Structure-Activity Relationship Study Enables the Discovery of a Novel Berberine Analogue as the RXRα Activator to Inhibit Colon Cancer.

We reported recently that berberine (Ber), a traditional oriental medicine to treat gastroenteritis, binds and activates retinoid X receptor α (RXRα) for suppressing the growth of colon cancer cells. Here, we extended our studies based on the binding mode of Ber with RXRα by design, synthesis, and biological evaluation of a focused library of 15 novel Ber analogues. Among them, 3,9-dimethoxy-5,6-dihydroisoquinolino[3,2-a]isoquinolin-7-ium chloride (B-12) was identified as the optimal RXRα activator. More efficiently than Ber, B-12 bound and altered the conformation of RXRα/LBD, thereby suppressing the Wnt/ß-catenin pathway and colon cancer cell growth via RXRα mediation. In addition, B-12 not only preserved Ber's tumor selectivity but also greatly improved its bioavailability. Remarkably, in mice, B-12 did not show obvious side effects including hypertriglyceridemia as other RXRα agonists or induce hepatorenal toxicity. Together, our study describes an approach for the rational design of Ber-derived RXRα activators as novel effective antineoplastic agents for colon cancer.

miR-191 promotes radiation resistance of prostate cancer through interaction with RXRA.

Radiation therapy is a common treatment for prostate cancer, however recurrence remains a problem. MicroRNA expression is altered in prostate cancer and may promote therapy resistance. Through bioinformatic analyses of TCGA and CPC-GENE patient cohorts, we identified higher miR-191 expression in tumor versus normal tissue, and increased expression in higher Gleason scores. In vitro and in vivo experiments demonstrated that miR-191 overexpression promotes radiation survival, and contributes to a more aggressive phenotype. Retinoid X receptor alpha, RXRA, was discovered to be a novel target of miR-191, and knockdown recapitulated radioresistance. Furthermore, treatment of prostate cancer cells with the RXRA agonist 9-cis-retinoic acid restored radiosensitivity. Supporting this relationship, patients with high miR-191 and low RXRA abundance experienced quicker biochemical recurrence. Reduced RXRA translated to a higher risk of distant failure after radiotherapy. Notably, this miR-191/RXRA interaction was conserved in a novel primary cell line derived from radiorecurrent prostate cancer. Together, our findings demonstrate that miR-191 promotes prostate cancer survival after radiotherapy, and highlights retinoids as a potential option to improve radiotherapy response.

Methods to Assess Activity and Potency of Rexinoids Using Rapid Luciferase-Based Assays: A Case Study with NEt-TMN.

This chapter outlines the materials, methods, and procedures for the in vitro biological evaluation of retinoid-X-receptor (RXR) agonists including 6-(ethyl(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino)nicotinic acid (NEt-TMN), as well as several NEt-TMN analog compounds recently reported by our group. These methods have general applicability beyond this NEt-TMN case study, and can be employed to characterize and biologically evaluate other putative RXR agonists (rexinoids), and benchmarked against perhaps the most common rexinoid known as bexarotene (Bex), a drug awarded FDA approval for the treatment of cutaneous T-cell lymphoma in 1999 but that is also prescribed for non-small cell lung cancer and continues to be explored in multiple human cancer types. The side-effect profile of Bex treatment includes hypothyroidism and hypertriglyceridemia arising from the inhibition or activation of additional nuclear receptors that partner with RXR. Because rexinoids often exhibit selectivity for RXR activation, versus activating the retinoic-acid-receptor (RAR), rexinoid treatment avoids the cutaneous toxicity commonly associated as a side effect with retinoids. There are many examples of other potent rexinoids, where biological evaluation has contributed useful insight into qSAR studies on these compounds, often also benchmarked to Bex, as potential treatments for cancer. Because of differential pleiotropy in other pathways, even closely related rexinoids display unique side-effect and activity profiles. Notable examples of potent rexinoids in addition to Bex and NEt-TMN include CD3254, LGD100268, and 9-cis-UAB30. Indeed, the methods described herein to evaluate NEt-TMN and analogous rexinoids are generally applicable to a wider variety of potent, moderate, and even weak RXR ligands.In terms of in vitro biological evaluation, methods for a rapid and preliminary assessment of rexinoid activity are described by employing a biologically relevant, RXR-responsive element (RXRE)-mediated transcription assay in mammalian cells. In addition, a second, more sensitive assay is also detailed that utilizes activation of RXR-RXR homodimers in the context of a mammalian two-hybrid (M2H) luciferase assay. Methods for applying the M2H assay at different rexinoid concentrations are further described for the determination of EC50 values for rexinoids from dose-response curves.

Tetrazolone as an acid bioisostere: application to marketed drugs containing a carboxylic acid.

Matched molecular pair analysis was used to evaluate the ability of a tetrazolone group to act as a bioisostere of a carboxylic acid. Compound 7, a tetrazolone of the anti-hypertensive drug, telmisartan 6, was shown to be a potent AT1 antagonist (Kb = 0.14 nM), with activity comparable to telmisartan itself (Kb = 0.44 nM). Additionally, compound 9, a tetrazolone congener of the marketed anti-cancer agent, bexarotene 8, was shown to be an agonist at the retinoid X receptor alpha (EC50 = 64 nM). Compounds containing a tetrazolone group showed similar microsomal stability and plasma protein binding to marketed acid counterparts, while also reducing the value for clog P. Furthermore, compound 7 displayed an improved rat pharmacokinetic profile cf. telmisartan 6. Taken together, the results demonstrate that a tetrazolone group may serve as a bioisostere for a carboxylic acid.

Trimodal Therapy: Combining Hyperthermia with Repurposed Bexarotene and Ultrasound for Treating Liver Cancer.

Repurposing of existing cancer drugs to overcome their physical limitations, such as insolubility, represents an attractive strategy to achieve enhanced therapeutic efficacy and broaden the range of clinical applications. Such an approach also promises to offer substantial cost savings in drug development efforts. Here we repurposed FDA-approved topical agent bexarotene (Targretin), currently in limited use for cutaneous manifestations of T-cell lymphomas, and re-engineer it for use in solid tumor applications by forming self-assembling nanobubbles. Physico-chemical characterization studies of the novel prodrug nanobubbles demonstrated their stability, enhanced target cell internalization capability, and highly controlled release profile in response to application of focused ultrasound energy. Using an in vitro model of hepatocellular carcinoma and an in vivo large animal model of liver ablation, we demonstrate the effectiveness of bexarotene prodrug nanobubbles when used in conjunction with catheter-based ultrasound, thereby highlighting the therapeutic promise of this trimodal approach.

Identification of a naturally occurring retinoid X receptor agonist from Brazilian green propolis.

BACKGROUND: Brazilian green propolis (BGP), a resinous substance produced from Baccharis dracunculifolia by Africanized honey bees (Apis mellifera), is used as a folk medicine. Our present study explores the retinoid X receptor (RXR) agonistic activity of BGP and the identification of an RXR agonist in its extract. METHODS: RXRα agonistic activity was evaluated using a luciferase reporter gene assay. Isolation of the RXRα agonist from the ethanolic extract of BGP was performed using successive silica gel and a reversed phase column chromatography. The interaction between the isolated RXRα agonist and RXRα protein was predicted by a receptor-ligand docking simulation. The nuclear receptor (NR) cofactor assay was used to estimate whether the isolated RXRα agonist bound to various NRs, including RXRs and peroxisome proliferator-activated receptors (PPARs). We further examined its effect on adipogenesis in 3T3-L1 fibroblasts. RESULTS: We identified drupanin as an RXRα agonist with an EC50 value of 4.8 ± 1.0 µM. Drupanin activated three RXR subtypes by a similar amount and activated PPARγ moderately. Additionally, drupanin induced adipogenesis and elevated aP2 mRNA levels in 3T3-L1 fibroblasts. CONCLUSIONS: Drupanin, a component of BGP, is a novel RXR agonist with slight PPARγ agonistic activity. GENERAL SIGNIFICANCE: This study revealed for the first time that BGP activates RXR and drupanin is an RXR agonist in its extract.

Mechanism for antioxidative effects of thiazolidinediones in pancreatic ß-cells.

Thiazolidinediones (TZDs) are synthetic ligands of peroxisome proliferator-activated receptor-γ (PPARγ), a member of the nuclear receptor superfamily. TZDs are known to increase insulin sensitivity and also to have an antioxidative effect. In this study, we tested whether TZDs protect pancreatic ß-cells from oxidative stress, and we investigated the mechanism involved in this process. To generate oxidative stress in pancreatic ß-cells (INS-1 and ßTC3) or isolated islets, glucose oxidase was added to the media. The extracellular and intracellular reactive oxygen species (ROS) were measured to directly determine the antioxidant effect of TZDs. The phosphorylation of JNK/MAPK after oxidative stress was detected by Western blot analysis, and glucose-stimulated insulin secretion and cell viability were also measured. TZDs significantly reduced the ROS levels that were increased by glucose oxidase, and they effectively prevented ß-cell dysfunction. The antioxidative effect of TZDs was abolished in the presence of a PPARγ antagonist, GW9662. Real-time PCR was used to investigate the expression levels of antioxidant genes. The expression of catalase, an antioxidant enzyme, was increased by TZDs in pancreatic ß-cells, and the knockdown of catalase significantly inhibited the antioxidant effect of TZDs. These results suggest that TZDs effectively protect pancreatic ß-cells from oxidative stress, and this effect is dependent largely on PPARγ. In addition, the expression of catalase is increased by TZDs, and catalase, at least in part, mediates the antioxidant effect of TZDs in pancreatic ß-cells.

Isoform-selective activation of human constitutive androstane receptor by Ginkgo biloba extract: functional analysis of the SV23, SV24, and SV25 splice variants.

Naturally occurring splice variants of human constitutive androstane receptor (hCAR) exist, including hCAR-SV23 (insertion of amino acids SPTV), hCAR-SV24 (APYLT), and hCAR-SV25 (SPTV and APYLT). An extract of Ginkgo biloba was reported to activate hCAR-SV24 and the wild type (hCAR-WT). However, it is not known whether it selectively affects hCAR splice variants, how it activates hCAR isoforms, and which chemical is responsible for the effects of the extract. Therefore, we evaluated the impact of G. biloba extract on the functionality of hCAR-SV23, hCAR-SV24, hCAR-SV25, and hCAR-WT and compared it with that of phenobarbital, di-(2-ethylhexyl)phthalate (DEHP), 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO), and 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) in cell-based reporter gene assays. Among the hCAR splice variants investigated, only hCAR-SV23 was activated by G. biloba extract, and this required cotransfection of a retinoid X receptor α (RXRα) expression plasmid. The extract activated hCAR-SV23 to a lesser extent than hCAR-WT, but ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, and bilobalide were not responsible for the effects of the extract. CITCO activated hCAR-SV23, hCAR-SV24, and hCAR-WT. By comparison, phenobarbital activated hCAR-WT, whereas DEHP activated hCAR-SV23, hCAR-SV24 (with exogenous RXRα supplementation), and hCAR-WT. TCPOBOP did not affect the activity of any of the isoforms. G. biloba extract and phenobarbital did not bind or recruit coactivators to the ligand-binding domains of hCAR-WT and hCAR-SV23, whereas positive results were obtained with the controls (CITCO for hCAR-WT and DEHP for hCAR-SV23). In conclusion, G. biloba extract activates hCAR in an isoform-selective manner, and hCAR-SV23, hCAR-SV24, and hCAR-WT have overlapping, but distinct, sets of ligands.

Furocoumarin derivatives from radix Angelicae dahuricae and their effects on RXRα transcriptional regulation.

A novel furocoumarin derivative named oxyalloimperatorin (1), together with seventeen furocoumarins 2-18 were isolated from the radix of Angelica dahurica. The chemical structure of new metabolite was characterized by analysis of IR, NMR, and HR-ESI-MS spectroscopic data. Among the isolated compounds, 13, 16, and 18 (each at 20 µM) could significantly promote the gene transcriptional function of nuclear receptor RXRα. While 7-9, 13, 14, and the new structure 1 (each at 20 µM) showed significant reduction in RXRα gene transcriptional activities induced by 9-cis-retinoid acid. The findings indicated that these furocoumarin skeleton derivatives might hold beneficial effects on many intractable diseases, such as cancer and metabolic diseases, due to their potential activities on regulating the transcriptional activation function of RXRα.