Structural Basis for Molecular Recognition of Cannabinoids by Inhibitory Cys-Loop Channels.

Cannabis sativa has a long history of medicinal use, dating back to ancient times. This plant produces cannabinoids, which are now known to interact with several human proteins, including Cys-loop receptors for glycine (GlyR) and gamma-aminobutyric acid (GABAAR). As these channels are the primary mediators of inhibitory signals, they contribute to the diverse effects of cannabinoids on the nervous system. Evidence suggests that cannabinoid binding sites are located within the transmembrane domain, although their precise location has remained undetermined for over a decade. The process of identification of the binding site and the computational approaches employed are the main subjects of this Perspective, which includes an analysis of the most recently resolved cryo-EM structures of zebrafish GlyR bound to Δ9-tetrahydrocannabinol and the THC-GlyR complex obtained through molecular dynamics simulations. With this work, we aim to contribute to guiding future studies investigating the molecular basis of cannabinoid action on inhibitory channels.

Molecular determinants of tetrahydrocannabinol binding to the glycine receptor.

The recognition of Cannabis as a source of new compounds suitable for medical use has attracted strong interest from the scientific community in its research, and substantial progress has accumulated regarding cannabinoids' activity; however, a thorough description of their molecular mechanisms of action remains a task to complete. Highlighting their complex pharmacology, the list of cannabinoids' interactors has vastly expanded beyond the canonical cannabinoid receptors. Among those, we have focused our study on the glycine receptor (GlyR), an ion channel involved in the modulation of nervous system responses, including, to our interest, sensitivity to peripheral pain. Here, we report the use of computational methods to investigate possible binding modes between the GlyR and Δ9 -tetrahydrocannabinol (THC). After obtaining a first pose for the THC binding from a biased molecular docking simulation and subsequently evaluating it by molecular dynamic simulations, we found a dynamic system with an identifiable representative binding mode characterized by the specific interaction with two transmembrane residues (Phe293 and Ser296). Complementarily, we assessed the role of membrane cholesterol in this interaction and positively established its relevance for THC binding to GlyR. Lastly, the use of restrained molecular dynamics simulations allowed us to refine the description of the binding mode and of the cholesterol effect. Altogether, our findings contribute to the current knowledge about the GlyR-THC mode of binding and propose a new starting point for future research on how cannabinoids in general, and THC in particular, modulate pain perception in view of its possible clinical applications.

Insights into estrogen receptor alpha modulation by cholestenoic acids.

Oxysterols are a family of over 25 cholesterol metabolites naturally produced by enzymatic or radical oxidation. They are involved in many physiological and pathological pathways. Although their activity has been mainly attributed to the modulation of the Liver X Receptors (LXR), it is currently accepted that oxysterols are quite promiscuous compounds, acting at several targets at the same time. The promiscuity of the oxysterols with the Estrogen Receptor α (ERα) is crucial in several pathologies such as ER+ breast cancer, inflammation and atherosclerosis. Regarding this matter, we have previously reported the synthesis, LXR activity and binding mode of a family of cholestenoic acid analogs with a modified side chain. Here we report the transcriptional activity on the ERα triggered by these compounds and details on the molecular determinants involved in their activities in order to establish structure-activity relationships to shed light over the molecular basis of the promiscuity of these compounds on ER/LXR responses. Our results show that 3ß-hydroxy-5-cholestenoic acid can interact with the ERα receptor in a way similar to 26-hydroxycholesterol and is an agonist of the receptor. Using molecular dynamics simulations, we were able to predict the ERα activity of a set of cholestenoic acid analogs with changes in the flexibility and/or steric requirements of the side chain, some of which exhibited selective activation of ERα or LXR.

Cholestenoic acid analogues as inverse agonists of the liver X receptors.

Liver X Receptors (LXRs) are ligand dependent transcription factors activated by oxidized cholesterol metabolites (oxysterols) that play fundamental roles in the transcriptional control of lipid metabolism, cholesterol transport and modulation of inflammatory responses. In the last decade, LXRs have become attractive pharmacological targets for intervention in human metabolic diseases and thus, several efforts have concentrated on the development of synthetic analogues able to modulate LXR transcriptional response. In this sense, we have previously found that cholestenoic acid analogues with a modified side chain behave as LXR inverse agonists. To further investigate the structure-activity relationships and to explore how cholestenoic acid derivatives interact with the LXRs, we evaluated the LXR biological activity of new analogues containing a C24-C25 double bond. Furthermore, a microarray assay was performed to evaluate the recruitment of coregulators to recombinant LXR LBD upon ligand binding. Also, conventional and accelerated molecular dynamics simulations were applied to gain insight on the molecular determinants involved in the inverse agonism. As LXR inverse agonists emerge as very promising candidates to control LXR activity, the cholestenoic acid analogues here depicted constitute a new relevant steroidal scaffold to inhibit LXR action.

Structural Insights into the Ligand Binding Domain of the Glucocorticoid Receptor: A Molecular Dynamics Study.

The glucocorticoid receptor (GR) is a ligand-binding dependent transcription factor that ultimately regulates vital biological processes and inflammation response through specific gene expression control, thus representing a notable drug target to explore. Structurally, its ligand binding domain (LBD) harbors the region for the ligand-dependent transcriptional activation function 2 (AF-2), a majorly hydrophobic groove formed by residues from helices H3, H4, and H12, where the H12 position plays a critical role in AF-2 spatial conformation and GR function as a whole. However, the exact mechanisms underlying how regulatory ligands control the H12 structure and dynamics are yet to be elucidated. In this work, we have explored the correlation between ligand identity and GR LBD H12 behavior through different molecular dynamics (MD) simulations. After building diverse GR LBD systems in agonist and nonagonist states, we studied each system's response in the absence or the presence of an agonist ligand (dexamethasone) or an antagonist ligand (RU486) using classical MD simulations. We complemented them with steered MD for assessing the transition between those states and with the Umbrella Sampling method for free-energy evaluation. On the one hand, successfully obtaining fully folded nonagonist GR LBD states from the partially unfolded crystal GR LBD/RU486 underlines the role of the H1 in the GR LBD folding pathway. On the other hand, our results describe the H12 as a dynamic ensemble of conformations whose relative population is in the end determined by the interacting ligand: while dexamethasone privileges only a few poses (determined by a potential energy surface with a deep minimum), RU486 favors a wider H12 conformational amplitude, as indicated by a flatter potential landscape. By characterizing the H12 conformation in different conditions, we provide novel GR LBD models that represent potential targets for rational glucocorticoid drugs design, with the aim of accurately modulating GR activity.

Mapping the neurosteroid binding sites on glycine receptors.

Glycine is a major inhibitory neurotransmitter in the CNS, where it modulates both sensory and motor transduction throughout its binding to glycine receptors (GlyRs), pentameric chloride channels that share structural and functional properties with type A γ-aminobutyric acid receptors (GABAAR). A large number of structurally diverse organic compounds have been identified as GlyR and GABAAR allosteric modulators, making these receptors attractive pharmacological targets. Taking into account the recent resolved crystal structures of GABAAR/neurosteroid complexes, and due to the high sequence identity between the GABAAR and GlyR transmembrane domains, in this work we applied molecular modeling methods to explore the neurosteroid binding to GlyR. Our results indicated that neurosteroid binding sites of GABAARs are also conserved in the GlyRs. Furthermore, docking and molecular dynamics simulations predicted that neurosteroids are stably recognized at these sites, providing precise information on the molecular basis of the neurosteroid binding mode to GlyR. The comparison of how allopregnanolone and pregnanolone 3-OH moieties are recognized by the GlyR binding pocket revealed significant differences that may be associated to opposite effects of these isomers on the GlyR response.

In Search of GABAA Receptor's Neurosteroid Binding Sites.

Neurosteroids (NS) are the main modulators of γ-aminobutyric acid type A receptors (GABAARs), which are the ligand-gated channels target of the major inhibitory neurotransmitter in vertebrates. As a consequence of their ability to modify inhibitory functions in the brain, NS have high physiological and clinical relevance. Accumulated evidence has strongly suggested that NS binding sites were located in the GABAAR transmembrane domain; however the specific localization of these sites has remained an enigma for decades. Fortunately, recent resolution of GABAARs crystal structures, together with computational strategies applied to investigate the NS binding, has paved the way to rationalizing the molecular basis of NS modulation. This work reviews from a historical perspective the road followed for establishing the GABAAR/NS binding mode, from their initial molecular modeling to the latest findings. Furthermore, a comparative analysis describing the NS binding is provided, plus a preliminary analysis of putative NS sites in other assemblies.

Structure and dynamics of neurosteroid binding to the α1ß2γ2 GABAA receptor.

Neurosteroids are the principal endogenous modulators of the γ-Aminobutyric acid receptors (GABAARs), pentameric membrane-bound proteins that can be assembled from at least 19 subunits. In the most abundant GABAAR arrangement (α1ß2γ2), neurosteroids can potentiate the GABA action as well as produce a direct activation of the channel. The recent crystal structures of neurosteroids bound to α homopentameric GABAAR reveal binding to five equivalent sites. However, these results have been obtained using receptors that are not physiologically relevant, suggesting a need to investigate neurosteroid binding to heteropentameric receptors that exist in the central nervous system. In a previous work, we predicted the neurosteroid binding site by applying molecular modeling methods on the ß3 homopentamer. Here we construct a homology model of the transmembrane domain of the heteropentameric α1ß2γ2 receptor and then, by combining docking and molecular dynamics simulations, we analyzed neurosteroid binding. Results show that the five neurosteroid cavities are conserved in the α1ß2γ2 receptor and all of them are able to bind neurosteroids. Two different binding modes were detected depending on the identity of the residue at position 241 in the transmembrane helix 1. These theoretical findings provide microscopic insights into neurosteroid binding at the heteropentameric GABAAR. The existence of two classes of sites may be associated with how neurosteroids modulate GABAAR. Our finding would represent the essential first step to reach a comprehensive understanding of how these endogenous molecules regulate the central nervous system.

Synthesis and activity evaluation of a series of cholanamides as modulators of the liver X receptors.

The Liver X receptors (LXRs) are members of the nuclear receptor family, that play fundamental roles in cholesterol transport, lipid metabolism and modulation of inflammatory responses. In recent years, the synthetic steroid N,N-dimethyl-3ß-hydroxycholenamide (DMHCA) arised as a promising LXR ligand. This compound was able to dissociate certain beneficial LXRs effects from those undesirable ones involved in triglyceride metabolism. Here, we synthetized a series of DMHCA analogues with different modifications in the steroidal nucleus involving the A/B ring fusion, that generate changes in the overall conformation of the steroid. The LXRα and LXRß activity of these analogues was evaluated by using a luciferase reporter assay in BHK21 cells. Compounds were tested in both the agonist and antagonist modes. Results indicated that the agonist/antagonist profile is dependent on the steroid configuration at the A/B ring junction. Notably, in contrast to DMHCA, the amide derived from lithocholic acid (2) with an A/B cis configuration and its 6,19-epoxy analogue 4 behaved as LXRα selective agonists, while the 2,19-epoxy analogues with an A/B trans configuration were antagonists of both isoforms. The binding mode of the analogues to both LXR isoforms was assessed by using 50 ns molecular dynamics (MD) simulations. Results revealed conformational differences between LXRα- and LXRß-ligand complexes, mainly in the hydrogen bonding network that involves the C-3 hydroxyl. Overall, these results indicate that the synthetized DMHCA analogues could be interesting candidates for a therapeutic modulation of the LXRs.

21-Hydroxy-6,19-epoxyprogesterone: A Promising Therapeutic Agent and a Molecular Tool for Deciphering Glucocorticoid Action.

Glucocorticoids are steroid hormones that exert most of their effects through their binding to the glucocorticoid receptor (GR), a ligand regulated transcription factor. Although glucocorticoids are widely used in the clinic, their usage in chronic therapies provokes severe adverse reactions. In the quest for safer glucocorticoids a dissociated model was established that proposes a disconnection between GR activated pathways responsible of desired pharmacological effects and pathways involved in adverse GR reactions. Under this model, a myriad of steroidal and non-steroidal compounds has been characterized, with most of them still producing side effects. X-ray crystallographic studies followed by molecular dynamics analysis led research to insights on the receptor Ligand Binding Domain (LBD), which undergoes specific ligand dependent conformational changes that influence receptor activities. In this sense, the flexibility of the ligand structure would contribute to the final GR outcome. Here, we review different data of 21-hydroxy-6,19-epoxyprogesterone (21OH-6,19OP), a rigid steroid with potential pharmaceutical interest due to its anti-inflammatory and immunosuppressive activities, lacking several GR adverse reactions. The rigid structure endows this compound with an enhanced selectivity towards GR. Molecular characterization of the GR/21OH-6,19OP complex revealed specific intermediate conformations adopted by the receptor that would explain the influence on GR dimerization and the recruitment of a specific set of GR transcription modulators. We summarize recent data that will contribute to understand the complexity of glucocorticoid response.

Fluorinated oxysterol analogues: Synthesis, molecular modelling and LXRß activity.

Liver X receptors (LXRs) are nuclear receptors that play central roles in the transcriptional control of lipid metabolism. The ability of LXRs to integrate metabolic and inflammation signalling makes them attractive targets for intervention in human metabolic diseases. Several oxidized metabolites of cholesterol (oxysterols) are endogenous LXR ligands, that modulate their transcriptional responses. While 25R-cholestenoic acid is an agonist of the LXRs, the synthetic analogue 27-norcholestenoic acid that lacks the 25-methyl is an inverse agonist. This change in the activity profile is triggered by a disruption of a key interaction between residues His435 and Trp457 that destabilizes the H11-H12 region of the receptor and favors the binding of corepressors. The introduction of fluorine atoms on the oxysterol side chain can favor both hydrophobic interactions as well as hydrogen bonds with the fluorine atoms and may thus induce changes in the receptor that may lead to changes in the activity profile. To evaluate these effects we have synthesized two fluorinated 27-nor-steroids, analogues of 27-norcholestenoic acid, the 25,25-difluoroacid and the corresponding 26-alcohol. The key step was a Reformatsky reaction on the C-24 cholenaldehyde, with ethyl bromodifluoroacetate under high intensity ultrasound (HIU) irradiation, followed by a Barton-McCombie type deoxygenation. Activity was evaluated in a luciferase reporter assay in the human HEK293T cells co-transfected with full length human LXRß expression vector. The 25,25-difluoro-27-norcholestenoic acid was an inverse agonist and antagonist similar to its non-fluorinated analogue while its reduced derivative 25,25-difluoro-27-norcholest-5-ene-3ß,26-diol was an agonist. Molecular dynamics simulation of the ligand-receptor complexes showed that the difluoroacid disrupted the His435-Trp457 interaction although the resulting conformational changes were different from those induced by the non-fluorinated analogue. In the case of the difluoroalcohol, the fluorine atoms actively participated in the interaction with several residues in the ligand binding pocket leading to a stabilization of the active receptor conformation.

Destabilization of the torsioned conformation of a ligand side chain inverts the LXRß activity.

BACKGROUND: Liver X receptors (LXRs) are transcription factors activated by cholesterol metabolites containing an oxidized side chain. Due to their ability to regulate lipid metabolism and cholesterol transport, they have become attractive pharmacological targets. LXRs are closely related to DAF-12, a nuclear receptor involved in nematode lifespan and regulated by the binding of C-27 steroidal acids. Based on our recent finding that the lack of the C-25 methyl group does not abolish their DAF-12 activity, we evaluated the effect of removing it from the (25R)-cholestenoic acid, a LXR agonist. METHODS: The binding mode and the molecular basis of action of 27-nor-5-cholestenoic acid were evaluated using molecular dynamics simulations. The biological activity was investigated using reporter gene expression assays and determining the expression levels of endogenous target genes. The in vitro MARCoNI assay was used to analyze the interaction with cofactors. RESULTS: 27-Nor-5-cholestenoic acid behaves as an inverse agonist. This correlates with the capacity of the complex to better bind corepressors rather than coactivators. The C-25 methyl moiety would be necessary for the maintenance of a torsioned conformation of the steroid side chain that stabilizes an active LXRß state. CONCLUSION: We found that a 27-nor analog is able to act as a LXR ligand. Interestingly, this minimal structural change on the steroid triggered a drastic change in the LXR response. GENERAL SIGNIFICANCE: Results contribute to improve our understanding on the molecular basis of LXRß mechanisms of action and provide a new scaffold in the quest for selective LXR modulators.

Exploring the molecular basis of neurosteroid binding to the ß3 homopentameric GABAA receptor.

Neurosteroids are the principal endogenous modulators of GABA(A) receptors (GABA(A)Rs), which are pentameric membrane-bound proteins that regulate the passage of chloride ions from the extracellular to the intracellular compartment. As consequence of their ability to modify inhibitory functions in the brain, neurosteroids have high physiological and clinical importance and may act as anesthetic, anticonvulsant and anxiolytic drugs. Despite their relevance, essential issues regarding neurosteroid action on GABA(A)Rs are still unsettled. In particular, residues taking part of the steroid recognition are not definitely identified. Taking as starting point the first reported crystal structure of a human GABAA receptor (a ß3 homopentamer), we have explored through a combination of computational methods (a cavity-detection algorithm, docking and molecular dynamics simulations) the binding mode of two structurally different representative neurosteroids, pregnanolone and allopregnanolone. We have identified a neurosteroid binding site between the TM3 of one subunit and TM1 and TM4 of the adjacent subunit that is consistent with the set of experimental data reported for the action of neurosteroids on ß3 homopentamers. These sites are able to properly accommodate both overall torsioned and flat steroidal structures and they specifically recognize the 3-OH group, explaining the requirement of a 3α-configuration for the activity. We believe that this work provides for first time convincing information about the molecular interaction between neurosteroids and a GABA(A)R. This information largely increases our understanding of this fundamental ligand-receptor system.

Exploring the molecular basis of action of ring D aromatic steroidal antiestrogens.

Salpichrolides are natural plant steroids that contain an unusual six-membered aromatic ring D. We recently reported that some of these compounds, and certain analogs with a simplified side chain, exhibited antagonist effects toward the human estrogen receptor (ER), a nuclear receptor whose endogenous ligand has an aromatic A ring (estradiol). Drugs acting through the inhibition or modulation of ERs are frequently used as a hormonal therapy for ER(+) breast cancer. Previous results suggested that the aromatic D ring was a key structural motif for the observed activity; thus, this modified steroid nucleus may provide a new scaffold for the design of novel antiestrogens. Using molecular dynamics (MD) simulation we have modeled the binding mode of the natural salpichrolide A and a synthetic analog with an aromatic D ring within the ERα. These results taken together with the calculated energetic contributions associated to the different ligand-binding modes are consistent with a preferred inverted orientation of the steroids in the ligand-binding pocket with the aromatic ring D occupying a position similar to that observed for the A ring of estradiol. Major changes in both dynamical behavior and global positioning of H11 caused by the loss of the ligand-His524 interaction might explain, at least in part, the molecular basis of the antagonism exhibited by these compounds. Using steered MD we also found a putative unbinding pathway for the steroidal ligands through a cavity formed by residues in H3, H7, and H11, which requires only minor changes in the overall receptor conformation.

Synthetic DAF-12 modulators with potential use in controlling the nematode life cycle.

Dafachronic acids (DAs) are 3-keto cholestenoic acids bearing a carboxylic acid moiety at the end of the steroid side chain. These compounds interact with the DAF-12 receptor, a ligand-dependent transcription factor that acts as a molecular switch mediating the choice between arrest at diapause or progression to reproductive development and adult lifespan in different nematodes. Recently, we reported that the 27-nor-Δ4-DA was able to directly activate DAF-12 in a transactivation cell-based luciferase assay and rescued the Mig phenotype of daf-9(rh50) Caenorhabditis elegans mutants. In the present paper, to investigate further the relationship between the structure of the steroid side chain and DAF-12 activity, we evaluated the in vitro and in vivo activity of Δ4-DA analogues with modified side chains using transactivation cell-based assays and daf-9(dh6) C. elegans mutants. Our results revealed that introduction of a 24,25-double bond on the cholestenoic acid side chain did not affect DAF-12 activity, whereas shortening the side chain lowered the activity. Most interestingly, the C24 alcohol 24-hydroxy-4-cholen-3-one (6) was an antagonist of the DAF-12 receptor both in vitro and in vivo.

Synthesis and biological evaluation of salpichrolide analogs as antiestrogenic agents.

The antiestrogenic activity of three natural salpichrolides A, G and B (1, 3 and 4) and of five synthetic analogs containing an aromatic D ring and a simplified side chain (5-9), was evaluated on MCF-7 cells. The 2,3-ene-1-keto steroids 8 and 9 were obtained from 3ß-acetoxy-17(13→18)-abeo-5αH-pregna-13,15,17-trien-20-one, the key step for these syntheses being a Wharton carbonyl rearrangement of a 1,2-epoxy-3-keto steroid to the allylic alcohol using hydrazine hydrate. The antiestrogenic activity was evaluated by performing dose-response experiments in ER(+) MCF-7 breast cancer cells. Dose-dependent proliferation was quantified via [(3)H]-thymidine incorporation after 3 days treatment. Salpichrolides A, G and B and analogs 5, 8 and 9 were active as antiestrogens with compound 9 being the most active of the synthetic analogs. Compounds 5 and 9 were also evaluated against the ER(-) cell line MDA-MB-231 and shown to be inactive.

Live cell imaging unveils multiple domain requirements for in vivo dimerization of the glucocorticoid receptor.

Glucocorticoids are essential for life, but are also implicated in disease pathogenesis and may produce unwanted effects when given in high doses. Glucocorticoid receptor (GR) transcriptional activity and clinical outcome have been linked to its oligomerization state. Although a point mutation within the GR DNA-binding domain (GRdim mutant) has been reported as crucial for receptor dimerization and DNA binding, this assumption has recently been challenged. Here we have analyzed the GR oligomerization state in vivo using the number and brightness assay. Our results suggest a complete, reversible, and DNA-independent ligand-induced model for GR dimerization. We demonstrate that the GRdim forms dimers in vivo whereas adding another mutation in the ligand-binding domain (I634A) severely compromises homodimer formation. Contrary to dogma, no correlation between the GR monomeric/dimeric state and transcriptional activity was observed. Finally, the state of dimerization affected DNA binding only to a subset of GR binding sites. These results have major implications on future searches for therapeutic glucocorticoids with reduced side effects.

Neuroprotective action of synthetic steroids with oxygen bridge. Activity on GABAA receptor.

Allopregnanolone (A) and pregnanolone (P) are able to modify neural activities acting through the GABAA receptor complex. This capacity makes them useful as anticonvulsant, anxiolytic, or anti-stress compounds. In this study, the performance of seven synthetic steroids (SS) analogous of A or P containing an intramolecular oxygen bridge was evaluated using different assays. Competition assays showed that compounds 1, 5, 6 and 7 affected the binding of specific ligands for the GABAA receptor in a way similar to that of A and P, whereas compounds 3 and 4 stimulated [(3)H]-flunitrazepam and reduced [(35)S]-TBPS binding. The enzyme 3ß-hydroxysteroid dehydrogenase (3ß-HSD) produces the precursor for A and P, and its activity is regulated by steroids. The action of several SS on 3ß-HSD activity was tested in different tissues. All SS analyzed inhibit its activity, but compound 5 was the least effective. Finally, the neuroprotective role of two SS was evaluated in cerebral cortex and hippocampus cultures subjected to hypoxia. Glial fibrillary acidic protein (GFAP) increase was prevented by A, P, and compounds 3 and 5. Only A, P and compound 5 prevented neurofilament (NF160/200) decrease in hippocampus cultures, whereas A and compound 5 partially prevented NF200 and NF160 decreases respectively in cerebral cortex cultures. A prevented microtubule associated protein (MAP 2b) decrease in cerebral cortex cultures, while in hippocampus cultures only compounds 3 and 5 had effect. All steroids prevented MAP 2c decrease in both brain regions.

27-Nor-Δ(4)-dafachronic acid is a synthetic ligand of Caenorhabditis elegans DAF-12 receptor.

27-Nor-Δ(4)-dafachronic acid was prepared in nine steps and 14% overall yield by two sequential 2-carbon homologations from 20ß-carboxyaldehyde-4-pregnen-3-one. Its activity was evaluated in vivo, where it rescued the Mig phenotype of daf-9(rh50) Caenorhabditis elegans mutants and restored their normal resistance to oxidative stress. 27-Nor-Δ(4)-dafachronic acid was also able to directly bind and activate DAF-12 in a transactivation cell-based luciferase reporter assay, although it was less active than the corresponding 25R-and 25S dafachronic acids. The binding mode of the 27-Nor steroid was studied by molecular dynamics using a homology model of the CeDAF-12 receptor.

The Caenorhabditis elegans DAF-12 nuclear receptor: structure, dynamics, and interaction with ligands.

A structure for the ligand binding domain (LBD) of the DAF-12 receptor from Caenorhabditis elegans was obtained from the X-ray crystal structure of the receptor LBD from Strongyloides stercoralis bound to (25R)-Δ(7)-dafachronic acid (DA) (pdb:3GYU). The model was constructed in the presence of the ligand using a combination of Modeller, Autodock, and molecular dynamics (MD) programs, and then its dynamical behavior was studied by MD. A strong ligand binding mode (LBM) was found, with the three arginines in the ligand binding pocket (LBP) contacting the C-26 carboxylate group of the DA. The quality of the ceDAF-12 model was then evaluated by constructing several ligand systems for which the experimental activity is known. Thus, the dynamical behavior of the ceDAF-12 complex with the more active (25S)-Δ(7)-DA showed two distinct binding modes, one of them being energetically more favorable compared with the 25R isomer. Then the effect of the Arg564Cys and Arg598Met mutations on the (25R)-Δ(7)-DA binding was analyzed. The MD simulations showed that in the first case the complex was unstable, consistent with the lack of transactivation activity of (25R)-Δ(7)-DA in this mutant. Instead, in the case of the Arg598Met mutant, known to produce a partial loss of activity, our model predicted smaller effects on the LBM with a more stable MD trajectory. The model also showed that removal of the C-25 methyl does not impede the simultaneous strong interaction of the carboxylate with the three arginines, predicting that 27-nor-DAs are putative ceDAF-12 ligands.