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.