. (2017) 'The molecular basis of the interactions between synthetic retinoic acid analogues and the retinoic acid receptors. ', MedChemComm., 8 (3). pp. 578-592. Further information on publisher's website:https://doi.org/10.1039/C6MD00680APublisher's copyright statement:Additional information: Use policyThe full-text may be used and/or reproduced, and given to third parties in any format or medium, without prior permission or charge, for personal research or study, educational, or not-for-pro t purposes provided that:• a full bibliographic reference is made to the original source • a link is made to the metadata record in DRO • the full-text is not changed in any way The full-text must not be sold in any format or medium without the formal permission of the copyright holders.Please consult the full DRO policy for further details. All-trans-retinoic acid (ATRA) and its synthetic analogues EC23 and EC19 direct cellular differentiation by interacting as ligands for the retinoic acid receptor (RARα, β and γ) family of nuclear receptor proteins. To date, a number of crystal structures of natural and synthetic ligands complexed to their target proteins have been solved, providing molecular level snap-shots of ligand binding. However, a deeper understanding of receptor and ligand flexibility and conformational freedom is required to develop stable and effective ATRA analogues for clinical use. Therefore, we have used molecular modelling techniques to define RAR interactions with ATRA and two synthetic analogues, EC19 and EC23, and compared their predicted biochemical activities to experimental measurements of relative ligand affinity and recruitment of coactivator proteins. A comprehensive molecular docking approach that explored the conformational space of the ligands indicated that ATRA is able to bind the three RAR proteins in a number of conformations with one extended structure being favoured. In contrast the biologically-distinct isomer, 9-cis-retinoic acid (9CRA), showed significantly less conformational flexibility in the RAR binding pockets. These findings were used to inform docking studies of the synthetic retinoids EC23 and EC19, and their respective methyl esters. EC23 was found to be an excellent mimic for ATRA, and occupied similar binding modes to ATRA in all three target RAR proteins. In comparison, EC19 exhibited an alternative binding mode which reduces the strength of key polar interactions in RARα/γ but is well-suited to the larger RARβ binding pocket. In contrast, docking of the corresponding esters revealed the loss of key polar interactions which may explain the much reduced biological activity. Our computational results were complemented using an in vitro binding assay based on FRET measurements, which showed that EC23 was a strongly binding, pan-agonist of the RARs, while EC19 exhibited specificity for RARβ, as predicted by the docking studies. These findings can account for the distinct behaviour of EC23 and EC19 in cellular differentiation assays, and additionally, the methods described ...

Burkholderia cenocepacia (B. cenocepacia) is an opportunistic bacterium; causing severe life threatening systemic infections in immunocompromised individuals including cystic fibrosis patients. The lack of gasdermin D (GSDMD) protects mice against endotoxin lipopolysaccharide (LPS) shock. On the other hand, GSDMD promotes mice survival in response to certain bacterial infections. However, the role of GSDMD during B. cenocepacia infection is not yet determined. Our in vitro study shows that GSDMD restricts B. cenocepacia replication within macrophages independent of its role in cell death through promoting mitochondrial reactive oxygen species (mROS) production. mROS is known to stimulate autophagy, hence, the inhibition of mROS or the absence of GSDMD during B. cenocepacia infections reduces autophagy which plays a critical role in the restriction of the pathogen. GSDMD promotes inflammation in response to B. cenocepacia through mediating the release of inflammasome dependent cytokine (IL-1β) and an independent one (CXCL1) (KC). Additionally, different B. cenocepacia secretory systems (T3SS, T4SS, and T6SS) contribute to inflammasome activation together with bacterial survival within macrophages. In vivo study confirmed the in vitro findings and showed that GSDMD restricts B. cenocepacia infection and dissemination and stimulates autophagy in response to B. cenocepacia. Nevertheless, GSDMD promotes lung inflammation and necrosis in response to B. cenocepacia without altering mice survival. This study describes the double-edged functions of GSDMD in response to B. cenocepacia infection and shows the importance of GSDMD-mediated mROS in restriction of B. cenocepacia.

Autophagy is a proposed route of amyloid-β (Aβ) clearance by microglia that is halted in Alzheimer’s Disease (AD), though mechanisms underlying this dysfunction remain elusive. Here, primary microglia from adult AD (5xFAD) mice were utilized to demonstrate that 5xFAD microglia fail to degrade Aβ and express low levels of autophagy cargo receptor NBR1. In 5xFAD mouse brains, we show for the first time that AD microglia express elevated levels of microRNA cluster Mirc1/Mir17-92a, which is known to downregulate autophagy proteins. By in situ hybridization in post-mortem AD human tissue sections, we observed that the Mirc1/Mir17-92a cluster member miR-17 is also elevated in human AD microglia, specifically in the vicinity of Aβ deposits, compared to non-disease controls. We show that NBR1 expression is negatively correlated with expression of miR-17 in human AD microglia via immunohistopathologic staining in human AD brain tissue sections. We demonstrate in healthy microglia that autophagy cargo receptor NBR1 is required for Aβ degradation. Inhibiting elevated miR-17 in 5xFAD mouse microglia improves Aβ degradation, autophagy, and NBR1 puncta formation in vitro and improves NBR1 expression in vivo. These findings offer a mechanism behind dysfunctional autophagy in AD microglia which may be useful for therapeutic interventions aiming to improve autophagy function in AD.

(1) Background and Aim: All-trans retinoic acid (ATRA) induces differentiation and inhibits growth of many cancer cells. However, resistance develops rapidly prompting the urgent need for new synthetic and potent derivatives. EC19 and EC23 are two synthetic retinoids with potent stem cell neuro-differentiation activity. Here, these compounds were screened for their in vitro antiproliferative and cytotoxic activity using an array of different cancer cell lines. (2) Methods: MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, AV/PI (annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI)), cell cycle analysis, immunocytochemistry, gene expression analysis, Western blotting, measurement of glutamate and total antioxidant concentrations were recruited. (3) Results: HepG2, Caco-2, and MCF-7 were the most sensitive cell lines; HepG2 (ATRA; 36.2, EC19; 42.2 and EC23; 0.74 µM), Caco-2 (ATRA; 58.0, EC19; 10.8 and EC23; 14.7 µM) and MCF-7 (ATRA; 99.0, EC19; 9.4 and EC23; 5.56 µM). Caco-2 cells were selected for further biochemical investigations. Isobologram analysis revealed the combined synergistic effects with 5-fluorouracil with substantial reduction in IC50. All retinoids induced apoptosis but EC19 had higher potency, with significant cell cycle arrest at subG0-G1, -S and G2/M phases, than ATRA and EC23. Moreover, EC19 reduced cellular metastasis in a transwell invasion assay due to overexpression of E-cadherin, retinoic acid-induced 2 (RAI2) and Werner (WRN) genes. (4) Conclusion: The present study suggests that EC-synthetic retinoids, particularly EC19, can be effective, alone or in combinations, for potential anticancer activity to colorectal cancer. Further in vivo studies are recommended to pave the way for clinical applications.

All-trans retinoic acid (ATRA) plays key roles in neurogenesis mediated by retinoic acid receptors (RARs). RARs are important targets for the therapeutic regulation of neurogenesis but effective drug development depends on modelling-based strategies to design high-specificity ligands in combination with good biological assays to discriminate between target-specificity and off-target effects. Using neuronal differentiation as a model, the aim of this study was to test the hypothesis that responses across different temporal scales and assay platforms can be used as comparable measures of retinoid activity. In biological assays based on cell phenotype or behaviour, two structurally similar synthetic retinoids, differing in RAR affinity and specificity, retained their relative activities across different temporal scales. In contrast, assays based on the transcriptional activation of specific genes in their normal genomic context were less concordant with biological assays. Gene-induction assays for retinoid activity as modulators of neurogenesis require careful interpretation in the light of variation in ligand-receptor affinity, receptor expression and gene function. A better characterization of neuronal phenotypes and their regulation by retinoids is badly needed as a framework for understanding how to regulate neuronal development.