Jurutka Peter W, Wagner Carl E
School of Mathematical and Natural Sciences, New College of Interdisciplinary Arts and Sciences, Arizona State University, Glendale, AZ, USA.
Methods Mol Biol. 2019;2019:95-108. doi: 10.1007/978-1-4939-9585-1_7.
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 EC values for rexinoids from dose-response curves.
本章概述了用于视黄酸X受体（RXR）激动剂体外生物学评价的材料、方法和程序，这些激动剂包括6-（乙基（5,5,8,8-四甲基-5,6,7,8-四氢萘-2-基）氨基）烟酸（NEt-TMN），以及我们小组最近报道的几种NEt-TMN类似化合物。这些方法不仅适用于这个NEt-TMN案例研究，还具有普遍适用性，可用于表征和生物学评价其他假定的RXR激动剂（类视黄醇），并以1999年获得美国食品药品监督管理局（FDA）批准用于治疗皮肤T细胞淋巴瘤的药物贝沙罗汀（Bex）作为基准进行比较，Bex也被用于治疗非小细胞肺癌，并且仍在多种人类癌症类型中进行探索。Bex治疗的副作用包括因抑制或激活与RXR相互作用的其他核受体而导致的甲状腺功能减退和高甘油三酯血症。由于类视黄醇通常对RXR激活具有选择性，而不是激活视黄酸受体（RAR），因此类视黄醇治疗可避免通常与类维生素A相关的皮肤毒性副作用。还有许多其他强效类视黄醇的例子，其生物学评价为这些化合物的定量构效关系（qSAR）研究提供了有用的见解，这些研究通常也以Bex作为基准，作为癌症的潜在治疗方法。由于其他途径存在不同的多效性，即使是密切相关的类视黄醇也表现出独特的副作用和活性特征。除了Bex和NEt-TMN之外，强效类视黄醇的显著例子还包括CD3254、LGD100268和9-顺式-UAB30。实际上，本文所述的评估NEt-TMN和类似类视黄醇的方法通常适用于更广泛的各种强效、中等强度甚至弱效的RXR配体。在体外生物学评价方面，描述了通过在哺乳动物细胞中采用生物学相关的、RXR反应元件（RXRE）介导的转录测定法来快速初步评估类视黄醇活性的方法。此外，还详细介绍了第二种更灵敏的测定法，该方法利用哺乳动物双杂交（M2H）荧光素酶测定法中的RXR-RXR同二聚体激活。进一步描述了在不同类视黄醇浓度下应用M2H测定法从剂量反应曲线确定类视黄醇EC值的方法。
