Receptor Ligand Binding Domain Research Articles

Molecular Imaging of Retinoic Acids in Live Cells Using Single-Chain Bioluminescence Probes.

Retinoic acid (RA) is a key metabolite necessary for embryonic development and differentiation in vertebrates. We demonstrate the utility of genetically encoded, ligand-activatable single-chain bioluminescence probes for detecting RAs from different biological sources. We examined 13 different molecular designs to identify an efficient single-chain probe that can quantify RA with significant sensitivity. The optimal probe consisted of four components: the N- and C-terminal fragments of artificial luciferase variant-16 (ALuc16), the ligand binding domain of retinoic acid receptor α (RARα LBD), and an LXXLL interaction motif. This probe showed a 5.2-fold greater bioluminescence intensity in response to RA when compared to the vehicle control in live mammalian cells. The probe was highly selective to all-trans-RA (at-RA), and highly sensitive in determining at-RA levels in cells derived from tumor xenografts created using MDA-MB-231 cells engineered to stably express the probe. We also detected RA levels in serum and cerebrospinal fluid. Using this probe, the detection limit for at-RA was ∼10-9.5 M, with a linear range of two orders. We present a highly useful technique to quantitatively image endogenous at-RA levels in live mammalian cells expressing novel single-chain bioluminescence probes.

Novel Pyrimidine Derivatives from 2,5-Dichloro-3-acetylthienyl Chalcones as Antifungal, Antitubercular and Cytotoxic Agents: Design, Synthesis, Biological Activity and Docking Study

Twenty novel pyrimidine derivatives were synthesized from 2,5-dichloro-3-acetylthienyl chalcones by reacting with guanidine HCl in presence of KOH and ethanol under reflux for 6 h. Their structural characterizations were evaluated by ATR-FTIR, 1H NMR, 13C NMR, mass spectroscopy. They were also screened for antifungal, antitubercular and cytotoxicity activities. They were displayed good antifungal activity (MIC = 32-125 μg/mL) against Aspergillus niger and Candida tropicalis fungal species except compound 15 with 4"-pyridinyl moiety (MIC = 8.00 μg/mL) being more potent. Compound 5 with 2",4"-dichlorophenyl moiety was shown with good antitubercular activity (MIC = 6.2 μg/mL) against Mycobacterium tuberculosis H37Rv (MTB) stain. They have also tested for in vitro cytotoxicity activity against DU-145 prostate cancer cell lines. In which the compound 15 with 4"-pyridinyl moiety (IC50 = 2.0 ± 0.1 μg/mL) and compound 17 with 2"-pyrrolyl moiety (IC50 = 6.0 ± 0.1 μg/mL) possess highly potent antiprostate cancer properties. The molecular docking was done with the crystalline structure of mitochondrial 2-enoyl thioester reductase Etr1p/Etr2p heterodimer from Candida tropicalis fungal species with compound 15 (-7.80 kcal/mol) and shown greater binding affinity than fluconazole (-7.60 kcal/mol). Docking was performed with protein crystalline structure (PDB ID: 2WEE) of Mycobacterium tuberculosis H37Rv (MTB) stain and among all, compound 5 was exhibited good binding affinity (-6.90 kcal/mol), compared to pyrazinamide (-4.10 kcal/mol). The protein crystalline structure of a mutant androgen receptor (AR) ligand-binding domain (LBD) (PDB file: 1GS4) was tested with compounds 15 and 17 (-7.60 and -8.20 kcal/mol). They were exhibited good binding properties compared to methotrexate (-5.10 kcal/mol). Hence, these novel pyrimidine compounds are as lead compounds as antifungal, antitubercular and cytotoxic agents.

The first identification of complete Eph-ephrin signalling in ctenophores and sponges reveals a role for neofunctionalization in the emergence of signalling domains

BackgroundAnimals have a greater diversity of signalling pathways than their unicellular relatives, consistent with the evolution and expansion of these pathways occurring in parallel with the origin of animal multicellularity. However, the genomes of sponges and ctenophores – non-bilaterian basal animals – typically encode no, or far fewer, recognisable signalling ligands compared to bilaterians and cnidarians. For instance, the largest subclass of receptor tyrosine kinases (RTKs) in bilaterians, the Eph receptors (Ephs), are present in sponges and ctenophores, but their cognate ligands, the ephrins, have not yet been detected.ResultsHere, we use an iterative HMM analysis to identify for the first time membrane-bound ephrins in sponges and ctenophores. We also expand the number of Eph-receptor subtypes identified in these animals and in cnidarians. Both sequence and structural analyses are consistent with the Eph ligand binding domain (LBD) and the ephrin receptor binding domain (RBD) having evolved via the co-option of ancient galactose-binding (discoidin-domain)-like and monodomain cupredoxin domains, respectively. Although we did not detect a complete Eph-ephrin signalling pathway in closely-related unicellular holozoans or in other non-metazoan eukaryotes, truncated proteins with Eph receptor LBDs and ephrin RBDs are present in some choanoflagellates. Together, these results indicate that Eph-ephrin signalling was present in the last common ancestor of extant metazoans, and perhaps even in the last common ancestor of animals and choanoflagellates. Either scenario pushes the origin of Eph-ephrin signalling back much earlier than previously reported.ConclusionsWe propose that the Eph-LBD and ephrin-RBD, which were ancestrally localised in the cytosol, became linked to the extracellular parts of two cell surface proteins before the divergence of sponges and ctenophores from the rest of the animal kingdom. The ephrin-RBD lost the ancestral capacity to bind copper, and the Eph-LBD became linked to an ancient RTK. The identification of divergent ephrin ligands in sponges and ctenophores suggests that these ligands evolve faster than their cognate receptors. As this may be a general phenomena, we propose that the sequence-structure approach used in this study may be usefully applied to other signalling systems where no, or a small number of, ligands have been identified.

Structural and Dynamics of Glucocorticoid Receptor Ligand Binding Domain in Complex with PGC1α and Synthetic Glucocorticoids

Synthetic glucocorticoids are important pharmaceutical drugs that are known to bind to the ligand binding domain (LBD) of glucocorticoid receptor (GR), a member of the nuclear receptor superfamily. These binding will induce conformational changes within GR for the subsequent DNA binding and different co‐regulators recruitments, ultimately lead to activate or repress target gene expression. One of the crucial co‐regulators is peroxisome proliferator‐activated gamma coactivator 1‐α (PGC1α), known to regulates energy metabolism by direct interacting with GR to modulate gene expression. However, the mechanisms through which PGC1α changes GR conformation to drive transcription are unknown. Here, an ancestral variant of GR (GR2) was utilized as a tool to produce stable protein for biochemical and structural studies. We report the first high resolution X‐ray crystal structures of GR2 LBD in complexes with PGC1α and dexamethasone and hydrocortisone. These structural analyses reveal how distinct steroid drugs bind to GR with different affinities via unique hydrogen bonding and hydrophobic interactions. Hydrogen‐deuterium exchange mass spectrometry showed different effects on GR2 LBD allosteric communication after glucocorticoids binding. Molecular dynamics simulations and network analysis identified the key residues involved in the intramolecular communication between ligand binding site and activation function surface, which is the PGC1α binding site.Support or Funding InformationThis work was supported by W.M. Keck Foundation Medical Research Grant to E. A. O. X. L. was supported by an American Heart Association postdoctoral fellowship [17POST33660110].This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Effect of Structural Diversity on Ligand Specificity and Resulting Signaling Differences of Estrogen Receptor α.

Numerous chemicals have been reported to exert estrogen-like endocrine disrupting effects via a receptor binding mechanism that directly interacts with the ligand binding domain of estrogen receptor α (ERα). However, not only their binding affinities to ERα but also their interference in specific cell and tissue functions are clearly different. In this regard, significant regulation differences among three representative estrogenic chemicals (diethylstilbestrol (DES), bisphenol A (BPA), and diarylpropionitrile (DPN)), well-known ERα agonists with very similar structures, have been observed. Molecular dynamics simulation is used to explore the underlying mechanism of different regulation effects induced by the similar estrogen-like chemicals. The DES-induced 12 Å motion of the H9-H10 loop markedly expands the negative electrostatic potential surface of the AF-2 domain, which is consistent with the over-regulation effect of the agonist. In comparison, the 3 Å motion induced by BPA and DPN corresponds to the low-regulation effect of the chemicals. Cross-correlation analysis indicates that the different ERα motions and resulting surface feature of AF-2 domain are brought by the distinguished binding modes of the agonists. Moreover, only hydrophobic DES with estrogen-like size and flexibility has a high binding affinity of -23.47 kcal/mol binding free energy. Both the hydrophilic group in DPN and the small molecular size of BPA dramatically decrease the agonist binding ability, and their binding free energies are only -12.43 kcal/mol and -11.82 kcal/mol, respectively. Our study demonstrates that similar chemicals interact differently with ERα and induce different allosteric effects, which explains the observed regulation diversity.

Comparison of the three SARMs RAD-140, GLPG0492 and GSK-2881078 in two different in vitro bioassays, and in an in silico androgen receptor binding assay

Selective androgen receptor modulators comprise compounds that bind as ligands to the androgen receptor and possess tissue-selective activities. Ideally, they show agonistic properties in anabolic target tissues, while inducing antagonistic or only weak agonistic effects in reproductive organs. Due to their myoanabolic effects, selective androgen receptor modulators are included in the list of prohibited substances and methods of the World Anti-Doping Agency.In the current investigation, the androgenic potential of RAD-140, GSK-2881078 and GLPG0492 was comparably investigated in two different in vitro bioassays. In the yeast androgen screen, the androgenic effects were lower than in the reporter gene assay in prostate carcinoma cells (e.g. for GSK-2881078, the EC50 values were 4.44 × 10−6M in the yeast screen and 3.99 × 10-9M in the prostate cells respectively). For future investigations, it is of importance whether the yeast androgen screen, which has been proven to detect androgenic compounds in urine, can detect an abuse of the selective androgen receptor modulators. Molecular modeling of the binding to the androgen receptor ligand binding domain suggests slight differences in the binding modes of RAD-140, GSK-2881078 and GLPG0492.In conclusion, androgenic activity of the three non-steroidal compounds in the two different in vitro test systems confirmed the results of the in silico modeling of the androgen receptor binding.

Abstract ES10-1: Recurrent ESR1 mutations activating mutations in breast cancer

Abstract Endocrine treatments are the cornerstone treatments in estrogen receptor positive (ER+) breast cancer. Despite their efficacy, resistance to endocrine treatments remains an important clinical challenge. A number of years ago, recurrent estrogen receptor ligand binding domain (LBD) mutations were identified as the most common genomic mechanism of acquired resistance to endocrine treatment in metastatic breast cancer. These mutations were found in about 20% of ER+ metastatic tissue specimens and in more than 30% of patients with metastatic ER+ breast cancer when testing circulating tumor DNA. Pre-clinical studies showed that these mutations lead to constitutional ligand independent transcriptional activity and tumor growth. Recently, we showed that the ER mutations have neomorphic properties that promote metastases. Clinical data, derived mainly from retrospective analyses of clinical trials, indicate that the ER mutations confer resistance to aromatase inhibitors and fulvestrant, and decreased overall survival. A recent study also showed that the Y537S LBD ER mutation is acquired during fulvestrant and palbociclib treatment. Collectively, the ER mutations are important drivers of resistance standard endocrine treatments and confer worse outcomes in ER+ metastatic breast cancer. Clinical trials stratifying patients by ER mutant status or dedicated to patients with the ER mutations are needed to identify therapeutic strategies to target these mutations. Citation Format: Jeselsohn R. Recurrent ESR1 mutations activating mutations in breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr ES10-1.

Discovery of deshydroxy bicalutamide derivatives as androgen receptor antagonists

Deshydroxy propioanilides were synthesised by Michael addition reaction between substituted thiophenols onto four different phenylacrylamide derivatives to give twenty-three novel deshydroxy bicalutamide derivatives lacking the central hydroxyl group. The antiproliferative activities of these compounds were evaluated against human prostate cancer cell lines and thirteen compounds showed better inhibitory activities (IC50 = 2.67–13.19 μM) compared to bicalutamide (IC50 = 20.44 μM) in LNCaP. Remarkably, novel double branched bicalutamide analogues (27 and 28) were isolated as major by-products and found to have the best activity across three human prostate cancer cell lines (LNCaP, VCaP and PC3). The most active compound 28 shows sub-micromolar activity (IC50 = 0.43 μM in LNCaP), which represents more than 40-fold improvement over the clinical anti-androgen bicalutamide (IC50 = 20.44 μM) and a more than 3 fold improvement over enzalutamide (IC50 = 1.36 μM). Moreover, strong reduction of PSA expression in LNCaP cells upon treatment with compounds 27, 28 and 33 was observed during qPCR analysis, confirming their AR antagonist activity. Molecular modelling studies revealed a novel binding mode of these structurally distinct double branched analogues within the ligand binding domain (LBD) of the androgen receptor.

Mechanism of AMPA Receptor Modulation by Gamma-8

Fast excitatory neurotransmission is mediated by ionotropic glutamate receptor (iGluR). AMPA-subtype ionotropic glutamate receptors (AMPARs) are associated with auxiliary subunits (transmembrane AMPA receptor regulatory proteins (TARPs), cornichons, cysteine knot proteins and GSG1); which regulate AMPAR assembly, trafficking, gating, and pharmacology. Of particular interest is the interactions with gamma-8 which results in re-sensitization of the currents where the transmembrane channel in the receptor initially undergoes closure due to desensitization but reopens at longer time and remain open in the continued presence of agonist. Here we have investigated the mechanism underlying this resensitization process using single molecule FRET and single channel functional recordings. Specifically we have determined changes at the AMPA receptor ligand binding domain dimer interface in presence of gamma-8 and compared it to the changes at this site observed in the presence of cyclothiazide, a drug that stabilizes the open channel state of the receptor. These studies show that while the overall states populated are similar in the two cases, the kinetics of transitions between states are different, with activation barrier between transitions being higher in the presence of gamma-8 . The single channel current recordings reflected these slower kinetics in the presence of gamma-8 and also showed very rapid channel openings and closing which were no reflected at the dimer interface suggesting that these may be localized changes at the transmembrane segments.

Detecting the Ligand-binding Domain Dimerization Activity of Estrogen Receptor Alpha Using the Mammalian Two-Hybrid Assay.

Estrogen receptor alpha (ERα) is an estrogenic ligand-dependent transcription regulator. The sequence of ERα protein is highly conserved among species. It has been thought that the function of human and mouse ERαs is identical. We demonstrate the differential 4-hydroxy-tamoxifen (4OHT) effect on mouse and human ERα ligand-binding domain (LBD) dimerization activity using the mammalian two-hybrid (M2H) assay. The M2H assay can demonstrate the efficiency of LBD homodimerization activity in mammalian cells, utilizing the transfection of two protein expression plasmids (GAL4 DNA-binding domain [DBD] fusion LBD and VP16 transactivation domain [VP16AD] fusion LBD) and a GAL4-responsive element (GAL4RE) fused luciferase reporter plasmid. When the GAL4DBD fusion LBD and the VP16AD fusion LBD make a dimer in the cells, this protein complex binds to the GAL4RE and, then, activates a luciferase gene expression through the VP16AD dependent transcription activity. The 4OHT-mediated luciferase activation is higher in the HepG2 cells that were transfected with the mouse ERα LBD fusion protein expression plasmids than in the human ERα LBD fusion protein expression plasmid transfected cells. This result suggests that the efficacy of the 4OHT-dependent mouse ERα LBD homodimerization activity is higher than human ERα LBD. In general, the utilization of the M2H assay is not ideal for the evaluation of nuclear receptor LBD dimerization activity, because agonistic ligands enhance the basal level of the LBD activity and that impedes the detection of LBD dimerization activity. We found that 4OHT does not enhance ERα LBD basal activity. That is a key factor for being able to determine and detect the 4OHT-dependent LBD dimerization activity for successfully using the M2H assay. ERα LBD-based M2H assays may be applied to study the partial agonist activity of selective estrogen receptor modulators (e.g., 4OHT) in various mammalian cell types.

In silico Molecular Modelling of Selected Natural Ligands and their Binding Features with Estrogen Receptor Alpha.

Breast cancer is one of the most common cancers diagnosed among women. It is now recognized that two receptors mediate estrogen action and the presence of estrogen receptor alpha (ERα) correlates with better prognosis and the likelihood of response to hormonal therapy. ERα is an attractive target for the treatment of breast cancer. Most of the drugs currently used for the breast cancer treatment have numerous side effects and they are often unsuccessful in removing the tumour completely. Hence, we focused on natural compounds like flavonoids, polyphenols, etc. which do not exhibit any high toxic effects against normal cells. To identify the potential natural inhibitors for BCa through an optimised in silico approach. Structural modification and molecular docking-based screening approaches were imposed to identify the novel natural compounds by using Schrödinger (Maestro 9.5). The Qikprop v3.5 was used for the evaluation of important ADME parameters and its permissible ranges. Cytotoxicity of the compounds was evaluated by MTT assay against MCF-7 Cell lines. From the docking studies, we found that the compounds, Myricetin, Quercetin, Apigenin, Luteolin and Baicalein showed the highest Glide Scores -10.78, -9.48, -8.92, -8.87 and -8.82 kcal mol-1 respectively. Of these, Luteolin and Baicalein showed the significant IC50 values (25 ± 4.0 and 58.3 ± 4.4 µM, respectively) against MCF-7 cell line. The ADME profiling of the test compounds was evaluated to find the drug-likeness and pharmacokinetic parameters. We mainly focused on in silico study to dock the compounds into the human estrogen receptor ligand binding domain (hERLBD) and compare their predicted binding affinity with known antiestrogens. Myricetin, Quercetin, Apigenin, Luteolin and Baicalein were identified as the most promising among all. Of these, Luteolin and Baicalein showed significant anticancer activities against MCF-7 cell line. These findings may provide basic information for the development of anti-breast cancer agents.

Activation of Hes1 and Msx1 in Transgenic Mouse Embryonic Stem Cells Increases Differentiation into Neural Crest Derivatives.

The neural crest (NC) comprises a multipotent cell population that produces peripheral neurons, cartilage, and smooth muscle cells, among other phenotypes. The participation of Hes1 and Msx1 when expressed in mouse embryonic stem cells (mESCs) undergoing NC differentiation is unexplored. In this work, we generated stable mESCs transfected with constructs encoding chimeric proteins in which the ligand binding domain of glucocorticoid receptor (GR), which is translocated to the nucleus by dexamethasone addition, is fused to either Hes1 (HGR) or Msx1 (MGR), as well as double-transgenic cells (HGR+MGR). These lines continued to express pluripotency markers. Upon NC differentiation, all lines exhibited significantly decreased Sox2 expression and upregulated Sox9, Snai1, and Msx1 expression, indicating NC commitment. Dexamethasone was added to induce nuclear translocation of the chimeric proteins. We found that Collagen IIa transcripts were increased in MGR cells, whereas coactivation of HGR+MGR caused a significant increase in Smooth muscle actin (α-Sma) transcripts. Immunostaining showed that activation in HGR+MGR cells induced higher proportions of β-TUBULIN III+, α-SMA+ and COL2A1+ cells. These findings indicate that nuclear translocation of MSX-1, alone or in combination with HES-1, produce chondrocyte-like cells, and simultaneous activation of HES-1 and MSX-1 increases the generation of smooth muscle and neuronal cells.

Ligand binding domain interface: A tipping point for pharmacological agents binding with GluN1/2A subunit containing NMDA receptors

N-methyl D-aspartate (NMDA) receptors play a crucial role in normal brain function, pathogenesis of neurodegenerative and psychiatric disorders. Functional tetra-heteromeric NMDA receptor contains two obligatory GluN1 subunits and two identical or different non-GluN1 subunits that evolve from six different genes including four GluN2 (A-D) and two GluN3 (A-B) subunits. Since NMDA receptors confer varied physiological properties and spatiotemporal distributions in the brain, pharmacological agents that target NMDA receptors with specific GluN2 subunits have significant potential for therapeutic applications. In the present work, by using electrophysiology techniques, we have studied the role of ligand binding domain (LBD) interactions in determining the effect of well-characterized pharmacological agents including agonists, competitive antagonists, channel blockers and an allosteric modulator. Remarkably, point mutations at the distal end (site-II&III) of GluN1 LBD interface increased memantine potency up to sevenfold when co-expressed with wild type GluN2A receptors but exhibit no effect on Mg2+ activity. Conversely, mutations at the proximal end (site-I) of the LBD interface did not affect the memantine but altered Zn2+ and Mg2+ potency towards opposite directions. These results indicate that GluN1/2A LBD interface interactions play a key role in determining channel function. Further, subtle changes in LBD interaction can be readily translated to the downstream extracellular vestibule of channel pore to adopt a conformation that may affect memantine, Zn2+ and Mg2+ binding. Further studies on NMDA receptor LBD to transmembrane domain signal propagation mechanisms will help develop GluN2 subunit selective biomolecules that can be used for the treatment of neurological and psychiatric disorders.

Comparative analysis of interactions between aryl hydrocarbon receptor ligand binding domain with its ligands: a computational study

BackgroundAryl hydrocarbon receptor (AhR) ligands may act as potential carcinogens or anti-tumor agents. Understanding how some of the residues in AhR ligand binding domain (AhRLBD) modulate their interactions with ligands would be useful in assessing their divergent roles including toxic and beneficial effects. To this end, we have analysed the nature of AhRLBD interactions with 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD), 6-formylindolo[3,2-b]carbazole (FICZ), indole-3-carbinol (I3C) and its degradation product, 3,3′-diindolylmethane (DIM), Resveratrol (RES) and its analogue, Piceatannol (PTL) using molecular modeling approach followed by molecular dynamic simulations.ResultsResults showed that each of the AhR ligands, TCDD, FICZ, I3C, DIM, RES and PTL affect the local and global conformations of AhRLBD.ConclusionThe data presented in this study provide a structural understanding of AhR with its ligands and set the basis for its functions in several pathways and their related diseases.

A New Tool for Inducible Gene Expression in Caenorhabditis elegans.

Controlling protein activity and localization is a key tool in modern biology. Mammalian steroid receptor ligand-binding domain (LBD) fusions have been used in a range of organisms and cell types to inactivate proteins of interest until the cognate steroid ligand is applied. Here, we demonstrate that the glucocorticoid receptor LBD confers ligand-gated control of a heterologous gene expression system (Q system) and the DAF-16 transcription factor in Caenorhabditis elegans These experiments provide a powerful tool for temporal control of protein activity, and will bolster existing tools used to modulate gene expression and protein activity in this animal.

Genes and Pathways Promoting Long-Term Liver Repopulation byEx VivohYAP-ERT2 Transduced Hepatocytes and Treatment of Jaundice in Gunn Rats.

Hepatocyte transplantation is an attractive alternative to liver transplantation. Thus far, however, extensive liver repopulation by adult hepatocytes has required ongoing genetic, physical, or chemical injury to host liver. We hypothesized that providing a regulated proliferative and/or survival advantage to transplanted hepatocytes should enable repopulation in a normal liver microenvironment. Here, we repopulated livers of DPPIV− (dipeptidyl peptidase‐4) rats and Ugt1a1 (uridinediphosphoglucuronate glucuronosyltransferase 1a1)‐deficient Gunn rats (model of Crigler‐Najjar syndrome type 1), both models without underlying liver injury, for up to 1 year by transplanting lenti‐hYAP‐ERT2 (mutated estrogen receptor ligand‐binding domain 2)‐transduced hepatocytes (YAP‐Hc). Yap (yes‐associated protein) nuclear translocation/function in YAP‐Hc was regulated by tamoxifen. Repopulating YAP‐Hc and host hepatocytes were fluorescence‐activated cell sorting–purified and their transcriptomic profiles compared by RNAseq. After 1 year of liver repopulation, YAP‐Hc clusters exhibited normal morphology, integration into hepatic plates and hepatocyte‐specific gene expression, without dysplasia, dedifferentiation, or tumorigenesis. RNAseq analysis showed up‐regulation of 145 genes promoting cell proliferation and 305 genes suppressing apoptosis, including hepatocyte growth factor and connective tissue growth factor among the top 30 in each category and provided insight into the mechanism of cell competition that enabled replacement of host hepatocytes by YAP‐Hc. In Gunn rats transplanted with YAP‐Hc+tamoxifen, there was a 65%‐81% decline in serum bilirubin over 6 months versus 8%‐20% with control‐Hc, representing a 3‐4‐fold increase in therapeutic response. This correlated with liver repopulation as demonstrated by the presence of Ugt1a1‐positive hepatocyte clusters in livers and western blot analysis of tissue homogenates. Conclusion: Tamoxifen‐regulated nuclear translocation/function of hYAP‐ERT2 enabled long‐term repopulation of DPPIV−/− and Gunn rat livers by hYAP‐ERT2‐transduced hepatocytes without tumorigenesis. This cell transplantation strategy may offer a potential therapy for most of the inherited monogenic liver diseases that do not exhibit liver injury.

Identification and Molecular Characterization of Peroxisome Proliferator-Activated Receptor δ as a Novel Target for Covalent Modification by 15-Deoxy-Δ12,14-prostaglandin J2.

PPARδ belongs to the peroxisome proliferator-activated receptor (PPAR) family of nuclear receptors. Upon activation by an agonist, PPARδ controls a variety of physiological processes via regulation of its target genes. 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) is a cyclopentenone prostaglandin that features an electrophilic, α,β-unsaturated ketone (an enone) in the cyclopentenone ring. Many of 15d-PGJ2's biological effects result from covalent interaction between C9 and the thiol group of a catalytic cysteine (Cys) in target proteins. In this study, we investigated whether 15d-PGJ2 activates PPARδ by forming a covalent adduct. Our data show that 15d-PGJ2 activates PPARδ's transcriptional activity through formation of a covalent adduct between its endocyclic enone at C9 and Cys249 in the receptor's ligand-binding domain. As expected, no adduct formation was seen following a Cys-to-Ser mutation at residue 249 (C249S) of PPARδ or with a PGD2/PGJ2 analogue that lacks the electrophilic C9. Furthermore, the PPARδ C249S mutation weakened induction of the receptor's DNA binding activity by 15d-PGJ2, which highlights the biological significance of our findings. Calculated chemical properties as well as data from molecular orbital calculations, reactive molecular dynamics simulations, and intrinsic reaction coordinate modeling also supported the selectivity of 15d-PGJ2's C9 toward PPARδ's Cys thiol. In summary, our results provide the molecular, chemical, and structural basis of 15d-PGJ2-mediated PPARδ activation, designating 15d-PGJ2 as the first covalent PPARδ ligand to be identified.

Sertad1 promotes prostate cancer progression through binding androgen receptor ligand binding domain.

Androgen receptor (AR) signaling is involved in the initiation and progression of prostate cancer (PCa), which is the most frequently diagnosed nonskin cancer and remains a leading cause of cancer-related death in men. Further investigation of the involvement of AR signaling in PCa progression is urgently needed. In the present study, we performed a yeast two-hybrid screen and demonstrated that SERTA domain-containing protein 1 (Sertad1) is a novel AR-binding protein that binds to the AR ligand binding domain (LBD). The binding between AR-LBD and Sertad1 was confirmed by glutathione S-transferase (GST) pull-down assays and immunoprecipitation (IP) and confocal immunofluorescence co-localization experiments. Furthermore, we demonstrated that DHT inhibited Sertad1 protein degradation in prostate cancer cell lines and that Sertad1 knockdown inhibited the proliferation of prostate cancer cells in vitro. In human PCa tumor tissues, Sertad1 expression is positively correlated with AR expression and the Gleason score. Taken together, this report is the first to show that Sertad1 is a novel AR-LBD-binding protein, and DHT-liganded AR-LBD inhibits Sertad1 degradation. Thus, Sertad1 may represent a novel therapeutic target for the treatment of AR-positive PCa.

Machine learning models for predicting endocrine disruption potential of environmental chemicals

We introduce here ML4Tox, a framework offering Deep Learning and Support Vector Machine models to predict agonist, antagonist, and binding activities of chemical compounds, in this case for the estrogen receptor ligand-binding domain. The ML4Tox models have been developed with a 10 × 5-fold cross-validation schema on the training portion of the CERAPP ToxCast dataset, formed by 1677 chemicals, each described by 777 molecular features. On the CERAPP “All Literature” evaluation set (agonist: 6319 compounds; antagonist 6539; binding 7283), ML4Tox significantly improved sensitivity over published results on all three tasks, with agonist: 0.78 vs 0.56; antagonist: 0.69 vs 0.11; binding: 0.66 vs 0.26.

Implication of OPRM1 A118G Polymorphism in Opioids Addicts in Pakistan: In vitro and In silico Analysis

Single nucleotide polymorphism in OPRM1 gene is associated with hedonic and reinforcing consequences of opioids. Risk and protective alleles may vary in different populations. One hundred healthy controls and 100 opioids (predominantly heroin) addicts from Pakistani origin were genotyped for A118G (N40D) polymorphism in OPRM1. Structural and functional impact of the polymorphism on encoded protein was predicted by in silico analysis. Results show significant association between homozygous GG genotype and opioid addiction in Pakistani population (p value = 0.016). In silico analysis by SIFT (TI = 0.61), PolyPhen (PISC = 0.227), PANTHER (subPSEC = −1.7171), and SNP effect predicted this SNP benign for encoded protein. Superimposing wild-type and mutated proteins by MODELLER shows no change (RMSD = 0.1) in extracellular ligand binding domain of μ-opioid receptor. However, Haploreg and RegulomeDB predicted OPRM1 gene repression by chromatin condensation and increased binding affinity of RXRA transcription factor that may reduce protein translation and hence the number of available receptors to bind with drugs, which may trigger underlying mechanisms for opioids addiction. Thus, this study outlines causal relationship between opioids addiction and genetic predisposition in Pakistani population.