Nuclear translocation of the membrane oxoeicosanoid/androgen receptor, OXER1: Possible mechanisms involved.

OXER1, the receptor for the arachidonic acid metabolite 5-οxo-eicosatetraenoic acid (5-oxo-ETE), has been reported to also bind and mediate the membrane-initiated actions of androgens. Indeed, androgens antagonize the 5-oxo-ETE effects through OXER1, affecting a number of signaling pathways and inhibiting cancer cell proliferation and migration. OXER1, being a GPCR, was classically described to be localized in the plasma membrane. However, for numerous GPCRs, there is now strong evidence that they can be also found in other cellular compartments, including the nucleus. The aim of the present work was to investigate OXER1's possible localization in the nucleus and identify the mechanism(s) involved. For this purpose, we verified OXER1's nuclear presence by immunofluorescence and western blot, in whole cells and nuclei of two different prostate cancer cell lines (DU-145 and LNCaP) and in CHO cells transfected with a GFP labelled OXER1, both in untreated and OXER1 ligands' treated cells. Mutated, OXER1-tGFP expressing, CHO cells were used to verify that OXER1 agonist (5-oxo-ETE) binding is necessary for OXER1 nuclear translocation. NLS sequences were in silico identified, and a specific inhibitor, as well as, specific importins' siRNAs were also utilized to explore the mechanism involved. Moreover, we examined the role of palmitoylation in OXER1 nuclear translocation by in silico identifying possible palmitoylation sites and using a palmitoylation inhibitor. Our results clearly show that OXER1 can be localized in the nucleus, in an agonist-dependent manner, that is inhibited by androgens. We also provide evidence for two possible mechanisms for its nuclear trafficking, that involve receptor palmitoylation and importin-mediated cytoplasmic-nuclear transport. In our knowledge, it is the first time that a membrane androgen receptor is identified into the nucleus, suggesting an alternative, more direct, mode of action, involving nuclear mechanisms. Therefore, our findings provide new insights on androgen-mediated actions and androgen-lipid interactions, and reveal new possible therapeutic targets, not only for cancer, but also for other pathological conditions in which OXER1 may have an important role.

Molecular Pathways of Genistein Activity in Breast Cancer Cells.

The most common malignancy in women is breast cancer. During the development of cancer, oncogenic transcription factors facilitate the overproduction of inflammatory cytokines and cell adhesion molecules. Antiapoptotic proteins are markedly upregulated in cancer cells, which promotes tumor development, metastasis, and cell survival. Promising findings have been found in studies on the cell cycle-mediated apoptosis pathway for medication development and treatment. Dietary phytoconstituents have been studied in great detail for their potential to prevent cancer by triggering the body's defense mechanisms. The underlying mechanisms of action may be clarified by considering the role of polyphenols in important cancer signaling pathways. Phenolic acids, flavonoids, tannins, coumarins, lignans, lignins, naphthoquinones, anthraquinones, xanthones, and stilbenes are examples of natural chemicals that are being studied for potential anticancer drugs. These substances are also vital for signaling pathways. This review focuses on innovations in the study of polyphenol genistein's effects on breast cancer cells and presents integrated chemical biology methods to harness mechanisms of action for important therapeutic advances.

Mining the ZINC database of natural products for specific, testosterone-like, OXER1 antagonists.

OXER1, the receptor for the oxidized arachidonic acid metabolite 5-oxo-ETE has been reported to play a significant role in inflammatory responses, being responsible for leucocyte chemotactic responses. Recently, we have identified OXER1 (GPR170) as a membrane receptor for androgens in prostate and breast cancer cells. Testosterone action via OXER1 induces specific Ca2+ release from intracellular organelles, modifies polymerized actin distribution induces apoptosis and decreases cancer cell migration. These actions are antagonized by 5-oxo-ETE. In addition, 5-oxo-ETE through a Gαi protein decreases cAMP, an action antagonized by testosterone. In this work, we mined the ZINC15 database, using QSAR, for natural compounds able to signal through Gαi and Gßγ simultaneously, mimicking testosterone actions, as well as for specific Gßγ interactors, inhibiting 5-oxo-ETE tumor promoting actions. We were able to identify four druggable Gαßγ and seven Gßγ specific OXER1 interactors. We further confirmed by bio-informatic methods their binding to the 5-oxo-ETE/testosterone binding groove of the receptor, their ADME properties and their possible interaction with other receptor and/or enzyme targets. Two compounds, ZINC04017374 (Naphthofluorescein) and ZINC08589130 (Puertogaline A) were purchased, tested in vitro and confirmed their OXER1 Gßγ and Gαßγ activity, respectively. The methodology followed is useful for a better understanding of the mechanism by which OXER1 mediates its actions, it has the potential to provide structural insights, in order to design small molecular specific interactors and ultimately design new anti-inflammatory and anti-cancer agents. Finally, the methodology may also be useful for identifying specific agonists/antagonists of other GPCRs.

Corrigendum to "Recognition motifs for importin 4 [(L)PPRS(G/P)P] and importin 5 [KP(K/Y)LV] binding, identified by bio-informatic simulation and experimental in vitro validation" [Comput Struct Biotechnol J 20 (2022) 5952-5961].

[This corrects the article DOI: 10.1016/j.csbj.2022.10.015.].

Molecular Pathways of Rosmarinic Acid Anticancer Activity in Triple-Negative Breast Cancer Cells: A Literature Review.

Breast cancer is the most frequent type of cancer in women. Oncogenic transcription factors promote the overproduction of cellular adhesion molecules and inflammatory cytokines during cancer development. Cancer cells exhibit significant upregulation of antiapoptotic proteins, resulting in increased cell survival, tumor growth, and metastasis. Research on the cell cycle-mediated apoptosis pathway for drug discovery and therapy has shown promising results. In fact, dietary phytoconstituents have been extensively researched for anticancer activity, providing indirect protection by activating endogenous defense systems. The role of polyphenols in key cancer signaling pathways could shed light on the underlying mechanisms of action. For instance, Rosmarinic Acid, a polyphenol constituent of many culinary herbs, has shown potent chemoprotective properties. In this review, we present recent progress in the investigation of natural products as potent anticancer agents, with a focus on the effect of Rosmarinic Acid on triple-negative BC cell lines resistant to hormone therapy. We highlight a variety of integrated chemical biology approaches aimed at utilizing relevant mechanisms of action that could lead to significant clinical advances in BC treatment.

Recognition motifs for importin 4 [(L)PPRS(G/P)P] and importin 5 [KP(K/Y)LV] binding, identified by bio-informatic simulation and experimental in vitro validation.

Nuclear translocation of large proteins is mediated through karyopherins, carrier proteins recognizing specific motifs of cargo proteins, known as nuclear localization signals (NLS). However, only few NLS signals have been reported until now. In the present work, NLS signals for Importins 4 and 5 were identified through an unsupervised in silico approach, followed by experimental in vitro validation. The sequences LPPRS(G/P)P and KP(K/Y)LV were identified and are proposed as recognition motifs for Importins 4 and 5 binding, respectively. They are involved in the trafficking of important proteins into the nucleus. These sequences were validated in the breast cancer cell line T47D, which expresses both Importins 4 and 5. Elucidating the complex relationships of the nuclear transporters and their cargo proteins is very important in better understanding the mechanism of nuclear transport of proteins and laying the foundation for the development of novel therapeutics, targeting specific importins.

Importins involved in the nuclear transportation of steroid hormone receptors: In silico and in vitro data.

The nuclear receptor superfamily (NRS) consists of 48 receptors for lipophilic substances and is divided into 7 different subfamilies, with subfamily 3 comprising steroid hormone receptors. Several nuclear receptors usually bind their cognate ligands in the cytosol and the complex (mono- or dimerized) is transported to the nucleus, where it acts as a transcription initiating factor for a number of genes. The general structure of nuclear receptors consists of an N-terminal activating domain (A/B), important for the binding of activating or inhibitory co-factors, the DNA-binding domain (C), responsible for the association of the receptor-ligand-co-factor complex to the nucleus, the ligand-AF2 domain (E/F), where ligand binding occurs as well as that of ligand-dependent activating/inhibiting factors, and a flexible/non-structured domain (D), linking the DBD and LBD, called hinge region, on which a significant number of post-translational modifications occur. This hinge domain, for the sub-class of steroid receptors, is a non-structured domain and was reported as mainly responsible for the nuclear transport of steroid receptors, since it contains a specific amino acid sequence (Nuclear Localization Signal-NLS), recognized by importin α. In addition to the importin α/ß complex, a number of other importins have been discovered and reported to be responsible for the nuclear transport of a number of significant proteins; however, the corresponding recognition sequences for these importins have not been identified. Recently, we have reported the identification of the NLS sequences for importins 4, 5 and 7. In this work, we provide in silico data, followed by experimental in vitro validation, showing that these alternative importins are responsible for the nuclear transportation of steroid hormone receptors such as ERα, AR and PR, and therefore they may consist of alternative targets for the pharmacological manipulation of steroid hormone actions. Moreover, we provide additional in silico data for the hinge region of steroid hormone receptors which is highly enriched with NLS sequences for importins 4, 5 and 7, in addition to the recognition NLS for importin α/ß.

OXER1 mediates testosterone-induced calcium responses in prostate cancer cells.

In prostate cancer, calcium homeostasis plays a significant role in the disease's development and progression. Intracellular calcium changes are an important secondary signal, triggered by a variety of extracellular stimuli, that controls many cellular functions. One of the main events affecting calcium is androgen signaling. Indeed, via calcium changes, androgens regulate cell processes like cell growth, differentiation and motility. In the present work we explored the nature of the receptor involved in calcium response induced by membrane-acting testosterone in prostate cancer cells. We report that testosterone, independently of the presence of the classical androgen receptor, can rapidly increase intracellular calcium from calcium stores, through the oxoeicosanoid receptor 1 (OXER1) and a specific signaling cascade that triggers calcium release from the endoplasmic reticulum. These findings reveal for the first time the receptor involved in the rapid calcium changes induced by androgens. Moreover, they further support the notion that androgens, even in the absence of AR, can still exert specific effects that regulate cancer cell fate.

New Antagonists of the Membrane Androgen Receptor OXER1 from the ZINC Natural Product Database.

OXER1 (oxoeicosanoid receptor 1) was deorphanized in 1993 and found to be the specific receptor for the arachidonic acid metabolite 5-oxo-ETE. Recently, we have reported that androgen binds to this receptor also, being a membrane androgen receptor, triggering a number of its membrane-mediated actions (cell migration, apoptosis, cell proliferation, Ca2+ movements). In addition, our previous work suggested that a number of natural monomeric and oligomeric polyphenols interact with OXER1, acting similar to testosterone. Here, we interrogated the natural product chemical space and identified nine polyphenolic molecules with interesting in silico pharmacological activities as putative OXER1 antagonists. The molecule with the best pharmacokinetic-pharmacodynamic properties (ZINC15959779) was purchased and tested on OXER1, in prostate cancer cell cultures. It showed that it has actions similar to those of testosterone in inhibiting cAMP, while it had no action in intracellular Ca2+ mobilization or actin cytoskeleton rearrangement/migration. These results are discussed under the prism of structure-activity relationships and in silico models of the OXER1 binding groove. We suggest that these compounds, together with the previously reported (poly)phenolic compounds, can be lead structures for the exploration of the anti-inflammatory and antiproliferative effects of OXER1 antagonists.

Natural Polyphenols Inhibit the Dimerization of the SARS-CoV-2 Main Protease: The Case of Fortunellin and Its Structural Analogs.

3CL-Pro is the SARS-CoV-2 main protease (MPro). It acts as a homodimer to cleave the large polyprotein 1ab transcript into proteins that are necessary for viral growth and replication. 3CL-Pro has been one of the most studied SARS-CoV-2 proteins and a main target of therapeutics. A number of drug candidates have been reported, including natural products. Here, we employ elaborate computational methods to explore the dimerization of the 3CL-Pro protein, and we formulate a computational context to identify potential inhibitors of this process. We report that fortunellin (acacetin 7-O-neohesperidoside), a natural flavonoid O-glycoside, and its structural analogs are potent inhibitors of 3CL-Pro dimerization, inhibiting viral plaque formation in vitro. We thus propose a novel basis for the search of pharmaceuticals as well as dietary supplements in the fight against SARS-CoV-2 and COVID-19.

The sequence [EKRKI(E/R)(K/L/R/S/T)] is a nuclear localization signal for importin 7 binding (NLS7).

BACKGROUND: Nuclear translocation of large proteins is mediated through specific protein carriers, collectively named karyopherins (importins, exportins and adaptor proteins). Cargo proteins are recognized by importins through specific motifs, known as nuclear localization signals (NLS). However, only the NLS recognized by importin α and transportin (M9 NLS) have been identified so far METHODS: An unsupervised in silico approach was used, followed by experimental validation. RESULTS: We identified the sequence EKRKI(E/R)(K/L/R/S/T) as an NLS signal for importin 7 recognition. This sequence was validated in the breast cancer cell line T47D, which expresses importin 7. Finally, we verified that importin 7-mediated nuclear protein transport is affected by cargo protein phosphorylation. CONCLUSIONS: The NLS sequence for importin 7 was identified and we propose this approach as an identification method of novel specific NLS sequences for ß-karyopherin family members. GENERAL SIGNIFICANCE: Elucidating the complex relationships of the nuclear transporters and their cargo proteins may help in laying the foundation for the development of novel therapeutics, targeting specific importins, with an immediate translational impact.

A simple open source bioinformatic methodology for initial exploration of GPCR ligands' agonistic/antagonistic properties.

Drug development is an arduous procedure, necessitating testing the interaction of a large number of potential candidates with potential interacting (macro)molecules. Therefore, any method which could provide an initial screening of potential candidate drugs might be of interest for the acceleration of the procedure, by highlighting interesting compounds, prior to in vitro and in vivo validation. In this line, we present a method which may identify potential hits, with agonistic and/or antagonistic properties on GPCR receptors, integrating the knowledge on signaling events triggered by receptor activation (GPCRs binding to Gα,ß,γ proteins, and activating Gα , exchanging GDP for GTP, leading to a decreased affinity of the Gα for the GPCR). We show that, by integrating GPCR-ligand and Gα -GDP or -GTP binding in docking simulation, which correctly predicts crystallographic data, we can discriminate agonists, partial agonists, and antagonists, through a linear function, based on the ΔG (Gibbs-free energy) of liganded-GPCR/Gα -GDP. We built our model using two Gαs (ß2-adrenergic and prostaglandin-D2 ), four Gαi (µ-opioid, dopamine-D3, adenosine-A1, rhodopsin), and one Gαo (serotonin) receptors and validated it with a series of ligands on a recently deorphanized Gαi receptor (OXER1). This approach could be a valuable tool for initial in silico validation and design of GPRC-interacting ligands.

Membrane androgen receptors (OXER1, GPRC6A AND ZIP9) in prostate and breast cancer: A comparative study of their expression.

Accumulating evidence during the last decades revealed that androgens exert membrane-initiated actions leading to the modulation of significant cellular processes, important for cancer cell growth and metastasis (including prostate and breast), that involve signaling via specific kinases. Collectively, many nonclassical, cell surface-initiated androgen actions are mediated by novel membrane androgen receptors (mARs), unrelated to nuclear androgen receptors. Recently, our group identified the G protein coupled oxo-eicosanoid receptor 1 (OXER1) (a receptor of the arachidonic acid metabolite, 5-oxoeicosatetraenoic acid, 5-oxoETE) as a novel mAR involved in the rapid effects of androgens. However, two other membrane proteins, G protein-coupled receptor family C group 6 member A (GPRC6A) and zinc transporter member 9 (ZIP9) have also been portrayed as mARs, related to the extranuclear action of androgens. In the present work, we present a comparative study of in silico pharmacology, gene expression and immunocytochemical data of the three receptors in various prostate and breast cancer cell lines. Furthermore, we analyzed the immunohistochemical expression of these receptors in human tumor and non-tumoral specimens and provide a pattern of expression and intracellular distribution.