is also drawn to the divergent residence times of hydrophobic antagonists like haloperidol when comparing radioligand binding data on cell membranes with those on intact cells and clinical data.

Interaction of 5‐HT3R ligands with acetylcholine binding protein by surface plasmon resonance

The field of Ligand Gated Ion Channels (LGIC) has benefited greatly over the last several years due to the discovery of the Acetylcholine Binding Protein (AChBP) from snail Lymnaea stagnalis and Aplysia californica. Studies have utilized docking simulations, crystal structure and radio ligand binding data of the AChBP to better understand the function of ligand binding. The AChBP has also been used to characterize other cys‐loop super family LIGC including the serotonin type 3 receptor (5‐HT3R). Although homology models for the AChBP and 5‐HT3R exist there is a lack of information describing how 5‐HT ligands interact with the AChBP.Understanding how 5‐HTR ligands interact with the AChBP binding pocket is crucial knowledge for comparing the functional relationship between AChBP and 5‐HT3R ligand binding site. The goal of this work was to obtain binding data for 5‐HT3R ligands using radio‐ligand binding experiments and Surface Plasmon Resonance (SPR) as well as confirm validity of 5‐HT3R antagonist binding models based on AChBP. We evaluated the binding of 5‐HT3R agonists, partial agonists and antagonists to the AChBP. Results showed binding affinities for 5‐HT3R agonist were 500–30,000 times lower on the AChBP compared to 5‐HT3R. Binding affinities for 5‐HT3R antagonists were 2–30 lower on the AChBP compared to the 5‐HT3R (&gt;KD). The interaction of antagonists to AChBP is similar to those present in 5‐HT3R while those residues interacting with agonists show less homology. Our docking studies supported these conclusions.