Carl E. Wagner

Modeling, synthesis and biological evaluation of potential retinoid X receptor (RXR) selective agonists: novel analogues of 4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethynyl]benzoic acid (bexarotene).

This report describes the synthesis of analogues of 4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethynyl]benzoic acid (1), commonly known as bexarotene, and their analysis in acting as retinoid X receptor (RXR)-specific agonists. Compound 1 has FDA approval to treat cutaneous T-cell lymphoma (CTCL); however, its use can cause side effects such as hypothyroidism and increased triglyceride concentrations, presumably by disruption of RXR heterodimerization with other nuclear receptors. The novel analogues in the present study have been evaluated for RXR activation in an RXR mammalian-2-hybrid assay as well as an RXRE-mediated transcriptional assay and for their ability to induce apoptosis as well as for their mutagenicity and cytotoxicity. Analysis of 11 novel compounds revealed the discovery of three analogues that best induce RXR-mediated transcriptional activity, stimulate apoptosis, have comparable K(i) and EC(50) values to 1, and are selective RXR agonists. Our experimental approach suggests that rational drug design can develop new rexinoids with improved biological properties.

Modeling, synthesis and biological evaluation of potential retinoid X receptor-selective agonists: novel halogenated analogues of 4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethynyl]benzoic acid (bexarotene).

The synthesis of halogenated analogues of 4‐[1‐(3,5,5,8,8‐pentamethyl‐5,6,7,8‐tetrahydro‐2‐naphthyl)ethynyl]benzoic acid (1), known commonly as bexarotene, and their evaluation for retinoid X receptor (RXR)‐specific agonist performance is described. Compound 1 is FDA approved to treat cutaneous T‐cell lymphoma (CTCL); however, bexarotene treatment can induce hypothyroidism and elevated triglyceride levels, presumably by disrupting RXR heterodimer pathways for other nuclear receptors. The novel halogenated analogues in this study were modeled and assessed for their ability to bind to RXR and stimulate RXR homodimerization in an RXRE‐mediated transcriptional assay as well as an RXR mammalian‐2‐hybrid assay. In an array of eight novel compounds, four analogues were discovered to promote RXR‐mediated transcription with EC50 values similar to that of 1 and are selective RXR agonists. Our approach also uncovered a periodic trend of increased binding and homodimerization of RXR when substituting a halogen atom for a proton ortho to the carboxylic acid on 1.

Aspartate β‐hydroxylase modulates cellular senescence through glycogen synthase kinase 3β in hepatocellular carcinoma

Aspartate β‐hydroxylase (ASPH) is an enzyme overexpressed in human hepatocellular carcinoma (HCC) tumors that participates in the malignant transformation process. We determined if ASPH was a therapeutic target by exerting effects on cellular senescence to retard HCC progression. ASPH knockdown or knockout was achieved by short hairpin RNAs or the CRISPR/Cas9 system, respectively, whereas enzymatic inhibition was rendered by a potent second‐generation small molecule inhibitor of ASPH. Alterations of cell proliferation, colony formation, and cellular senescence were evaluated in human HCC cell lines. The potential mechanisms for activating cellular senescence were explored using murine subcutaneous and orthotopic xenograft models. Inhibition of ASPH expression and enzymatic activity significantly reduced cell proliferation and colony formation but induced tumor cell senescence. Following inhibition of ASPH activity, phosphorylation of glycogen synthase kinase 3β and p16 expression were increased to promote senescence, whereas cyclin D1 and proliferating cell nuclear antigen were decreased to reduce cell proliferation. The mechanisms involved demonstrate that ASPH binds to glycogen synthase kinase 3β and inhibits its subsequent interactions with protein kinase B and p38 upstream kinases as shown by coimmunoprecipitation. In vivo experiments demonstrated that small molecule inhibitor treatment of HCC bearing mice resulted in significant dose‐dependent reduced tumor growth, induced phosphorylation of glycogen synthase kinase 3β, enhanced p16 expression in tumor cells, and promoted cellular senescence. Conclusions: We have identified a new mechanism that promotes HCC growth and progression by modulating senescence of tumor cells; these findings suggest that ASPH enzymatic activity is a novel therapeutic target for HCC. (Hepatology 2016;63:1213–1226)

Analysis of differential secondary effects of novel rexinoids: Select rexinoid X receptor ligands demonstrate differentiated side effect profiles

In order to determine the feasibility of utilizing novel rexinoids for chemotherapeutics and as potential treatments for neurological conditions, we undertook an assessment of the side effect profile of select rexinoid X receptor (RXR) analogs that we reported previously. We assessed pharmacokinetic profiles, lipid and thyroid‐stimulating hormone (TSH) levels in rats, and cell culture activity of rexinoids in sterol regulatory element‐binding protein (SREBP) induction and thyroid hormone inhibition assays. We also performed RNA sequencing of the brain tissues of rats that had been dosed with the compounds. We show here for the first time that potent rexinoid activity can be uncoupled from drastic lipid changes and thyroid axis variations, and we propose that rexinoids can be developed with improved side effect profiles than the parent compound, bexarotene (1).

Synthesis and biological evaluation of halogenated curcumin analogs as potential nuclear receptor selective agonists.

This report describes the synthesis of analogs of curcumin, and their analysis in acting as nuclear receptor specific agonists. Curcumin (CM), a turmeric-derived bioactive polyphenol found in curry, has recently been identified as a ligand for the vitamin D receptor (VDR), and it is possible that CM exerts some of its bioeffects via direct binding to VDR and/or other proteins in the nuclear receptor superfamily. Using mammalian-two-hybrid (M2H) and vitamin D responsive element (VDRE) biological assay systems, we tested CM and 11 CM synthetic analogs for their ability to activate VDR signaling. The M2H assay revealed that RXR and VDR association was induced by CM and several of its analogs. VDRE-based assays demonstrated that pure curcumin and eight CM analogs activated transcription of a luciferase plasmid at levels approaching that of the endocrine 1,25 dihydroxyvitamin D(3) (1,25D) ligand in human colon cancer cells (HCT-116). Additional experiments were performed in HCT-116 utilizing various nuclear receptors and hormone responsive elements to determine the receptor specificity of curcumin binding. CM did not appear to activate transcription in a glucocorticoid responsive system. However, CM along with several analogs elicited transcriptional activation in retinoic acid and retinoid X receptor (RXR) responsive systems. M2H assays using RXR-RXR, VDR-SRC1 and VDR-DRIP revealed that CM and select analogs stimulate RXR homodimerization and VDR-coactivator interactions. These studies may lead to the discovery of novel curcumin analogs that activate nuclear receptors, including RXR, RAR and VDR, resulting in similar health benefits as those for vitamins A and D, such as lowering the risk of epithelial and colon cancers.

Modeling, synthesis, and biological evaluation of potential retinoid X receptor (RXR) selective agonists: Novel analogues of 4-[1-(3,5,5,8,8- Pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethynyl]benzoic Acid (Bexarotene) and (E)-3-(3-(1,2,3,4-tetrahydro-1,1,4,4,6-pentamethylnaphthalen-7-yl) -4-hydroxyphenyl)acrylic acid (CD3254)

Three unreported analogues of 4-[1-(3,5,5,8,8-pentamethyl-5-6-7-8-tetrahydro-2-naphthyl)ethynyl]benzoic acid (1), otherwise known as bexarotene, as well as four novel analogues of (E)-3-(3-(1,2,3,4-tetrahydro-1,1,4,4,6-pentamethylnaphthalen-7-yl)-4-hydroxyphenyl)acrylic acid (CD3254), are described and evaluated for their retinoid X receptor (RXR) selective agonism. Compound 1 has FDA approval as a treatment for cutaneous T-cell lymphoma (CTCL), although treatment with 1 can elicit side-effects by disrupting other RXR-heterodimer receptor pathways. Of the seven modeled novel compounds, all analogues stimulate RXR-regulated transcription in mammalian 2 hybrid and RXRE-mediated assays, possess comparable or elevated biological activity based on EC50 profiles, and retain similar or improved apoptotic activity in CTCL assays compared to 1. All novel compounds demonstrate selectivity for RXR and minimal crossover onto the retinoic acid receptor (RAR) compared to all-trans-retinoic acid, with select analogues also reducing inhibition of other RXR-dependent pathways (e.g., VDR-RXR). Our results demonstrate that further improvements in biological potency and selectivity of bexarotene can be achieved through rational drug design.

A novel gene expression analytics-based approach to structure aided design of rexinoids for development as next-generation cancer therapeutics

HIGHLIGHTSCTCL cells reveal differential gene expression when treated with chemically distinct rexinoids.Rexinoids can be partitioned into discrete classes employing gene expression profiles.Minor variations in rexinoid structure lead to unique gene expression signatures.Divergence scoring using expression profiles can lead to valuable structural and decision making insights in rexinoid design.Five of the twelve studied bexarotene analogs show promise for further analysis/drug development. ABSTRACT Rexinoids are powerful ligands that bind to retinoid‐X‐receptors (RXRs) and show great promise as therapeutics for a wide range of diseases, including cancer. However, only one rexinoid, bexarotene (Targretin TM) has been successfully transitioned from the bench to the clinic and used to treat cutaneous T‐cell lymphoma (CTCL). Our goal is to develop novel potent rexinoids with a less untoward side effect profile than bexarotene. To this end, we have synthesized a wide array of rexinoids with EC50 values and biological activity similar to bexarotene. In order to determine their suitability for additional downstream analysis, and to identify potential candidate analogs for clinical translation, we treated human CTCL cells in culture and employed microarray technology to assess gene expression profiles. We analyzed twelve rexinoids and found they could be stratified into three distinct categories based on their gene expression: similar to bexarotene, moderately different from bexarotene, and substantially different from bexarotene. Surprisingly, small changes in the structure of the bexarotene parent compound led to marked differences in gene expression profiles. Furthermore, specific analogs diverged markedly from our hypothesis in expression of genes expected to be important for therapeutic promise. However, promoter analysis of genes whose expression was analyzed indicates general regulatory trends along structural frameworks. Our results suggest that certain structural motifs, particularly the basic frameworks found in analog 4 and analog 9, represent important starting points to exploit in generating additional rexinoids for future study and therapeutic applications.

Abstract 2245: Evaluation of new rexinoids for lung cancer

Lung cancer remains the leading cause of cancer deaths around the world. It is estimated that there will be over 150,000 deaths in the United States alone from this disease every year. With no significant improvement in 5 year survival rates over the past 30 years, effective prevention or early intervention is a promising approach to reduce the high mortality of lung cancer. Rexinoids are selective ligands for retinoid X receptors (RXR), which regulate the expression of a wide variety of genes. As transcription factors, rexinoids play important roles in proliferation, differentiation, and apoptosis, which are highly relevant in cancer. Bexarotene is the only synthetic rexinoid that has been approved by the FDA and is used for the treatment of refractory cutaneous T-cell lymphoma. It has also been tested in various clinical trials for lung cancer and breast cancer. However, Bexarotene is not potent or selective enough for RXRs, causing limited efficacy and unacceptable toxicities. Therefore, we synthesized a series of new rexinoids and screened them for their ability to inhibit nitric oxide (NO) production in RAW264.7 macrophage-like cells stimulated with LPS. Several of these compounds inhibited NO production at nanomolar concentrations. Based on this screening and other in vitro assays, two new rexinoids were chosen for in vivo testing. Female A/J mice were injected i.p. with vinyl carbamate (16 mg/kg). One week later, the mice were fed control diet or rexinoids in diet (40-80 mg/kg diet) for 16 weeks. Tumor number, size and histopathology were then evaluated. All of the rexinoids reduced the number and size of the lung tumors. However, Bexarotene only reduced the average number of tumors by 8-17%, while a new pyrimidine-analog of Bexarotene—henceforth, pyrimidine-Bexarotene—reduced the number of tumors by 24-28%. The average tumor burden on lung sections was also reduced by pyrimidine-Bexarotene by 59% compared to the controls (from 0.47 ± 0.19 mm 3 in the control group to 0.19 ± 0.05 mm 3 in the treated group). In contrast, average tumor burden was 0.35 ± 0.08 mm 3 in the group treated with Bexarotene, a reduction of 26%. Notably, the percentage of high grade tumors (both histological and nuclear characteristics) were significantly (p Citation Format: Di Zhang, Ana S. Leal, Sarah E. Carapellucci, Kayla Zydeck, Nicole Chaaban, Michael B. Sporn, Carl E. Wagner, Karen T. Liby. Evaluation of new rexinoids for lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2245. doi:10.1158/1538-7445.AM2017-2245

Distinct functional modes of SUMOylation for retinoid X receptor alpha.

The present study investigated human retinoid X receptor alpha (hRXRα) as a substrate for modification with small ubiquitin like modifier (SUMO) and how members of the protein inhibitor of activated STAT (PIAS) family may impact upon this process. In agreement with a previous study, we validate Ubc9 to facilitate SUMOylation of hRXRα at lysine 108 but note this modification to occur for all isoforms rather than specifically with SUMO1 and to preferentially occur with the unliganded form of hRXRα. SUMOylation of hRXRα is significantly enhanced through PIAS4-mediated activity with lysine 245 identified as a specific SUMO2 acceptor site modified in a PIAS4-dependent fashion. While individual mutations at lysine 108 or 245 modestly increase receptor activity, the combined loss of SUMOylation at both sites significantly potentiates the transcriptional responsiveness of hRXRα suggesting both sites may cooperate in a DNA element-dependent context. Our findings highlight combinatorial effects of SUMOylation may regulate RXRα-directed signalling in a gene-specific fashion.

Dataset on the response of Hut78 cells to novel rexinoids

This article presents the experimental data supporting analysis of differential gene expression of human cutaneous T cell lymphoma (CTCL) cell culture cells (Hut78) treated with bexarotene or a variety of rexinoids, in conjunction with “A Novel Gene Expression Analytics-based Approach to Structure Aided Design of Rexinoids for Development as Next-Generation Cancer Therapeutics” (Hanish et al. 2018). Data presented here include microarray gene expression analysis of a subset of genes. A novel method for analyzing gene expression in the context of a model of ligand mechanism, called the Divergence Score, is described. Analysis to identify the presence of potential retinoid response elements in putative promoter regions of the study genes is also presented.