Receptor Ligand Binding Domain Research Articles

Abstract 3104: Stapled peptide inhibitors of the estrogen receptor/steroid receptor coactivator interaction

Abstract Despite undeniable successes, one of the major unsolved problems in estrogen receptor-positive breast cancer therapy is resistance to endocrine therapy. One-third of breast cancer patients who are given tamoxifen will develop recurrent cancer within 15 years. Importantly, the estrogen receptor is still present and active in 80-85% of these recurrent cases, but it is no longer sensitive to current therapies, so new ways of targeting the estrogen receptor are needed. Because selective estrogen receptor modulators indirectly block estrogen receptor/coactivator interactions, our hypothesis is that directly blocking the estrogen receptor/coactivator interaction will provide a more robust blockade that will antagonize forms of estrogen receptor that are active in both wild-type and some endocrine-resistant breast cancers. As our starting point for inhibiting estrogen receptor/coactivator interactions, we have chosen to use a class of compounds that mimic α-helices: “stapled peptides.” These non-natural, synthetic peptides have a key alkene bond that resembles a staple. Typically, peptides make poor drugs because they are rapidly hydrolyzed by proteases and are poorly cell-permeable. Because of their non-natural linkage, stapled peptides are poor substrates for proteases, so they are metabolically stable. Some stapled peptides are also cell-penetrant. This effect is highly sequence-specific, but it seems to be aided when peptides have a relatively high formal positive charge. Stapled peptides have been used to inhibit a variety of important protein-protein interactions in cell culture and animal models. In this work, we have created a library of stapled peptides that inhibit the interaction of the estrogen receptor with the steroid receptor coactivator in vitro. The best peptides show nanomolar IC50 values in a time-resolved fluorescence resonance energy transfer assay. They show high helical content according to circular dichroism studies. We have solved x-ray crystal structures of these molecules bound to the estrogen receptor mutant Y537S, which demonstrate their binding mode, and these studies are in agreement with molecular dynamics simulations of these molecules bound to the estrogen receptor ligand binding domain. Citation Format: Terry W. Moore, Thomas E. Speltz, Sean W. Fanning, Christopher G. Mayne, Jonna Frasor, Geoffrey L. Greene, Emad Tajkhorshid. Stapled peptide inhibitors of the estrogen receptor/steroid receptor coactivator interaction. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3104.

A parallel panning scheme used for selection of a GluA4-specific Fab targeting the ligand-binding domain

A method for development of murine Fab fragments towards extracellular domains of a surface receptor is presented. The GluA4 ionotropic glutamate receptor is used as a model system. Recombinant GluA4 ectodomain comprising both the N-terminal domain (NTD) and the ligand-binding domain (LBD) in one molecule was used for immunization. A Fab-phage library was constructed and a parallel panning approach enabled selection of murine Fab fragments towards either intact ectodomain or the isolated LBD of the GluA4 receptor. One LBD-Fab (FabL9) showed exclusive selectivity for the GluA4 LBD, over a panel of LBDs from GluA2, GluK1, GluK2 and GluD2. Soluble FabL9 was produced in amounts suitable for characterization. Competitive ELISA and rat-brain immunoprecipitation experiments confirmed that the FabL9 epitope is conserved in the LBD and in the intact native receptor. By an alignment of GluA2 and GluA4, the likely binding epitope for FabL9 was predicted.This study demonstrates a simple approach for development of antibody fragments towards specific sub-domains of a large ligand-gated ion channel, and this method could be utilized for all multi-domain surface receptors where antibody domain-selectivity may be desirable. Furthermore, we present for the first time a GluA4 subtype-specific murine Fab fragment targeting the LBD of the receptor.

Identification of Ecdysone Hormone Receptor Agonists as a Therapeutic Approach for Treating Filarial Infections.

BackgroundA homologue of the ecdysone receptor has previously been identified in human filarial parasites. As the ecdysone receptor is not found in vertebrates, it and the regulatory pathways it controls represent attractive potential chemotherapeutic targets.Methodology/ Principal FindingsAdministration of 20-hydroxyecdysone to gerbils infected with B. malayi infective larvae disrupted their development to adult stage parasites. A stable mammalian cell line was created incorporating the B. malayi ecdysone receptor ligand-binding domain, its heterodimer partner and a secreted luciferase reporter in HEK293 cells. This was employed to screen a series of ecdysone agonist, identifying seven agonists active at sub-micromolar concentrations. A B. malayi ecdysone receptor ligand-binding domain was developed and used to study the ligand-receptor interactions of these agonists. An excellent correlation between the virtual screening results and the screening assay was observed. Based on both of these approaches, steroidal ecdysone agonists and the diacylhydrazine family of compounds were identified as a fruitful source of potential receptor agonists. In further confirmation of the modeling and screening results, Ponasterone A and Muristerone A, two compounds predicted to be strong ecdysone agonists stimulated expulsion of microfilaria and immature stages from adult parasites.ConclusionsThe studies validate the potential of the B. malayi ecdysone receptor as a drug target and provide a means to rapidly evaluate compounds for development of a new class of drugs against the human filarial parasites.

Screening of saponins and sapogenins from Medicago species as potential PPARγ agonists and X-ray structure of the complex PPARγ/caulophyllogenin.

A series of saponins and sapogenins from Medicago species were tested for their ability to bind and activate the nuclear receptor PPARγ by SPR experiments and transactivation assay, respectively. The SPR analysis proved to be a very powerful and fast technique for screening a large number of compounds for their affinity to PPARγ and selecting the better candidates for further studies. Based on the obtained results, the sapogenin caulophyllogenin was proved to be a partial agonist towards PPARγ and the X-ray structure of its complex with PPARγ was also solved, in order to investigate the binding mode in the ligand binding domain of the nuclear receptor. This is the first known crystal structure of a sapogenin directly interacting with PPARγ. Another compound of the series, the echinocistic acid, showed antagonist activity towards PPARγ, a property that could be useful to inhibit the adipocyte differentiation which is a typical adverse effect of PPARγ agonists. This study confirms the interest on saponins and sapogenins as a valuable natural resource exploitable in the medical and food industry for ameliorating the metabolic syndrome.

In Vivo Performance and Properties of Tamoxifen Metabolites for CreERT2 Control.

Mutant Estrogen Receptor (ERT2) ligand-binding domain fusions with Cre recombinase are a key tool for spatio-temporally controlled genetic recombination with the Cre/lox system. CreERT2 is efficiently activated in a concentration-dependent manner by the Tamoxifen metabolite trans-4-OH-Tamoxifen (trans-4-OHT). Reproducible and efficient Cre/lox experimentation is hindered by the gradual loss of CreERT2 induction potency upon prolonged storage of dissolved trans-4-OHT, which potentially results from gradual trans-to-cis isomerization or degradation. Here, we combined zebrafish CreERT2 recombination experiments and cell culture assays to document the gradual activity loss of trans-4-OHT and describe the alternative Tamoxifen metabolite Endoxifen as more stable alternative compound. Endoxifen retains potent activation upon prolonged storage (3 months), yet consistently induces half the ERT2 domain fusion activity compared to fresh trans-4-OHT. Using 1H-NMR analysis, we reveal that trans-4-OHT isomerization is undetectable upon prolonged storage in either DMSO or Ethanol, ruling out isomer transformation as cause for the gradual loss of trans-4-OHT activity. We further establish that both trans-4-OHT and Endoxifen are insensitive to light exposure under regular laboratory handling conditions. We attribute the gradual loss of trans-4-OHT potency to precipitation over time, and show that heating of aged trans-4-OHT aliquots reinstates their CreERT2 induction potential. Our data establish Endoxifen as potent and reproducible complementary compound to 4-OHT to control ERT2 domain fusion proteins in vivo, and provide a framework for efficient chemically controlled recombination experiments.

High throughput microscopy identifies bisphenol AP, a bisphenol A analog, as a novel AR down-regulator.

Prostate cancer remains a deadly disease especially when patients become resistant to drugs that target the Androgen Receptor (AR) ligand binding domain. At this stage, patients develop recurring castrate-resistant prostate cancers (CRPCs). Interestingly, CRPC tumors maintain dependency on AR for growth; moreover, in CRPCs, constitutively active AR splice variants (e.g., AR-V7) begin to be expressed at higher levels. These splice variants lack the ligand binding domain and are rendered insensitive to current endocrine therapies. Thus, it is of paramount importance to understand what regulates the expression of AR and its splice variants to identify new therapeutic strategies in CRPCs. Here, we used high throughput microscopy and quantitative image analysis to evaluate effects of selected endocrine disruptors on AR levels in multiple breast and prostate cancer cell lines. Bisphenol AP (BPAP), which is used in chemical and medical industries, was identified as a down-regulator of both full length AR and the AR-V7 splice variant. We validated its activity by performing time-course, dose-response, Western blot and qPCR analyses. BPAP also reduced the percent of cells in S phase, which was accompanied by a ~60% loss in cell numbers and colony formation in anchorage-independent growth assays. Moreover, it affected mitochondria size and cell metabolism. In conclusion, our high content analysis-based screening platform was used to classify the effect of compounds on endogenous ARs, and identified BPAP as being capable of causing AR (both full-length and variants) down-regulation, cell cycle arrest and metabolic alterations in CRPC cell lines.

MiR-137 Modulates a Tumor Suppressor Network-Inducing Senescence in Pancreatic Cancer Cells.

Activating K-Ras mutations occurs frequently in pancreatic cancers and is implicated in their development. Cancer-initiating events, such as oncogenic Ras activation, lead to the induction of cellular senescence, a tumor suppressor response. During senescence, the decreased levels of KDM4A lysine demethylase contribute to p53 activation, however, the mechanism by which KDM4A is downregulated is unknown. We show that miR-137 targets KDM4A mRNA during Ras-induced senescence and activates both p53 and retinoblastoma (pRb) tumor suppressor pathways. Restoring the KDM4A expression contributed to bypass of miR-137-induced senescence and inhibition of endogenous miR-137 with an miRNA sponge-compromised Ras-induced senescence. miR-137 levels are significantly reduced in human pancreatic tumors, consistent with previous studies revealing a defective senescence response in this cancer type. Restoration of miR-137 expression inhibited proliferation and promoted senescence of pancreatic cancer cells. These results suggest that modulating levels of miR-137 may be important for triggering tumor suppressor networks in pancreatic cancer.

Rigorous Selection of Random Forest Models for Identifying Compounds that Activate Toxicity-Related Pathways

Random forest (RF) is a machine-learning ensemble method with high predictive performance. Majority voting in RF uses the discrimination results in numerous decision trees produced from bootstrapping data. For the same dataset, the bootstrapping process yields different predictive capacities in each generation. As participants in the Toxicology in the 21st Century (Tox21) DATA Challenge 2014, we produced numerous RF models for predicting the structures of compounds that can activate each toxicity-related pathway, and then selected the model with the highest predictive ability. Half of the compounds in the training dataset supplied by the competition organizer were allocated to the validation dataset. The remaining compounds were used in model construction. The charged and uncharged forms of each molecule were calculated using the molecular operating environment (MOE) software. Subsequently, the descriptors were computed using MOE, MarvinView, and Dragon. These combined methods yielded over 4,071 descriptors for model construction. Using these descriptors, pattern recognition analyses were performed by RF implemented in JMP Pro (a statistical software package). A hundred to two hundred RF models were generated for each pathway. The predictive performance of each model was tested against the validation dataset, and the best-performing model was selected. In the competition, the latter model selected a best-performing model from the 50% test set that best predicted the structures of compounds that activate the estrogen receptor ligand-binding domain (ER-LBD).

Positive Allosteric Modulators of GluN2A-Containing NMDARs with Distinct Modes of Action and Impacts on Circuit Function

To enhance physiological function of NMDA receptors (NMDARs), we identified positive allosteric modulators (PAMs) of NMDARs with selectivity for GluN2A subunit-containing receptors. X-ray crystallography revealed a binding site at the GluN1-GluN2A dimer interface of the extracellular ligand-binding domains (LBDs). Despite the similarity between the LBDs of NMDARs and AMPA receptors (AMPARs), GluN2A PAMs with good selectivity against AMPARs were identified. Potentiation was observed with recombinant triheteromeric GluN1/GluN2A/GluN2B NMDARs and with synaptically activated NMDARs in brain slices from wild-type (WT), but not GluN2A knockout (KO), mice. Individual GluN2A PAMs exhibited variable degrees of glutamate (Glu) dependence, impact on NMDAR Glu EC50, and slowing of channel deactivation. These distinct PAMs also exhibited differential impacts during synaptic plasticity induction. The identification of a new NMDAR modulatory site and characterization of GluN2A-selective PAMs provide powerful molecular tools to dissect NMDAR function and demonstrate the feasibility of a therapeutically desirable type of NMDAR enhancement.

Mechanisms of Glutamate Capture by a Clamshell Binding Domain

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels responsible for the majority of excitatory synaptic transmission in the brain. Glutamate binding into the clamshell-like ligand-binding domains (LBDs) triggers a conformational change that opens the channel pore, resulting in receptor activation. The stable glutamate-bound conformation of the LBD has been well-studied structurally, but the dynamic process of ligand binding is poorly understood. To elucidate this process, we performed microsecond-timescale molecular dynamics (MD) simulations of glutamate binding to an AMPA receptor LBD together with electrophysiological studies. Potential of mean force (PMF) calculations identify several metastable glutamate interaction regions outside of the binding pocket. Glutamate is observed to bind in two distinct conformations, one of which corresponds to the crystallographically-observed conformation. We evaluated the kinetics of various dynamical processes using a Markov state model analysis. Using the simulations as a guide, a battery of mutants was designed to target the metastable regions, replacing charged residues with either neutral ones or those of opposite charge. Some of these mutants slowed activation and deactivation of the receptor, consistent with slowed association and dissociation of glutamate, whereas off pathway mutations had little effect. Our results suggest that glutamate binding in iGluRs occurs through multiple, independent pathways, and that the ligand interacts strongly with several key residues along these pathways.

Conformational Dynamics of the GluK2 Ligand-Binding Domain

The kainate family of ionotropic glutamate receptors (GluK) mediates neurotransmission in pre- and post-synaptic neurons. Closure of the ligand-binding domain (LBD) in response to agonist binding is a necessary conformational transition to gate the ion channel. Previously computed free energy landscapes for iGluR LBDs revealed subtype-dependent conformational differences between AMPA and NMDA iGluR families, which may help to explain functional differences between iGluR subtypes. Here we report free energy landscapes, or potentials of mean force (PMFs), for the GluK2 LBD in apo and glutamate-bound states. GluK2 PMFs reveal both apo and glutamate-bound LBDs preferentially access a closed-cleft conformation, consistent with a conformational selection mechanism for binding. These results are also consistent with PMFs for NMDA but not AMPA receptor LBDs. Additionally, the open-cleft conformation is more accessible to the GluK2 apo state than the glutamate-bound state, consistent with previous results for both NMDA and AMPA receptors. Small-angle X-ray scattering profiles for the apo- and glutamate-bound states calculated from a Boltzmann-weighted ensemble of conformations are compared with experimental SAXS profiles. Finally, structural and biochemical properties of GluK2 in its apo state are probed using proteolytic protection assays, circular dichroism, and multi-angle light scattering.

Assessment of the contribution of chemoreceptor-based signalling to biofilm formation.

Although it is well established that one- and two-component regulatory systems participate in regulating biofilm formation, there also exists evidence suggesting that chemosensory pathways are also involved. However, little information exists about which chemoreceptors and signals modulate this process. Here we report the generation of the complete set of chemoreceptor mutants of Pseudomonas putida KT2440 and the identification of four mutants with significantly altered biofilm phenotypes. These receptors are a WspA homologue of Pseudomonas aeruginosa, previously identified to control biofilm formation by regulating c-di-GMP levels, and three uncharacterized chemoreceptors. One of these receptors, named McpU, was found to mediate chemotaxis towards different polyamines. The functional annotation of McpU was initiated by high-throughput thermal shift assays of the receptor ligand binding domain (LBD). Isothermal titration calorimetry showed that McpU-LBD specifically binds putrescine, cadaverine and spermidine, indicating that McpU represents a novel chemoreceptor type. Another uncharacterized receptor, named McpA, specifically binds 12 different proteinogenic amino acids and mediates chemotaxis towards these compounds. We also show that mutants in McpU and WspA-Pp have a significantly reduced ability to colonize plant roots. Data agree with other reports showing that polyamines are signal molecules involved in the regulation of bacteria-plant communication and biofilm formation.

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor.

Förster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. Biosensors relying on the principle of FRET have enabled real-time visualization of subcellular signaling events in live cells with high temporal and spatial resolution. Here, we describe the application of a genetically encoded Bile Acid Sensor (BAS) that consists of two fluorophores fused to the farnesoid X receptor ligand binding domain (FXR-LBD), thereby forming a bile acid sensor that can be activated by a large number of bile acids species and other (synthetic) FXR ligands. This sensor can be targeted to different cellular compartments including the nucleus (NucleoBAS) and cytosol (CytoBAS) to measure bile acid concentrations locally. It allows rapid and simple quantitation of cellular bile acid influx, efflux and subcellular distribution of endogenous bile acids without the need for labeling with fluorescent tags or radionuclei. Furthermore, the BAS FRET sensors can be useful for monitoring FXR ligand binding. Finally, we show that this FRET biosensor can be combined with imaging of other spectrally distinct fluorophores. This allows for combined analysis of intracellular bile acid dynamics and i) localization and/or abundance of proteins of interest, or ii) intracellular signaling in a single cell.

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

Förster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. Biosensors relying on the principle of FRET have enabled real-time visualization of subcellular signaling events in live cells with high temporal and spatial resolution. Here, we describe the application of a genetically encoded Bile Acid Sensor (BAS) that consists of two fluorophores fused to the farnesoid X receptor ligand binding domain (FXR-LBD), thereby forming a bile acid sensor that can be activated by a large number of bile acids species and other (synthetic) FXR ligands. This sensor can be targeted to different cellular compartments including the nucleus (NucleoBAS) and cytosol (CytoBAS) to measure bile acid concentrations locally. It allows rapid and simple quantitation of cellular bile acid influx, efflux and subcellular distribution of endogenous bile acids without the need for labeling with fluorescent tags or radionuclei. Furthermore, the BAS FRET sensors can be useful for monitoring FXR ligand binding. Finally, we show that this FRET biosensor can be combined with imaging of other spectrally distinct fluorophores. This allows for combined analysis of intracellular bile acid dynamics and i) localization and/or abundance of proteins of interest, or ii) intracellular signaling in a single cell.

Development of Inducible Molecular Switches Based on All-in-One Lentiviral Vectors Equipped with Drug Controlled FLP Recombinase.

Drug-inducible recombination based on flippase (FLP) is frequently used in animal models and in transgenic cell lines to initiate or to abrogate gene expression. Although the system is highly efficient, functional gene analyses depend on the availability of suitable animal models. In contrast, lentiviral vectors are readily available and versatile tools for the transfer of genetic information into a wide variety of target cells, and can be produced at high titer in a timely manner. To combine the advantages of both approaches, we generated a tight, drug-controlled FLP recombinase consisting of a 5' FKBP12 derived conditional destruction domain and a 3' estrogen receptor ligand binding (ERT2) domain. We successfully constructed lentiviral vectors expressing drug-controlled FLP in combination with a fluorescent reporter for recombination of FLP recognition target (FRT) sites located in trans as well as with target alleles located in cis (all-in-one configuration). In this chapter, we describe the design of the drug controlled FLP recombinase, the construction of molecular switches consisting of FLP expressing lentiviral vectors for inducible recombination of target sites located in cis and in trans, as well as the details for the characterization of lentiviral FLP vectors in cell lines.

Neutrophils Derived from Ezh2 -/- Progenitor Cells Demonstrate Aberrant Erythroid Lineage Gene Expression

Abstract Introduction: Ezh2 is the catalytic component of the polycomb repressive complex 2, which methylates lysine 27 of histone H3 (H3K27). Loss of function mutations in EZH2 are found in 6% of MDS patients and are independently associated with worse overall survival compared to patients with wildtype EZH2 (Bejar R. et al., 2011 and 2012). Our group has described that neutrophils derived from Ezh2-/- mice have functional defects (Perez-Ladaga et al., 2013), including decreased phagocytosis, aberrant migration and overproduction of reactive oxygen species (ROS). To determine how loss of Ezh2 might contribute to these functional deficits, we performed gene expression profiling on immortalized myeloid cell lines capable of neutrophilic differentiation. Methods: Bone marrow from Ezh2 null (Ezh2-/-) and littermate control mice (WT) were transduced with HOXB8 fused to the estrogen receptor ligand-binding domain to produce immortalized myeloid progenitor cells. Removal of estrogen from the media allows these cells differentiate into mature neutrophils (Wang G.G., 2006). RNA from progenitor and mature neutrophils (WT and Ezh2-/-) was extracted each condition in duplicate and subjected to gene expression profile (Affymetrix). Transcriptome analysis was conducted with TAC software from Affymetrix and gene set comparisons between the different phenotypes were analyzed with Gene Set Enrichment Analysis (GSEA). Rescue by lentiviral re-introduction of Ezh2 into Ezh2-/- cells is currently ongoing. Results: Estrogen withdrawal causes differentiation of WT and Ezh2-/- lines into mature neutrophils after six days. Interestingly, WT neutrophils lose Ezh2 mRNA and protein expression as soon as three days after estrogen withdrawal. WT mature neutrophils lack Ezh2 and trimethyl-H3K27 (me3H3K27), showing similar amounts as Ezh2-/- derived neutrophils. Gene expression profiling of 65956 transcripts demonstrated that 1953 of them were differentially expressed between WT and Ezh2-/- mature neutrophils. Nearly 65% of these genes were upregulated in Ezh2-/- derived neutrophils when compared to WT. As Ezh2 levels in mature neutrophils are similar in both conditions, gene expression differences are likely due to EZH2 and me3H3K27 differences in the progenitor state. Among the differentially expressed genes, the transcription factor GATA1 was found upregulated in Ezh2-/- derived neutrophils, a result confirmed by qPCR. GATA1 regulates the expression of hundreds of genes and is essential for erythropoiesis. GATA1 target erythroid genes were also found upregulated in Ezh2-/- derived neutrophils when compared to WT, while no significant differences in neutrophil gene expression were detected. Similarly, GSEA analysis of Ezh2-/- vs. WT confirmed strong enrichment for erythroid associated expression programs. A Heme Metabolism Signature based on a panel of 182 genes showed a strong correlation with Ezh2-/- derived neutrophils (Figure 1A). GSEA was used to examine possible mechanisms behind the functional defects previously reported in Ezh2-/- derived neutrophils such as overproduction of ROS and impaired migration. A gene set based on 192 genes encoding proteins involved in oxidative phosphorylation demonstrated a significant correlation between this pathway signature and Ezh2-/- derived neutrophils (Figure 1B).On the other hand, GSEA showed a positive correlation between WT differentiated neutrophils and a panel of 115 genes involved in leukocyte transendothelial migration (Figure 1C). Conclusion: Our results show that HOXB8-ER immortalized myeloid progenitor cells are able to produce mature neutrophils even in absence of Ezh2. The loss of Ezh2 in myeloid progenitor cells is associated with the differential expression of 1953 genes in mature neutrophils, including the upregulation of genes involved in erythroid differentiation programs and oxidative phosphorylation, and the downregulation of genes involved in leukocyte migration. Ongoing rescue experiments re-introducing Ezh2 into Ezh2-/- progenitor cells are being performed to determine if this restores normal neutrophil functions and silences the aberrant erythroid gene expression in Ezh2-/- derived neutrophils. Our findings may help explain how Ezh2 loss causes neutrophil dysfunction and contributes to the adverse prognosis associated with EZH2 mutations in MDS patients. Disclosures Orkin: Editas Inc.: Consultancy. Ebert:genoptix: Consultancy, Patents & Royalties; Celgene: Consultancy; H3 Biomedicine: Consultancy. Bejar:Alexion: Other: ad hoc advisory board; Celgene: Consultancy, Honoraria; Genoptix Medical Laboratory: Consultancy, Honoraria, Patents & Royalties: MDS prognostic gene signature.

ChemInform Abstract: Recent Developments in Androgen Receptor Antagonists

AbstractReview: 78 refs.

Relationship between Structure and Conformational Change of the Vitamin D Receptor Ligand Binding Domain in 1α,25-Dihydroxyvitamin D3 Signaling.

Vitamin D Receptor (VDR) belongs to the nuclear receptor (NR) superfamily. Whereas the structure of the ligand binding domain (LBD) of VDR has been determined in great detail, the role of its amino acid residues in stabilizing the structure and ligand triggering conformational change is still under debate. There are 13 α-helices and one β-sheet in the VDR LBD and they form a three-layer sandwich structure stabilized by 10 residues. Thirty-six amino acid residues line the ligand binding pocket (LBP) and six of these residues have hydrogen-bonds linking with the ligand. In 1α,25-dihydroxyvitamin D3 signaling, H3 and H12 play an important role in the course of conformational change resulting in the provision of interfaces for dimerization, coactivator (CoA), corepressor (CoR), and hTAFII 28. In this paper we provide a detailed description of the amino acid residues stabilizing the structure and taking part in conformational change of VDR LBD according to functional domains.

Partial association of restriction polymorphism of the ligand binding domain of human androgen receptor in prostate cancer

BackgroundHuman androgen receptor (AR) functions as a steroid-hormone activated transcription factor. The receptor binds to its ligand (testosterone or dihydrotestosterone) and is translocated to the nucleus to stimulate the transcription of androgen responsive genes. Mutations in the ligand binding domain (LBD) impair the receptor activity and play a crucial role in the development and progression of prostate cancer (PCa). Materials and methodsThis work was designated to investigate the restriction integrity of the LBD and its association with benign prostatic hyperplasia (BPH) and prostate cancer. Exons of this domain (exons: 4–8) were amplified from prostate tissue of BPH and PCa patients and the restriction polymorphism was investigated by SmlI, HphI and Tsp45I restriction enzymes in both BPH and PCa groups. ResultsData revealed the integrity of exons 4–6 in both BPH and PCa patients. Exons 7 and 8, however have kept their constitutional pattern only in BPH patients. Hph1 site showed an abnormal restriction pattern in 40% and 26.7% of PCa patients. Also, Tsp45I demonstrated restriction polymorphism in 20% and 13% of PCa patients. ConclusionOur results indicate that the loss of the restriction integrity in the C-terminal part (exons: 7 and 8) of the LBD is associated with the progression of benign prostatic hyperplasia to prostate cancer.

Recent Developments in Androgen Receptor Antagonists

The androgen receptor (AR), a ligand-dependent transcription factor that regulates the expression of a series of downstream target genes after the binding of androgens, has been a target for the discovery of drugs used to treat prostate cancer. Prostate cancer always progresses to castration-resistant prostate cancer after a period of androgen deprivation therapy. Thus, developing potent androgen receptor antagonists for the therapy of castration-resistant prostate cancer possesses great significance. This review summarizes the preclinical development of androgen receptor antagonists, conventional androgen receptor antagonists that competitively bind to the ligand binding domain of the androgen receptor and coactivator antagonists of the androgen receptor, including both activation function-2 antagonists and binding function-3 antagonists. We hope that this review can help other researchers find new scaffolds and sites for the treatment of prostate cancer.