Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Mangelsdorf DJ, Evans RM (1994) The Retinoids. Academic Press, Orlando, FL
Leid M, Kastner P, Chambon P (1992) Multiplicity generates diversity in the retinoic acid signalling pathways. Trends Biochem Sci 17:427–433
Lala DS, Mukherjee R, Schulman IG, Koch SSC, Dardashti LJ, Nadzan AM et al (1996) Activation of specific RXR heterodimers by an antagonist of RXR homodimers. Nature 383:450
Mangelsdorf DJ, Ong ES, Dyck JA, Evans RM (1990) Nuclear receptor that identifies a novel retinoic acid response pathway. Nature 345:224
Jong L, Lehmann JM, Hobbs PD, Harlev E, Huffman JC, Pfahl M et al (1993) Conformational effects on retinoid receptor selectivity. 1. Effect of 9-double bond geometry on retinoid X receptor activity. J Med Chem 36:2605–2613
Dawson MI, Jong L, Hobbs PD, Cameron JF, Chao W-r, Pfahl M et al (1995) Conformational effects on retinoid receptor selectivity. 2. Effects of retinoid bridging group on retinoid X receptor activity and selectivity. J Med Chem 38:3368–3383
Boehm MF, Zhang L, Badea BA, White SK, Mais DE, Berger E et al (1994) Synthesis and structure-activity relationships of novel retinoid X receptor-selective retinoids. J Med Chem 37:2930–2941
Boehm MF, Zhang L, Zhi L, McClurg MR, Berger E, Wagoner M et al (1995) Design and synthesis of potent retinoid X receptor selective ligands that induce apoptosis in leukemia cells. J Med Chem 38:3146–3155
Liby K, Rendi M, Suh N, Royce DB, Risingsong R, Williams CR et al (2006) The combination of the rexinoid, LG100268, and a selective estrogen receptor modulator, either arzoxifene or acolbifene, synergizes in the prevention and treatment of mammary tumors in an estrogen receptor–negative model of breast cancer. Clin Cancer Res 12:5902
Cao M, Royce DB, Risingsong R, Williams CR, Sporn MB, Liby KT (2016) The rexinoids LG100268 and LG101506 inhibit inflammation and suppress lung carcinogenesis in A/J mice. Cancer Prev Res 9:105
Yen W-C, Corpuz MR, Prudente RY, Cooke TA, Bissonnette RP, Negro-Vilar A et al (2004) A selective retinoid x receptor agonist bexarotene (targretin) prevents and overcomes acquired paclitaxel (taxol) resistance in human non–small cell lung cancer. Clin Cancer Res 10:8656
Dragnev KH, Petty WJ, Shah SJ, Lewis LD, Black CC, Memoli V et al (2007) A proof-of-principle clinical trial of bexarotene in patients with non–small cell lung cancer. Clin Cancer Res 13:1794
Heo J-C, Jung T-H, Lee S, Kim HY, Choi G, Jung M et al (2016) Effect of bexarotene on differentiation of glioblastoma multiforme compared with ATRA. Clin Exp Metastasis 33:417–429
W-c Y, Prudente RY, Lamph WW (2004) Synergistic effect of a retinoid X receptor-selective ligand bexarotene (LGD1069, Targretin) and paclitaxel (Taxol) in mammary carcinoma. Breast Cancer Res Treat 88:141–148
Cramer PE, Cirrito JR, Wesson DW, Lee CYD, Karlo JC, Zinn AE et al (2012) ApoE-directed therapeutics rapidly clear β-amyloid and reverse deficits in AD mouse models. Science 335:1503
Tai LM, Bilousova T, Jungbauer L, Roeske SK, Youmans KL, Yu C et al (2013) Levels of soluble apolipoprotein E/amyloid-β (Aβ) complex are reduced and oligomeric Aβ increased with APOE4 and alzheimer disease in a transgenic mouse model and human samples. J Biol Chem 288:5914–5926
McFarland K, Spalding TA, Hubbard D, Ma J-N, Olsson R, Burstein ES (2013) Low dose bexarotene treatment rescues dopamine neurons and restores behavioral function in models of Parkinson’s disease. ACS Chem Nerosci 4:1430–1438
Cummings JL, Zhong K, Kinney JW, Heaney C, Moll-Tudla J, Joshi A et al (2016) Double-blind, placebo-controlled, proof-of-concept trial of bexarotene Xin moderate Alzheimer’s disease. Alzheimer's Res Ther 8(4)
Wagner CEJ, Jurutka PW Methods to generate an array of novel rexinoids by SAR on a potent retinoid X receptor agonist: a case study with NEt-TMN. Methods Mol Biol. https://doi.org/10.2174/1568026616666160617091559
Fujii S, Ohsawa F, Yamada S, Shinozaki R, Fukai R, Makishima M et al (2010) Modification at the acidic domain of RXR agonists has little effect on permissive RXR-heterodimer activation. Bioorg Med Chem Lett 20:5139–5142
Ohsawa F, Morishita K-i, Yamada S, Makishima M, Kakuta H (2010) Modification at the lipophilic domain of RXR agonists differentially influences activation of RXR heterodimers. ACS Med Chem Lett 1:521–525
Kakuta H, Yakushiji N, Shinozaki R, Ohsawa F, Yamada S, Ohta Y et al (2012) RXR partial agonist CBt-PMN exerts therapeutic effects on type 2 diabetes without the side effects of RXR full agonists. ACS Med Chem Lett 3:427–432
Kakuta H, Ohsawa F, Yamada S, Makishima M, Tai A, Yasui H et al (2012) Feasibility of structural modification of retinoid X receptor agonists to separate blood glucose-lowering action from adverse effects: studies in KKAy type 2 diabetes model mice. Biol Pharm Bull 35:629–633
Ohsawa F, Yamada S, Yakushiji N, Shinozaki R, Nakayama M, Kawata K et al (2013) Mechanism of retinoid X receptor partial agonistic action of 1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)-1H-benzotriazole-5-carboxylic acid and structural development to increase potency. J Med Chem 56:1865–1877
Heck MC, Wagner CE, Shahani PH, MacNeill M, Grozic A, Darwaiz T et al (2016) Modeling, synthesis, and biological evaluation of potential retinoid X receptor (RXR)-selective agonists: analogues of 4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethynyl]benzoic acid (Bexarotene) and 6-(ethyl(5,5,8,8-tetrahydronaphthalen-2-yl)amino)nicotinic acid (NEt-TMN). J Med Chem 59:8924–8940
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Jurutka, P.W., Wagner, C.E. (2019). Methods to Assess Activity and Potency of Rexinoids Using Rapid Luciferase-Based Assays: A Case Study with NEt-TMN. In: Ray, S. (eds) Retinoid and Rexinoid Signaling . Methods in Molecular Biology, vol 2019. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9585-1_7
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9585-1_7
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9584-4
Online ISBN: 978-1-4939-9585-1
eBook Packages: Springer Protocols