In the search for novel potent immunomodulatory nuclear factor-erythroid 2 related factor 2 (Nrf2) activators, a derivative of cholic bile acid, SB140, was synthesized. The synthesis of SB140 aimed to increase the electrophilic functionality of the compound, enhancing its ability to activate Nrf2. Effects of SB140 on microglial cells, myeloid-derived cells (MDC), and T cells were explored in the context of (central nervous system) CNS autoimmunity. SB140 potently activated Nrf2 signaling in MDC and microglia. It was efficient in reducing the ability of microglial cells to produce inflammatory nitric oxide, interleukin (IL)-6, and tumor necrosis factor (TNF). Also, SB140 reduced the proliferation of encephalitogenic T cells and the production of their effector cytokines: IL-17 and interferon (IFN)-γ. On the contrary, the effects of SB140 on anti-inflammatory IL-10 production in microglial and encephalitogenic T cells were limited or absent. These results show that SB140 is a potent Nrf2 activator, as well as an immunomodulatory compound. Thus, further research on the application of SB140 in the treatment of neuroinflammatory diseases is warranted. Animal models of multiple sclerosis and other inflammatory neurological disorders will be a suitable choice for such studies.

The necessity of anti-inflammatory drugs such as glucocorticoids has been evident during the COVID-19 pandemic. Glucocorticoids, are the standard therapy for the treatment of moderate and severe COVID-19 patients. However, serious side effects limit the use of these drugs, and anti-inflammatory drugs with better pharmacological properties are urgently required. Bile acids are of interest, because of their anti-inflammatory and immunomodulatory properties, facilitated through an unclear mechanism involving transmembrane and nuclear receptors. In this work, we screened the binding activity of a number of bile acid derivatives, for the ligand-binding domain of glucocorticoid receptor (GR-LBD), the most important receptor for anti-inflammatory processes. Tested compounds include oximes, lactones, lactams, tetrazoles, dienones, C-24 alcohols and cholic acid amides. Cholic acid oxime, deoxycholic acid dienone, 3-keto-24-cholic alcohol and cholic acid amide showed best binding affinities for GR-LBD among tested compounds. The in silico molecular docking explanation is provided. SAR analysis showed that expansion of B and C steroid rings or attachment of heterocycle to C ring is not beneficial for binding; side chain should contain hydrogen donor group; the GR-LBD tolerate well different functionalities on C-3 position. These results provide valuable information toward synthesis of the new glucocorticoids based on bile acids.

Biosensors developed in yeast cells represent an attractive research area in biomedicine because they allow for the detection of molecules of various structures and biological activities, economically and simply, without the use of harmful radioactive materials. We focused our attention on the identification of androgen, glucocorticoid and estrogen receptor α/β ligands using fluorescent biosensors in yeast. Identification of compounds that modulate the activity of androgen (AR) or estrogen receptors (ER) is one of the major goals in the design of new treatments of hormone-dependent cancers. Similarly, glucocorticoid receptor (GR) ligands are used to treat autoimmune and inflammatory diseases, but due to a large number of side effects and drug resistance, great effort has been directed to finding new modulators. In this study, ligand-binding domains (LBDs) of AR, ERα, ERβ or GR fused with yellow fluorescent protein (YFP) were expressed in Saccharomyces cerevisiae. Recombinant yeast cells were treated with tested steroid hormone or bile acid derivatives, and, due to the fluorescence resonance energy transfer phenomenon following ligand binding, relative binding affinities were quantified fluorometrically. Our results show that some of the tested compounds have moderate to high binding affinity for particular steroid receptors, similar to natural ligands, while the affinities of other compounds were low or negligible. To elucidate the mechanisms of action for these compounds, additional experiments are necessary, and to better understand the molecular interactions within the ligand-binding pocket of the receptor, molecular docking analysis can be conducted. In summary, the yeast-based biosensors used in this work have proven to be very useful for in vitro screening of novel anticancer and anti-inflammatory drug candidates, as well as for the elimination of compounds that do not deserve further attention and resources due to their lack of desired bioactivities.