Receptor Ligand Binding Domain Research Articles

A partially open conformation of an androgen receptor ligand-binding domain with drug-resistance mutations.

Mutations in the androgen receptor (AR) ligand-binding domain (LBD) can cause resistance to drugs used to treat prostate cancer. Commonly found mutations include L702H, W742C, H875Y, F877L and T878A, while the F877L mutation can convert second-generation antagonists such as enzalutamide and apalutamide into agonists. However, pruxelutamide, another second-generation AR antagonist, has no agonist activity with the F877L and F877L/T878A mutants and instead maintains its inhibitory activity against them. Here, it is shown that the quadruple mutation L702H/H875Y/F877L/T878A increases the soluble expression of AR LBD in complex with pruxelutamide in Escherichia coli. The crystal structure of the quadruple mutant in complex with the agonist dihydrotestosterone (DHT) reveals a partially open conformation of the AR LBD due to conformational changes in the loop connecting helices H11 and H12 (the H11-H12 loop) and Leu881. This partially open conformation creates a larger ligand-binding site for AR. Additional structural studies suggest that both the L702H and F877L mutations are important for conformational changes. This structural variability in the AR LBD could affect ligand binding as well as the resistance to antagonists.

Study of novel androgen receptor V770 variant in androgen insensitivity syndrome patients reveals the transitional state of the androgen receptor ligand binding domain homodimer.

We report the discovery of the androgen receptor missense mutation V770D, that was found in two sisters suffering from complete androgen insensitivity. Experimental validation of AR V770 variants demonstrated that AR V770D was transcriptionally inactive due to the inability to dimerize and a reduced ligand binding affinity. The more conservative AR V770A variant showed a dimerization defect at low levels of DHT with a partial recovery of the transcriptional activity and of the receptor's ability to dimerize when increasing the DHT levels. With V770 located outside of the proposed LBD dimerization interface of the AR LBD homodimer crystal structure, the effects of the V770A mutation on AR dimerization were unexpected. We therefore explored whether the AR LBD dimerization interface would be better described by an alternative dimerization mode based on available human homodimeric LBD crystal structures of other nuclear receptors. Superposition of the monomeric AR LBD in the homodimeric crystal structures of GR, PR, ER, CAR, TRβ, and HNF-4α showed that the GR-like LBD dimer model was energetically the most stable. Moreover, V770 was a key energy residue in the GR-like LBD dimer while it was not involved in the stabilization of the AR LBD homodimer according to the crystal structure. Additionally, the observation that 4 AIS mutations impacted the stability of the AR LBD dimer while 16 mutations affected the GR-like LBD dimer, suggested that the AR LBD dimer crystal is a snapshot of a breathing AR LBD homodimer that can transition into the GR-like LBD dimer model.

The marionette mechanism of domain–domain communication in the antagonist, agonist, and coactivator responses of the estrogen receptor

The human estrogen receptor α (hERα) is involved in the regulation of growth, development, and tissue homeostasis. Agonists that bind to the receptor's ligand-binding domain (LBD) lead to recruitment of coactivators and the enhancement of gene expression. In contrast, antagonists bind to the LBD and block the binding of coactivators thus decreasing gene expressions. In this work, we carry out simulations using the AWSEM (Associative memory, Water mediated, Structure and Energy Model)-Suite force field along with the 3SPN.2C force field for DNA to predict the structure of hERα and study its dynamics when binding to DNA and coactivators. Using simulations of antagonist-bound hERα and agonist-bound hERα by themselves and also along with bound DNA and coactivators, principal component analyses and free energy landscape analyses capture the pathway of domain-domain communication for agonist-bound hERα. This communication is mediated through the hinge domains that are ordinarily intrinsically disordered. These disordered segments manipulate the hinge domains much like the strings of a marionette as they twist in different ways when antagonists or agonists are bound to the ligand-binding domain.

Screening the binding affinity of bile acid derivatives for the glucocorticoid receptor ligand-binding domain

The necessity of anti-inflammatory drugs such as glucocorticoids has been evident during the COVID-19 pandemic. Glucocorticoids, are the standard therapy for the treatment of moderate and severe COVID-19 patients. However, serious side effects limit the use of these drugs, and anti-inflammatory drugs with better pharmacological properties are urgently required. Bile acids are of interest, because of their anti-inflammatory and immunomodulatory properties, facilitated through an unclear mechanism involving transmembrane and nuclear receptors. In this work, we screened the binding activity of a number of bile acid derivatives, for the ligand-binding domain of glucocorticoid receptor (GR-LBD), the most important receptor for anti-inflammatory processes. Tested compounds include oximes, lactones, lactams, tetrazoles, dienones, C-24 alcohols and cholic acid amides. Cholic acid oxime, deoxycholic acid dienone, 3-keto-24-cholic alcohol and cholic acid amide showed best binding affinities for GR-LBD among tested compounds. The in silico molecular docking explanation is provided. SAR analysis showed that expansion of B and C steroid rings or attachment of heterocycle to C ring is not beneficial for binding; side chain should contain hydrogen donor group; the GR-LBD tolerate well different functionalities on C-3 position. These results provide valuable information toward synthesis of the new glucocorticoids based on bile acids.

The multivalency of the glucocorticoid receptor ligand-binding domain explains its manifold physiological activities.

The glucocorticoid receptor (GR) is a ubiquitously expressed transcription factor that controls metabolic and homeostatic processes essential for life. Although numerous crystal structures of the GR ligand-binding domain (GR-LBD) have been reported, the functional oligomeric state of the full-length receptor, which is essential for its transcriptional activity, remains disputed. Here we present five new crystal structures of agonist-bound GR-LBD, along with a thorough analysis of previous structural work. We identify four distinct homodimerization interfaces on the GR-LBD surface, which can associate into 20 topologically different homodimers. Biologically relevant homodimers were identified by studying a battery of GR point mutants including crosslinking assays in solution, quantitative fluorescence microscopy in living cells, and transcriptomic analyses. Our results highlight the relevance of non-canonical dimerization modes for GR, especially of contacts made by loop L1-3 residues such as Tyr545. Our work illustrates the unique flexibility of GR's LBD and suggests different dimeric conformations within cells. In addition, we unveil pathophysiologically relevant quaternary assemblies of the receptor with important implications for glucocorticoid action and drug design.

Novel Nanomolar Allosteric Modulators of AMPA Receptor of Bis(pyrimidine) Series: Synthesis, Biotesting and SAR Analysis

Positive allosteric modulators (PAMs) of AMPA receptors represent attractive candidates for the development of drugs for the treatment of cognitive and neurodegenerative disorders. Dimeric molecules have been reported to have an especially potent modulating effect, due to the U-shaped form of the AMPA receptor's allosteric binding site. In the present work, novel bis(pyrimidines) were studied as AMPA receptor modulators. A convenient and flexible preparative approach to bis(pyrimidines) containing a hydroquinone linker was elaborated, and a series of derivatives with varied substituents was obtained. The compounds were examined in the patch clamp experiments for their influence on the kainate-induced currents, and 10 of them were found to have potentiating properties. The best potency was found for 2-methyl-4-(4-((2-methyl-5,6,7,8-tetrahydroquinazolin-4-yl)oxy)phenoxy)-6,7,8,9-tetrahydro-5H-cyclohepta[d]pyrimidine, which potentiated the kainate-induced currents by up to 77% in all tested concentrations (10-12-10-6 M). The results were rationalized via the modeling of modulator complexes with the dimeric ligand binding domain of the GluA2 AMPA receptor, using molecular docking and molecular dynamics simulation. The prediction of ADMET, physicochemical, and PAINS properties of the studied bis(pyrimidines) confirmed that PAMs of this type may act as the potential lead compounds for the development of neuroprotective drugs.

Antitumor potential of novel 5α,6β-dibromo steroidal D-homo lactone

New steroidal D-homo androstane derivative, 5α,6β-dibromo-3β-hydroxy-17-oxa-17a-homoandrostan-16-one was synthesized and its structure was confirmed by NMR spectroscopy. In silico ADME properties of this compound were assessed using the SwissADME online prediction tool. Six human cancer cell lines (MDA-MB-231, MCF-7, PC3, HT-29, HeLa, and A549) and one human noncancerous cell line (MRC-5) were used for in vitro cytotoxicity testing. Novel steroidal dibromide was also tested for relative binding affinity for the ligand binding domain of estrogen receptor α and β or the androgen receptor using a published assay in yeast cells. Ligand binding domains of each steroid receptor were expressed in-frame with yellow fluorescent protein in yeast and the fluorescence intensity changes upon addition of test compound was measured. The new compound showed selective cytotoxic activity against HT-29 (colon adenocarcinoma) and A549 (lung adenocarcinoma) cell lines, as well as the potential to induce apoptosis in HT-29 cells, while results obtained from ligand binding assay in yeast suggested a lack of significant estrogenic or androgenic properties.

Deciphering the importance of MD descriptors in designing Vitamin D Receptor agonists and antagonists using machine learning

The Vitamin D Receptor (VDR) ligand-binding domain undergoes conformation change upon the binding of VDR agonists/antagonists. Helix 12 ((H)12) is one of the important helices at VDR ligand binding and its conformational changes are controlled by the binding of agonists and antagonists molecules. Various molecular modeling studies are available to explain the agonistic and antagonistic activity of vitamin D analogs. In this work, for the first time, we attempted to generate a machine learning model with fingerprints, 2D, 3D and MD descriptors that are specific to Vitamin D analogs and VDR. Initially, 2D and 3D descriptors and fingerprints of 1003 vitamin D analogs were calculated using CDK and RDKit. The machine learning model was generated using descriptors and fingerprints. Further, 80 Vitamin D analogs (40 VDR agonists + 40 VDR antagonists) were docked in the VDR active site. 50ns MD simulation was performed for each protein-ligand complex. Different MD descriptors such as Solvent Accessible Surface Area (SASA), radius of gyration, PC1 and PC2 were calculated and considered along with CDK and RDKit descriptors as features for machine learning calculations. A few other descriptors that are related to VDR conformational changes such as conformation of the (H)12, the angle at kink were considered for machine learning model generation. It was observed that the descriptors calculated from VDR conformational changes i) were able to distinguish between agonists and antagonists ii) provide key and comprehensive information about the unique binding characteristics of agonists and antagonists iii) provide a strong basis for the machine learning model generation. Overall, this study attempts the utilization of descriptors that are specific to a protein conformation will be helpful for the generation of an efficient machine learning model.

Interplay of receptor-ligand binding and lipid domain formation during cell adhesion.

Cell adhesion involved in biological processes such as cell migration, immune responses, and cancer metastasis, is mediated by the specific binding of receptor and ligand proteins. Some of these proteins exhibit affinity for nanoscale lipid clusters in cell membranes. A key question is how these nanoscale lipid clusters influence and react to the receptor-ligand binding during cell adhesion. In this article, we review recent computational studies that shed new light on the interplay of the receptor-ligand binding and the formation of lipid domains in adhering membranes. These studies indicate that the receptor-ligand binding promotes coalescence of lipid clusters into mesoscale domains, which, in turn, enhances both the affinity and cooperativity of the receptor-ligand binding in cell-cell adhesion with mobile ligands. In contrast, in the case of cell-extracellular matrix adhesion with immobile ligands, the receptor-ligand binding and the lipid cluster coalescence can be correlated or anti-correlated, depending strongly on the ligand distribution. These findings deepen our understanding of correlations between cell adhesion and membrane heterogeneities.

Abstract 2686: Is ARV7 a regulator of prostate cancer stemness and plasticity

Abstract Therapy options for advanced and castration-resistant prostate cancer (CRPC) include the use of drugs targeting directly/indirectly the ligand binding domain (LBD) of androgen receptor (AR) such as enzalutamide (Enza) and abiraterone acetate. However, primary or acquired treatment failures are frequently observed and have been associated with the expression of truncated AR variants devoid of the LBD. In particular, the AR variant AR3, also known as ARV7, is detected in over 80% of CRPC specimen and is increasingly expressed in metastatic lesions. In PC models ARV7 expression causes treatment resistance to Enza. Moreover, ARV7 expression leads to increased expression of mesenchymal markers and stem cell signature genes. CDH2 (N-Cadherin) expression is increased by ARV7 binding to regulatory elements in intron 1 of CDH2, which is antagonized by full length (FL) AR. Based on these observations we hypothesize that ARV7 is a marker and regulator of PC stemness and/or plasticity. ARFL or ARV7 mRNA and protein was detected in cell lines and tissue samples by standard procedures including qRT-PCR, RNA in situ hybridization (RISH), immunofluorescence (IF) and western blotting using the monoclonal ARV7 antibody RM7. ARFL or ARV7 stably expressing LNCaP were obtained by lentiviral transduction. LNCaP cells resistant to Enza (LNCaP-Enza) were generated by exposure to increasing concentrations (up to 5 µM) of the drug. Transcriptome analysis in LNCaP-Enza vs. LNCaP was done by RNAseq and Gene Set Enrichment Analysis. ARV7 mRNA was detectable in LNCaP, albeit gene expression was 2.3% - 2.6% of the known ARV7 positive cell lines 22Rv1 and VCaP, respectively. RISH and IF demonstrated that only a fraction (~10%) of LNCaP cells expressed ARV7 mRNA and nuclear localized ARV7 protein. ARV7 mRNA was also detected in isolated epithelial and stromal areas in treatment-naïve prostate cancer specimen by RISH. In contrast, all LNCaP-Enza cells were positive for nuclear ARV7 protein. Transcriptome analysis of LNCaP-Enza cells showed enrichments for published ARV7 gene expression signatures. Insular spots of ARV7 mRNA were detected in treatment-naïve tissue and in isolated cells of the castration-sensitive cell line LNCaP suggesting that these cells could be intrinsically resistant against LBD-targeting drugs and responsible for the failure of various endocrine therapies. This project will further analyze whether this subpopulation possesses stem cell (-like)/plasticity characteristics and whether these are regulated by ARV7. Citation Format: Tobias Furlan, Florian Handle, Marcus V. Cronauer, Frédéric R. Santer. Is ARV7 a regulator of prostate cancer stemness and plasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2686.

Characterization of a microglia-specific humanized P2X7 receptor knock-out mouse line: Implications for translational psychoneuroimmunology

IntroductionDepression is a common psychiatric disorder and chronic stress is considered its main environmental risk factor. Recently, immune processes including adenosine triphosphate mediated P2X7 receptor (P2X7R) signalling via microglia and macrophages (M/Ms) were found to play a critical role in depression genesis, by linking environmental stress to depression biology and symptoms.ObjectivesTo characterize the role of human P2X7R (hP2X7R) in psychosocial and immune stress conditions, both in vitro and in vivo.MethodsSeveral, custom designed mouse lines expressing the loxP-flanked, hP2X7R-sequence in the murine P2X7R locus were established. In addition, these mice possess a Cre-sensitive reporter and express a Cre recombinase fused to a mutant estrogen receptor ligand-binding domain in M/Ms. This enables conditional, tamoxifen-inducible hP2X7R inactivation and simultaneous tdTomato expression. First, we established primary microglia cell cultures and characterized them at baseline and following immune stimulation. Next, we performed behavioural assessment of hP2X7Rwt and microglia-specific hP2X7RKO mice following chronic psychosocial stress. Last, we developed a novel in vivo two-photon microscopy (TPM) approach by use of frontolimbic cranial windows.ResultsPrimary hP2X7RKO microglia displayed significantly lower IL-1β production, increased survival and decreased morphological activation upon immune stimulation. Although hP2X7RKO mice showed a significant increase of locomotor activity at baseline, there was no impact on anxiety- and depressive-like phenotypes. Longitudinal in vivo TPM enabled morphometric characterization of cortical M/Ms over several weeks.ConclusionsOur results illustrate the great potential of this humanized mouse line for translational psychiatry. In the future, this system could proof useful to evaluate immunomodulatory approaches in chronic stress and depression.DisclosureNo significant relationships.

Hormone receptor binding, selectivity and cytotoxicity of steroid D-homo lactone loaded chitosan nanoparticles for the treatment of breast and prostate cancer cells

Chemically modified steroids have a long history as anti-neoplastic drugs. Incorporation of a lactone moiety in the steroid nucleus, as in previously obtained 3β-acetoxy-17-oxa-17a-homoandrost-5-en-16-one (A) and 3β-hidroxy-17-oxa-17a-homoandrost-5-en-16-one (B), often results in enhanced anticancer properties. In this work, chitosan-based (Ch) nanoparticles were created and loaded with potent anticancer steroidal compounds, A and B. Changes to hormone receptor binding and cytotoxicity were then measured. In agreement with our previous results for A and B, A- and B-loaded Ch displayed cytotoxic properties against cancer cell lines. Both A-Ch and B-Ch showed activity toward estrogen negative breast cancer (MDA-MB-231) and androgen negative prostate cancer cell lines (PC-3). Greater selectivity toward cancer cells versus healthy lung fibroblast (MRC-5) was observed for B-Ch particles. Cell viability and cytotoxicity measurements after a recovery period indicate more robust recovery of healthy cells versus malignant cells. Compounds A and B or their Ch-encapsulated forms were shown to have negligible affinity for the ligand binding domain of estrogen receptor β or the androgen receptor in a fluorescent yeast screen, suggesting a lack of estrogenicity and androgenicity. Steroid-loaded chitosan nanoparticles display strong cytotoxicity towards MDA-MB-231 and PC-3 with a lack of hormone activity, indicating their safety and efficacy.

De novo missense mutation in GRIA2 in a patient with global developmental delay, autism spectrum disorder, and epileptic encephalopathy.

De novo variants are increasingly recognized as a common cause of early infantile epileptic encephalopathies. We present a 4-yr-old male with epileptic encephalopathy characterized by seizures, autism spectrum disorder, and global developmental delay. Whole-genome sequencing of the proband and his unaffected parents revealed a novel de novo missense variant in GRIA2 (c.1589A > T; p.Lys530Met; ENST00000264426.14). Variants in the GRIA2 gene were recently reported to cause an autosomal dominant neurodevelopmental disorder with language impairments and behavioral abnormalities (OMIM; MIM #618917), a condition characterized by intellectual disability and developmental delay in which seizures are a common feature. The de novo variant identified in our patient maps to the edge of a key ligand binding domain of the AMPA receptor and has not been previously reported in gnomAD or other public databases, making it novel. Our findings provided a long-sought diagnosis for this patient and support the link between GRIA2 and a dominant neurodevelopmental disorder.

Structural and Biophysical Mechanisms Driving Differential Hormone Response in Steroid Receptors

Steroid receptors (SRs) belong to the superfamily of ligand‐activated transcription factors that regulate a myriad of biological processes upon binding of cholesterol‐derived hormones. A poorly understood aspect of this transcriptional regulation is how diverse steroid hormones modulate the ligand binding domain (LBD) of steroid receptors to achieve selective transcriptional outcomes. Here we investigate hormone specificity in the ancestral steroid receptor 2 (ancSR2) which is transcriptionally activated by 3‐ketosteroid hormones and unresponsive to 3‐hydroxylated, A‐ring aromatized steroids (i.e., estrogens). Previous work uncovered evolutionarily conserved ligand sensing‐residues in the binding pocket of LBD which also enables hormone discrimination. Specifically, the L42 (Helix3)‐M75 (Helix5) interaction was predicted to enable discrimination between 3‐ketosteroids and estrogens. To further probe the role of these ligand sensors in mediating differential hormone responses, we mutated M75 to modulate the Helix3‐Helix5 interaction and determined how transcriptional function, protein structure and dynamics are impacted. Specifically, we have combined site‐directed mutagenesis with biophysical experiments, cell‐based assays, and molecular dynamics (MD) simulations. While our secondary structure measurements reveal minor impacts on structure, MD simulations reveal that M75 mutations modulate the status of the H3‐H5 interaction, subsequently affecting ancSR2 conformations in a hormone‐dependent manner. Additionally, hydrogen deuterium exchange‐mass spectrometry (HDX‐MS) measurements reveal conformational effects at regions distant from the mutation site that may modulate ancSR2 inter‐residue interaction networks for differential hormone response. The differential hormone response is supported in luciferase reporter assays as well as ligand binding assays. These studies will provide biophysical and structural insight into how steroid receptors achieve hormone‐specific transcriptional responses.

The switch from client holding to folding in the Hsp70/Hsp90 chaperone machineries is regulated by a direct interplay between co-chaperones

Folding of stringent clients requires transfer from Hsp70 to Hsp90. The co-chaperone Hop physically connects the chaperone machineries. Here, we define its role from the remodeling of Hsp70/40-client complexes to the mechanism of client transfer and the conformational switching from stalled to active client-processing states of Hsp90. We show that Hsp70 together with Hsp40 completely unfold a stringent client, the glucocorticoid receptor ligand-binding domain (GR-LBD) in large assemblies. Hop remodels these for efficient transfer onto Hsp90. As p23 enters, Hsp70 leaves the complex via switching between binding sites in Hop. Current concepts assume that to proceed to client folding, Hop dissociates and the co-chaperone p23 stabilizes the Hsp90 closed state. In contrast, we show that p23 functionally interacts with Hop, relieves the stalling Hsp90-Hop interaction, and closes Hsp90. This reaction allows folding of the client and is thus the key regulatory step for the progression of the chaperone cycle.

The T850D Phosphomimetic Mutation in the Androgen Receptor Ligand Binding Domain Enhances Recruitment at Activation Function 2.

Several key functions of the androgen receptor (AR) such as hormone recognition and co-regulator recruitment converge in the ligand binding domain (LBD). Loss- or gain-of-function of the AR contributes to pathologies such as the androgen insensitivity syndrome and prostate cancer. Here, we describe a gain-of-function mutation of the surface-exposed threonine at position 850, located at the amino-terminus of Helix 10 (H10) in the AR LBD. Since T850 phosphorylation was reported to affect AR function, we created the phosphomimetic mutation T850D. The AR T850D variant has a 1.5- to 2-fold increased transcriptional activity with no effect on ligand affinity. In the androgen responsive LNCaP cell line grown in medium with low androgen levels, we observed a growth advantage for cells in which the endogenous AR was replaced by AR T850D. Despite the distance to the AF2 site, the AR T850D LBD displayed an increased affinity for coactivator peptides as well as the 23FQNLF27 motif of AR itself. Molecular Dynamics simulations confirm allosteric transmission of the T850D mutation towards the AF2 site via extended hydrogen bond formation between coactivator peptide and AF2 site. This mechanistic study thus confirms the gain-of-function character of T850D and T850 phosphorylation for AR activity and reveals details of the allosteric communications within the LBD.

Androgen receptor mutations modulate activation by 11-oxygenated androgens and glucocorticoids.

Androgen receptor (AR) ligand-binding domain (LBD) mutations occur in ~20% of all castration-resistant prostate cancer (CRPC) patients. These mutations confer ligand promiscuity, but the affinity for many steroid hormone pathway intermediates is unknown. In this study, we investigated the stimulation of clinically relevant AR-LBD mutants by endogenous and exogenous steroid hormones present in CRPC patients to unravel their potential contribution to AR pathway reactivation. A meta-analysis of studies reporting untargeted analysis of AR mutants was performed to identify clinically relevant AR-LBD mutations. Using luciferase reporter and quantitative fluorescent microscopy, these AR mutants were screened for sensitivity for various endogenous steroids and synthetic glucocorticoids used in the treatment of CRPC. The meta-analysis revealed that ARL702H (3.4%), ARH875Y (4.9%), and ART878A (4.4%) were the most prevalent AR-LBD mutations across 1614 CRPC patients from 21 unique studies. Testosterone (EC50: 0.22 nmol/L) and 11-ketotestosterone (11KT, EC50: 0.74 nmol/L) displayed subnanomolar affinity for ARWT. The p.H875Y mutation selectively increased sensitivity of the AR for 11KT (EC50: 0.15 nmol/L, p < 0.05 vs ARWT), whereas p.L702H decreased sensitivity for 11KT by almost 50-fold. While cortisol and prednisolone both stimulate ARL702H, dexamethasone importantly does not. Both testosterone and 11KT effectively contribute to ARWT activation, while selective sensitization positions 11KT as a more prominent activator of ARH875Y. Dexamethasone may be a suitable alternative to prednisolone and should be explored in patients bearing the ARL702H.

Differential Gene Expression Profiles between N-Terminal Domain and Ligand-Binding Domain Inhibitors of Androgen Receptor Reveal Ralaniten Induction of Metallothionein by a Mechanism Dependent on MTF1.

Simple SummaryInhibition of the androgen receptor (AR) remains the mainstay treatment for prostate cancer. All current therapies involving AR inhibition either directly or indirectly target its ligand-binding domain (LBD). We have developed the first novel compounds which target the N-terminal domain, (NTD) a region which is essential for AR transcriptional activity. First-generation ralaniten (NCT02606123), and second-generation EPI-7386 (NCT04421222) remain the only AR-NTD inhibitors to progress to clinical trials. Here we aim to characterize differences between different classes of AR antagonists targeting the AR-LBD and the AR-NTD, as well as next generation AR-NTD inhibitors. An incidental finding was that ralaniten was uniquely associated with increased metallothionein expression which was independent of AR activity. Instead, expression of metallothionein genes was driven by MTF-1 indicating a potential off-target effect. Neither AR-LBD inhibitor enzalutamide nor second-generation AR-NTD inhibitor EPI-7170 had this effect. This work has important implications for the development of novel AR-NTD inhibitors.Hormonal therapies for prostate cancer target the androgen receptor (AR) ligand-binding domain (LBD). Clinical development for inhibitors that bind to the N-terminal domain (NTD) of AR has yielded ralaniten and its analogues. Ralaniten acetate is well tolerated in patients at 3600 mgs/day. Clinical trials are ongoing with a second-generation analogue of ralaniten. Binding sites on different AR domains could result in differential effects on AR-regulated gene expression. Here, we provide the first comparison between AR-NTD inhibitors and AR-LBD inhibitors on androgen-regulated gene expression in prostate cancer cells using cDNA arrays, GSEA, and RT-PCR. LBD inhibitors and NTD inhibitors largely overlapped in the profile of androgen-induced genes that they each inhibited. However, androgen also represses gene expression by various mechanisms, many of which involve protein–protein interactions. De-repression of the transcriptome of androgen-repressed genes showed profound variance between these two classes of inhibitors. In addition, these studies revealed a unique and strong induction of expression of the metallothionein family of genes by ralaniten by a mechanism independent of AR and dependent on MTF1, thereby suggesting this may be an off-target. Due to the relatively high doses that may be encountered clinically with AR-NTD inhibitors, identification of off-targets may provide insight into potential adverse events, contraindications, or poor efficacy.

Human-like performance umami electrochemical biosensor by utilizing co-electrodeposition of ligand binding domain T1R1-VFT and Prussian blue

Over the past decades, due to the desire for artificial umami flavors, apparatuses for detecting the umami taste have constantly been developed. Nevertheless, most information on umami is still acquired through human sensory assessment, which makes it difficult to establish an umami standard or quantify the umami flavor. In this study, the ligand binding domain called venus flytrap (VFT) domain of the umami taste receptor protein T1R1 was used as a recognition element, and an electrochemical biosensor based on a double-signal amplification strategy was constructed using single-walled carbon nanotubes (SWCNTs) and Prussian blue (PB). Moreover, the umami taste of four representative umami substances, inosine-5′-monophosphate (IMP), monosodium L-glutamate (MSG), beefy meaty peptide (BMP), and sodium succinate (WSA), were successfully quantitatively measured using differential pulse voltammetry (DPV) at an electrochemical workstation. Based on an equation (S/N = 3), the low detection limits (LODs) of IMP, MSG, BMP, and WSA were 0.1, 0.1, 0.1, and 0.01 pM, respectively. Meanwhile, a normalized signal intensity of more than 90% was kept for 4 days. The results showed that the biosensor could be used to detect umami substances with high sensitivity and selectivity, and was shown to have human-like performance. To develop the T1R1-VFT biosensor using the above-mentioned method, we utilized the ligand binding domain of the human umami receptor, rather than the entire umami receptor protein, which had a complex structure, having the following advantages: volume reduction, simplicity, and stability. This method has great potential for the detection of umami tastes, instead of using sensory evaluation, and for the development of new artificial flavorings.

Dual-mechanism estrogen receptor inhibitors

Efforts to improve estrogen receptor-α (ER)-targeted therapies in breast cancer have relied upon a single mechanism, with ligands having a single side chain on the ligand core that extends outward to determine antagonism of breast cancer growth. Here, we describe inhibitors with two ER-targeting moieties, one of which uses an alternate structural mechanism to generate full antagonism, freeing the side chain to independently determine other critical properties of the ligands. By combining two molecular targeting approaches into a single ER ligand, we have generated antiestrogens that function through new mechanisms and structural paradigms to achieve antagonism. These dual-mechanism ER inhibitors (DMERIs) cause alternate, noncanonical structural perturbations of the receptor ligand-binding domain (LBD) to antagonize proliferation in ER-positive breast cancer cells and in allele-specific resistance models. Our structural analyses with DMERIs highlight marked differences from current standard-of-care, single-mechanism antiestrogens. These findings uncover an enhanced flexibility of the ER LBD through which it can access nonconsensus conformational modes in response to DMERI binding, broadly and effectively suppressing ER activity.