Receptor Ligand Binding Domain Research Articles

Species traits predict the aryl hydrocarbon receptor 1 (AHR1) subtypes responsible for dioxin sensitivity in birds

Differences in avian sensitivity to dioxin-like compounds (DLCs) are directly attributable to the identities of amino acids at two sites within the ligand binding domain (LBD) of the aryl hydrocarbon receptor 1 (AHR1). Recent work suggests that by influencing avian exposure to naturally occurring dioxins, differences in diet, habitat, and migration may have influenced the evolution of three AHR1 LBD genotypes in birds: type 1 (high sensitivity), type 2 (moderate sensitivity), and type 3 (low sensitivity). Using a boosted regression tree (BRT) analysis, we built on previous work by examining the relationship between a comprehensive set of 17 species traits, phylogeny, and the AHR1 LBD across 89 avian species. The 17 traits explained a combined 74% of the model deviance, while phylogenetic relatedness explained only 26%. The strongest predictors of AHR1 LBD were incubation period and habitat type. We found that type 3 birds tended to occupy aquatic habitats, and, uniquely, we also found that type 3 birds tended to have slower developmental rates. We speculate that this reflects higher evolutionary exposure to naturally occurring dioxins in waterbirds and species with K-selected life histories. This study highlights the value of trait-based approaches in helping to understand differing avian species sensitivities to environmental contaminants.

SUN-LB138 Dynamic Structural Model of Testosterone Entry Into the Unliganded Androgen Receptor

Background: Crystallographic structures of nuclear receptor ligand binding domains provide a static model of a receptor stably wrapped around an internalized ligand. Understanding the dynamics of a receptor at different stages of ligand binding has been hampered by the paucity of crystal structures for unliganded nuclear receptors. Molecular dynamic models have been constructed for some nuclear receptors to fill that void. Methods: The molecular simulation docking program MORDOR (MOlecular Recognition with a Driven dynamics OptimizeR)(1) was used to study the structural dynamics of the androgen receptor ligand binding domain (AR LBD) modeled from the static structure of the AR LBD bound to testosterone (T) (PDB ID: 2AM9). The goals of the study were to understand a) the dynamic interaction of the T in its binding pocket, b) AR LBD structural flexibilities that permit T entry/exit from the binding pocket and c) a model of the unliganded AR LBD. Results: Modeling AR LBD structure flexibility over time revealed possible alternative dynamic structures, including those without ligand, overlaid against the canonical nuclear receptor structure. The model dynamically tracks the structural changes as a ligand enters into the ligand binding domain and nestles into the ligand binding pocket. The model predicted the appearance of alpha helices within the AR LBD that transiently fold/unfold during the ligand entry phases. Once in the pocket, the ligand itself remains very dynamic in a still flexible pocket. The model predicted also AR LBD amino acids that sequentially interact with the ligand during its dynamic entry into the AR LBD. Intriguingly, those AR amino acids include those mutated in castration-resistant prostate tumors that continue to grow during androgen suppression therapy. Functional studies showed those mutant ARs had a primary consequence of enhancing response to lower level T, and other androgens, consistent with their role in creating a higher affinity AR that can scavenge low-level androgens in an androgen-suppressed patient. Conclusions: The molecular model of T binding to the AR LBD suggests a degree of structural dynamism not evident in the crystallographic structures commonly associated with nuclear receptors. Some AR mutations activating prostate tumor growth may do so by impacting androgen entry/exit, rather than by altering androgen fit into the ligand binding pocket. Reference: (1) Guilbert C, James TL (2008) J Chem Inf Model. 2008 48(6): 1257-1268. doi: 10.1021/ci8000327

SUN-LB134 Androgen Receptor Phosphorylated at Serine 815 in Mouse and Human Prostates

Androgen receptor (AR) regulates male sexual development and maintenance. AR forms a homodimer in the cytoplasm and monomerizes following hormonal activation, translocating to the nucleus in Cos-1 cells (Shizu et al. Scientific reports. 2019). Utilizing Ser815 of AR, the conserved phosphorylation residue within the ligand binding domains of steroid hormone receptors (NR3C), whether and how this phosphorylation regulates AR functions was investigated. While, like AR WT, a phosphomimic AR S815D mutant formed a homodimer in the cytoplasm, unlike the WT, this mutant remained as a homodimer in the cytoplasm even after hormone treatment. Apparently, Ser815 phosphorylation disabled AR’s capability to monomerize and nuclear translocate in Cos-1 cells. A phospho-Ser815 peptide antibody was used to detect phosphorylation of endogenous AR in mouse as well as human prostates. Immunohistochemistry showed phosphorylation present in both the cytoplasm and nucleus. Mouse prostates were cell fractionated in cell membrane, mitochondria, endoplasmic reticulum (ER) and cytosolic fractions for subsequent Western blot analysis. While AR was found in all of these fractions, phosphorylated AR was only detected in the ER and cytosolic fractions. A cDNA microarray analysis of PC-3 cells with ectopic expression of AR S815D suggested that phosphorylated AR may regulate ER stress.

OR12-07 Full Antagonism of Breast Cancer Cell Proliferation Can Result from Many Ligand-Induced Conformational Distortions of the Estrogen Receptor Ligand Binding Domain

Although most estrogen receptor alpha (ERα)-positive breast cancers initially respond well to endocrine therapies using aromatase inhibitors (AIs) or antiestrogens, after varying time periods the cancer frequently recurs as metastatic disease. A significant fraction of these recurrences are driven by ERs that have acquired activating mutations in their ligand binding domains (LBDs), giving them constitutive activity and thus resistance to AIs. Because these mutations also reduce the affinity and potency of SERMs and SERDs, expanded efforts have been made to vary the structure of antiestrogens to make them more potent.Typical antiestrogens are comprised of a core element that binds securely in the ligand binding pocket and from which extends a single ring (ring E) having a side chain that sterically interferes with the position of helix-12 by direct antagonism, reorienting it so that it occludes the activation function 2 (AF2) hydrophobic groove for coactivator binding. Through structural studies, we found that bridged oxabicycloheptene-sulfonamide (OBHS-N) core ligands have two rings (E and F) that can be poised to engage in both “direct antagonism” and “indirect antagonism”, the latter of which disrupts the orientation of helix-12 by impinging on helix-11 and the helix-11–12 loop.In this study, we have placed typical antiestrogen side chains on either the E or the F ring of OBHS-N core ligands and characterized their activities in ERα-positive breast cancer cells. All compounds have full antiproliferative activity and reverse estrogen-regulated gene expression, with the antiproliferative potency of each type of side chain having a distinct preference for E- vs F-ring attachment. Conformational analysis using a multiplexed coregulator peptide interaction assay shows that compounds with an E-ring substitution have interaction profiles similar to 4-hydroxytamoxifen and fulvestrant, whereas the F-ring substitution gives a very different pattern, suggesting that the antagonist activity of the two classes rely on different sets of coregulator proteins. A large number of high resolution (better than 2 Å) X-ray crystal structures reveal that this set of novel ER antagonists disrupt the conformation of the ER LBD in a variety of ways, several of which are distinct from those seen with previous antiestrogens such as Tamoxifen and Fulvestrant.Our findings expand design concepts by which ERα ligands can block the activity of this receptor and illustrate how direct and indirect modes of ER antagonism can be combined to facilitate the development of more efficacious antiestrogens for breast cancer treatment and possibly for regulating ER-mediated activities in other estrogen target tissues.

The Structure-Function Relationship of Angular Estrogens and Estrogen Receptor Alpha to Initiate Estrogen-Induced Apoptosis in Breast Cancer Cells.

High-dose synthetic estrogen therapy was the standard treatment of advanced breast cancer for three decades until the discovery of tamoxifen. A range of substituted triphenylethylene synthetic estrogens and diethylstilbestrol were used. It is now known that low doses of estrogens can cause apoptosis in long-term estrogen deprived (LTED) breast cancer cells resistant to antiestrogens. This action of estrogen can explain the reduced breast cancer incidence in postmenopausal women over 60 who are taking conjugated equine estrogens and the beneficial effect of low-dose estrogen treatment of patients with acquired aromatase inhibitor resistance in clinical trials. To decipher the molecular mechanism of estrogens at the estrogen receptor (ER) complex by different types of estrogens—planar [17β-estradiol (E2)] and angular triphenylethylene (TPE) derivatives—we have synthesized a small series of compounds with either no substitutions on the TPE phenyl ring containing the antiestrogenic side chain of endoxifen or a free hydroxyl. In the first week of treatment with E2 the LTED cells undergo apoptosis completely. By contrast, the test TPE derivatives act as antiestrogens with a free para-hydroxyl on the phenyl ring that contains an antiestrogenic side chain in endoxifen. This inhibits early E2-induced apoptosis if a free hydroxyl is present. No substitution at the site occupied by the antiestrogenic side chain of endoxifen results in early apoptosis similar to planar E2. The TPE compounds recruit coregulators to the ER differentially and predictably, leading to delayed apoptosis in these cells.SIGNIFICANCE STATEMENTIn this paper we investigate the role of the structure-function relationship of a panel of synthetic triphenylethylene (TPE) derivatives and a novel mechanism of estrogen-induced cell death in breast cancer, which is now clinically relevant. Our study indicates that these TPE derivatives, depending on the positioning of the hydroxyl groups, induce various conformations of the estrogen receptor’s ligand-binding domain, which in turn produces differential recruitment of coregulators and subsequently different apoptotic effects on the antiestrogen-resistant breast cancer cells.

Effectiveness of the Qube in Studying the Rapidly-Desensitizing Alpha7 Nicotinic Acetylcholine Receptor

Alpha7 nicotinic acetylcholine receptor (α7 nAChR) is an integral part of the excitatory cholinergic nervous system and must be maintained in humans. α7 nAChRs serve as a primary immune response and are highly expressed in the nervous system. Moreover, the ion channel has a direct link to schizophrenia and Alzheimer's disease including several other classes of neurological ailments such as cognitive and memory impairment, therefore, making α7 a critical target of ion channel drug discovery. Several different subunits and combinations can comprise the pentameric ligand-gated ion channel. In the case of α7, five identical α7 homomeric subunits form a functional channel comprised of an extracellular ligand-binding domain, a transmembrane domain, containing the ion gate and the pore, and an intracellular regulatory domain. Binding of a single ligand is enough to activate the channel. α7 contain three major conformational states: closed (C), open (O), and desensitized (D). In the open state, the channel conducts Na+, K+, and Ca2+ ions with Ca2+ displaying highest permeability. Historically, the very rapid agonist-induced channel activation and desensitization make α7 a challenging target to study where the data are limited by the temporal resolution of the protocol and the proper solution exchange rate used to measure current. This contrasts with chimeras comprising the ligand binding domain of α7 receptors and the ion pore domain of the homomeric α1 glycine receptors, which desensitize to a lesser degree than homomeric α7 counterparts. Our results demonstrated the reliability of the Qube, our high-throughput 384-well automated patch-clamp (APC), in recording the α7 nAChR and α7-nAChR/GlyR chimeras in recombinantly expressed HEK293 cells with our microfluidic QChips that allow complete solution exchange coupled to customized rapid-stacked solution-exchange.

Amyloid Fibril Formation of α‐Synuclein Is Modulated via the Estrogen Receptor Ligand Binding Domain of Estrogen Receptor α Bound with Tamoxifen‐based Small Molecules

Alpha‐synuclein (αSyn) forms amyloid fibrils in the brain of patients suffering from Parkinson's disease. Aiming at the development of a new therapy via regulating amyloid fibril formation of αSyn, we studied estrogen receptor ligand‐binding domain (ERLBD) of estrogen receptor α (ΕRα). The ERLBD encoding gene was cloned, expressed by IPTG induction, and successfully purified by Ni2+‐affinity chromatography followed by anion‐exchange chromatography. In the thioflavin T (ThT) fluorescence assay, we examined the kinetics of amyloid fibril formation of αSyn in the presence of ERLBD protein. Dose‐dependent inhibition of amyloid fibril formation of αSyn by ERLBD protein was observed, indicating that ERLBD protein binds to αSyn, thereby blocking amyloid fibril formation. The kinetics of amyloid fibril formation of αSyn were compared in the absence and presence of a small molecule, such as 17β‐estradiol (E2, agonist) or tamoxifen (TMX, antagonist) of ΕRα. Regardless of the presence of E2, we found that ERLBD protein inhibits αSyn from forming amyloid fibrils. On the other hand, TMX reversed the inhibition effect of ERLBD protein on amyloid fibril formation of αSyn. Interestingly, the photo‐reactive hybrid ligand 2‐phenylquinoline‐4‐hydroxytamoxifen (PQ‐OHT) was expected to exhibit the anti‐inhibitory effect of ERLBD on amyloid fibril formation of αSyn. Upon illuminating with a UV lamp, PQ‐OHT was found to degrade ERLBD protein as well. These observations suggest the examination of the possibility that a complex of ERLBD‐αSyn amyloid fibril can be eliminated via the ligand PQ‐OHT upon exposure to light for therapeutic purposes.

Prevalence of ESR1 Mutation in Chinese ER-Positive Breast Cancer.

BackgroundESR1 mutation and its possible relation to endocrine therapy resistance in ER-positive breast cancers have been studied with respect to genetic sequencing data from Western patients but rarely from Chinese patients. This study aimed to investigate the prevalence of ESR1 mutation in Chinese primary and metastatic ER-positive breast cancer.MethodsTumor samples from 297 primary breast cancer (PBC) patients and blood samples from 43 metastatic breast cancer (MBC) patients were obtained to perform whole exon sequencing of the ESR1 gene through next-generation sequencing (NGS). Clinicopathological features of MBC patients were listed and grouped to explore potential factors in ESR1 mutations.ResultsA total of 15 ESR1 variations, including 11 point mutations, 1 in-frame deletion mutation, 1 synonymous mutation, and 2 amplifications were identified in 13 patients. The ESR1 mutation rate was 1% (3/297) in PBC patients and 18.6% (8/43) in MBC patients. All ESR1 point mutations occurred in the estrogen receptor ligand-binding domain. Six (54.5%) of the 11 point mutations were hotspot mutations. Among all MBC patients, the ESR1 mutation rate in those who had a treatment history using aromatase inhibitors (AI) was significantly higher than those who did not (25.8% versus 0%, P=0.015). Moreover, the ESR1 mutation rate in those who received AI treatment over a period of 12 months was significantly higher than in those whose treatment lasted less than 12 months [36.3% versus 0%, P<0.001].ConclusionESR1 mutations were more frequently observed in the circulating cell-free DNA of MBC patients than in PBC patients among the Chinese cohort, and higher among those pretreated with AI, suggesting that such mutations may undergo selection during AI treatment.

Atomistic simulations shed new light on the activation mechanisms of ROR\u03b3 and classify it as Type III nuclear hormone receptor regarding ligand-binding paths

The molecular recognition of the RORγ nuclear hormone receptor (NHR) ligand-binding domain (LBD) has been extensively studied with numerous X-ray crystal structures. However, the picture afforded by these complexes is static and does not fully explain the functional behavior of the LBD. In particular, the apo structure of the LBD seems to be in a fully active state, with no obvious differences to the agonist-bound structure. Further, several atypical in vivo inverse agonists have surprisingly been found to co-crystallize with the LBD in agonist mode (with co-activator), leading to a disconnection between molecular recognition and functional activity. Moreover, the experimental structures give no clues on how RORγ LBD binders access the interior of the LBD. To address all these points, we probe here, with a variety of simulation techniques, the fine structural balance of the RORγ LBD in its apo vs. holo form, the differences in flexibility and stability of the LBD in complex with agonists vs. inverse agonists and how binders diffuse in and out of the LBD in unbiased simulations. Our data conclusively point to the stability afforded by the so-called “agonist lock” between H479 and Y502 and the precise location of Helix 12 (H12) for the competence of the LBD to bind co-activator proteins. We observe the “water trapping” mechanism suggested previously for the atypical inverse agonists and discover a different behavior for the latter when co-activator is present or absent, which might help explain their conflicting data. Additionally, we unveil the same entry/exit path for agonists and inverse agonist into and out of the LBD for RORγ, suggesting it belongs to the type III NHR sub-family.

Virtual Prediction of Thymoquinone and β-Glucan Interaction as Estrogen Receptor-Alpha (ER-α receptor) May Reducing on The Breast Cancer Signaling Pathway

Many cases of chemical drug treatment in breast cancer patients result in a negative impact on drug resistance. Therefore the use of natural compounds such as β-glucan on ajwa dates and thymoquinone on black cumin was expected to stop the process of cancer cell proliferation and to consume in long-term safety. This study aimed to predict the effect of thymoquinone and β-glucan in reducing breast cancer cascade with molecular docking. This research was carried out in silico . The ligand and protein preparations were done using Discovery Studio 2016. We used Hex 8.0.0 for docking. Visualization was established using Discovery Studio 2016 as well. The results showed that thymoquinone and β-glucan can undergo conformational changes in ligand binding domain (LBD) of ER-α receptor. The interaction between ER-α receptor with β-estradiol (inhibited by thymoquinone-β-glucan) was suggested to be the best solution in downregulating breast cancer signaling pathway. This interaction showed more stable conformation with the smallest binding energy (-332,84 kcal/mol). The thymoquinone-β-glucan complex could block the His476 and Met438.

Molecular, chemical, and structural characterization of prostaglandin A2 as a novel agonist for Nur77.

Nur77 is a transcription factor belonging to the NR4A subfamily of nuclear hormone receptors. Upon induction, Nur77 modulates the expression of its target genes and controls a variety of biological and pathophysiological processes. Prior research that revealed a structurally atypical ligand-binding domain (LBD) and failed to locate an endogenous ligand had led to a classification of Nur77 as an orphan receptor. However, several more recent studies indicate that small synthetic molecules and unsaturated fatty acids can bind to Nur77. Discovery of additional endogenous ligands will facilitate our understanding of the receptor's functions and regulatory mechanisms. Our data have identified prostaglandin A2 (PGA2), a cyclopentenone prostaglandin (PG), as such a ligand. Cyclopentenone PGs exert their biological effects primarily by forming protein adducts via the characteristic electrophilic β-carbon(s) located in their cyclopentenone rings. Our datashow that PGA2 induces Nur77 transcriptional activity by forming a covalent adduct between its endocyclic β-carbon, C9, and Cys566 in the receptor's LBD. The importance of this endocyclic β-carbon was substantiated by the failure of PGs without such electrophilic properties to react with Nur77. Calculated chemical properties and data from reactive molecular dynamic simulations, intrinsic reaction co-ordinate modeling, and covalent molecular docking also corroborate the selectivity of PGA2's C9 β-carbon towards Nur77's Cys. In summary, our molecular, chemical, and structural characterization of the PGA2-Nur77 interaction provides the first evidence that PGA2 is an endogenous Nur77 agonist.

An AND-Gated Drug and Photoactivatable Cre-loxP System for Spatiotemporal Control in Cell-Based Therapeutics.

While engineered chimeric antigen receptor (CAR) T cells have shown promise in detecting and eradicating cancer cells within patients, it remains difficult to identify a set of truly cancer-specific CAR-targeting cell surface antigens to prevent potentially fatal on-target off-tumor toxicity against other healthy tissues within the body. To help address this issue, we present a novel tamoxifen-gated photoactivatable split-Cre recombinase optogenetic system, called TamPA-Cre, that features high spatiotemporal control to limit CAR T cell activity to the tumor site. We created and optimized a novel genetic AND gate switch by integrating the features of tamoxifen-dependent nuclear localization and blue-light-inducible heterodimerization of Magnet protein domains (nMag, pMag) into split Cre recombinase. By fusing the cytosol-localizing mutant estrogen receptor ligand binding domain (ERT2) to the N-terminal half of split Cre(2-59aa)-nMag, the TamPA-Cre protein ERT2-CreN-nMag is physically separated from its nuclear-localized binding partner, NLS-pMag-CreC(60-343aa). Without tamoxifen to drive nuclear localization of ERT2-CreN-nMag, the typically high background of the photoactivation system was significantly suppressed. Upon blue light stimulation following tamoxifen treatment, the TamPA-Cre system exhibits sensitivity to low intensity, short durations of blue light exposure to induce robust Cre-loxP recombination efficiency. We finally demonstrate that this TamPA-Cre system can be applied to specifically control localized CAR expression and subsequently T cell activation. As such, we posit that CAR T cell activity can be confined to a solid tumor site by applying an external stimulus, with high precision of control in both space and time, such as light.

Revealing a Mutant-Induced Receptor Allosteric Mechanism for the Thyroid Hormone Resistance.

SummaryResistance to thyroid hormone (RTH) is a clinical disorder without specific and effective therapeutic strategy, partly due to the lack of structural mechanisms for the defective ligand binding by mutated thyroid hormone receptors (THRs). We herein uncovered the prescription drug roxadustat as a novel THRβ-selective ligand with therapeutic potentials in treating RTH, thereby providing a small molecule tool enabling the first probe into the structural mechanisms of RTH. Despite a wide distribution of the receptor mutation sites, different THRβ mutants induce allosteric conformational modulation on the same His435 residue, which disrupts a critical hydrogen bond required for the binding of thyroid hormones. Interestingly, roxadustat retains hydrophobic interactions with THRβ via its unique phenyl extension, enabling the rescue of the activity of the THRβ mutants. Our study thus reveals a critical receptor allosterism mechanism for RTH by mutant THRβ, providing a new and viable therapeutic strategy for the treatment of RTH.

875P - Multicenter phase I trial of a DNA vaccine encoding the androgen receptor ligand binding domain (pTVG-AR, MVI-118) in patients with metastatic prostate cancer

BackgroundPreclinical studies have demonstrated that a DNA vaccine encoding the androgen receptor ligand-binding domain (AR LBD) can elicit CD8+ T cells specific for the AR LBD, delay disease progression and prolong survival of prostate tumor-bearing animals. Vaccination of tumor-bearing mice treated with androgen deprivation (AD) led to a delay in the emergence of castration-resistant disease. This same DNA vaccine (pTVG-AR, MVI-118) was evaluated in a phase I clinical trial in men with metastatic prostate cancer (mPC) on AD therapy. Methods40 patients with mPC, within 6 months of beginning standard AD therapy, were randomly assigned to receive pTVG-AR on one of two different treatment schedules, and with or without GM-CSF used as an adjuvant. Ten total vaccinations were given over 1 year, and patients were followed for 18 months. The primary objectives were safety and immune activity. Secondary objectives were to evaluate different schedules, the requirement for GM-CSF, and whether treatment was associated with PSA progression-free survival (PSA PFS). ResultsBetween 2015 and 2017, 40 patients were enrolled at 3 centers. 27 patients completed treatment and 18 months of follow-up. Eleven patients (28%) had a PSA progression event. No grade 3 or 4 adverse events were observed. Samples from 30 patients were available for immune analysis. 14/30 (47%) developed Th1 biased immunity to the AR LBD as determined by IFNγ or granzyme B ELISPOT that was detectable at least twice after beginning treatment. Differences in immune responses were observed with different schedules, but not with GM-CSF adjuvant. Patients who developed immunity had a significantly prolonged PSA PFS compared to patients without immunity (HR=0.18, 95% CI: 0.04-0.92, p=0.040). ConclusionsThis trial showed that pTVG-AR was safe and immunologically active in patients with mPC, and identified a preferred schedule of administration. Association between immunity and PSA PFS suggests treatment may delay the time to castration resistance, similar to observations in preclinical studies. Future studies will evaluate pTVG-AR in earlier stages of disease, and in combination with other agents. Clinical trial identificationNCT02411786. Legal entity responsible for the studyDouglas McNeel. FundingMadison Vaccines, Inc. DisclosureD.G. McNeel: Advisory / Consultancy, Research grant / Funding (self), Travel / Accommodation / Expenses, Shareholder / Stockholder / Stock options, Licensing / Royalties, Officer / Board of Directors: Madison Vaccines Inc; Research grant / Funding (institution): BMS; Research grant / Funding (institution): Janssen; Research grant / Funding (institution): Novartis; Research grant / Funding (institution): Merck. R. Lesniewski: Leadership role, Travel / Accommodation / Expenses, Shareholder / Stockholder / Stock options, Full / Part-time employment, Officer / Board of Directors: Madison Vaccines Inc. B. Olson: Licensing / Royalties: Madison Vaccines Inc. C. Kyriakopoulos: Research grant / Funding (institution): Madison Vaccines Inc. All other authors have declared no conflicts of interest.

&lt;i&gt;N&lt;/i&gt;-Glycans on the Ligand Binding Domain of NMDA-type Glutamate Receptor Interact with Nearby Peptides and Glycans

&lt;i&gt;N&lt;/i&gt;-Glycans on the Ligand Binding Domain of NMDA-type Glutamate Receptor Interact with Nearby Peptides and Glycans

&lt;i&gt;N&lt;/i&gt;-Glycans on the Ligand Binding Domain of NMDA-type Glutamate Receptor Interact with Nearby Peptides and Glycans

&lt;i&gt;N&lt;/i&gt;-Glycans on the Ligand Binding Domain of NMDA-type Glutamate Receptor Interact with Nearby Peptides and Glycans

Stronger estrogenic and antiandrogenic effects on zebrafish larvae displayed by 6:2 polyfluoroalkyl phosphate diester than the 8:2 congener at environmentally relevant concentrations

Polyfluoroalkyl phosphate esters (PAPs) are one kind of emerging polyfluoroalkyl substances in the environment. However, their in vivo toxicities are largely unknown, especially at environmental relevant concentrations. To fill this gap, zebrafish embryos were exposed to 6:2 or 8:2 diPAP at environmentally relevant concentrations (0.5, 5, 50 ng/L) for 7 d. 6:2 and 8:2 diPAPs upregulated the mRNA and protein levels of aromatase in the exposed larvae, and elevated estradiol (E2) and vitellogenin (VTG) levels, but reduced testosterone (T) and 11-ketotestosterone (11-KT) levels, demonstrating estrogenic and antiandrogenic effects. Among the three ER subtypes, ERβ2 displayed the highest in vivo mRNA expression and the lowest in silico binding energies, suggesting that it was the main target ER subtype responsible for the estrogenic effect. Molecular simulation results indicated that diPAPs and E2 could bind to one common residue, arginine (Arg) 87, in the binding pocket of ERβ2, inducing similar estrogenic disruption mechanisms as E2. Both compounds could form hydrophobic interaction with glutamic acid (Glu) 12 and tryptophan (Trp) 80 and two hydrogen bonds with Arg81 of androgen receptor (AR) ligand-binding domains (LBDs) in antagonistic mode, resulting in a reduced level of AR upon exposure. The in silico binding energies of 6:2 diPAP with both ER and AR were lower than 8:2 diPAP, explaining the observed greater in vivo estrogenic and antiandrogenic activities of 6:2 diPAP. This study provided the first line of evidences that diPAPs could display adverse effects on the endocrine functions of fish species.

Deciphering the structural basis for glucocorticoid resistance caused by missense mutations in the ligand binding domain of glucocorticoid receptor

The glucocorticoid resistance hereditary condition may emerge from the occurrence of point mutations in the glucocorticoid receptor (GR), which could impair its functionality. Because the main feature of such pathology is the resistance of the hypothalamic-pituitary-adrenal axis to the hormone cortisol, we used the GR ligand binding domain three-dimensional structure to perform computational analysis for eight variants known to cause this clinical condition (I559 N, V571A, D641V, G679S, F737L, I747 M, L753F and L773P), aiming to understand, on the atom scale, how they cause glucocorticoid resistance. We observed that the mutations generated a reduced affinity to cortisol and they alter some loop conformations, which could be a consequence from changes in protein motion, which in turn could result from the reduced stability of mutant GR structures. Therefore, the analyzed mutations compromise the GR ligand binding domain structure and cortisol binding, which could characterize the glucocorticoid resistance phenotype.

Modeling the binding of diverse ligands within the Ah receptor ligand binding domain

The Ah receptor (AhR) is a ligand-dependent transcription factor belonging to the basic helix-loop-helix Per-Arnt-Sim (bHLH-PAS) superfamily. Binding to and activation of the AhR by a variety of chemicals results in the induction of expression of diverse genes and production of a broad spectrum of biological and toxic effects. The AhR also plays important roles in several physiological responses, which has led it to become a novel target for the development of therapeutic drugs. Differences in the interactions of various ligands within the AhR ligand binding domain (LBD) may contribute to differential modulation of AhR functionality. We combined computational and experimental analyses to investigate the binding modes of a group of chemicals representative of major classes of AhR ligands. On the basis of a novel computational approach for molecular docking to the homology model of the AhR LBD that includes the receptor flexibility, we predicted specific residues within the AhR binding cavity that play a critical role in binding of three distinct groups of chemicals. The prediction was validated by site-directed mutagenesis and evaluation of the relative ligand binding affinities for the mutant AhRs. These results provide an avenue for understanding ligand modulation of the AhR functionality and for rational drug design.

Comparative in Vitro and in Vivo Evaluation of the Estrogenic Effect of Hexafluoropropylene Oxide Homologues.

As alternatives to perfluorooctanoic acid (PFOA), hexafluoropropylene oxide (HFPO) homologues, including hexafluoropropylene oxide dimer acid (HFPO-DA), hexafluoropropylene oxide trimer acid (HFPO-TA), and hexafluoropropylene oxide tetramer acid (HFPO-TeA), have been used in the fluoropolymer industry for a long period of time. These compounds have attracted widespread attention in recent years due to their environmental ubiquity and high bioaccumulation capability, as well as their toxicity. In our study, we evaluated the potential estrogenic effects of HFPOs in comparison to PFOA by ligand binding, transcriptional activity, and in vivo assays. Fluorescence ligand binding assays showed that both HFPO-TA and HFPO-TeA exhibited higher binding affinity to estrogen receptor ligand binding domains (ER-LBDs) than PFOA, with 2.5- and 57.5-fold higher affinity to ERα-LBD and 2.6- and 41.8-fold higher affinity to ERβ-LBD, respectively, whereas HFPO-DA exhibited weaker binding affinity than PFOA. Unlike PFOA, HFPO-TA and HFPO-TeA exhibited antagonistic activity toward the ERs' signaling pathway, with HFPO-TeA displaying the strongest potency. In silico study revealed that while PFOA binds with ERs in a similar fashion as 17β-estradiol, the HFPOs display an antagonistic binding mode. Using a zebrafish model, we further found that exposure to HFPO homologues significantly altered the levels of sex steroid hormones and vitellogenin. In general, both in vivo and in vitro results indicate that HFPO homologues might exert higher estrogenic effects than PFOA.