Identification and characterization of novel splice variants of human farnesoid X receptor.

Farnesoid X receptor (FXR, NR1H4) is a ligand-activated nuclear receptor, which regulates bile acid, lipid and glucose metabolism. Due to these functions, FXR has been investigated as a potential drug target for the treatment of liver diseases, such as primary biliary cholangitis and non-alcoholic steatohepatitis. Based on the previously described four splice variants, it has been suggested that alternative promoter usage and splicing may have an impact on total FXR activity as a result of encoding functionally diverse variants. Here we aimed for a systematic analysis of human hepatic FXR splice variants. In addition to the previously described FXRα1-4, we identified four novel splice variants (FXRα5-8) in human hepatocytes, which resulted from previously undetected exon skipping events. These newly identified isoforms displayed diminished DNA binding and impaired transactivation activities. Isoform FXRα5, which suppressed the transactivation activity of the functional isoform FXRα2, was further characterized as deficient in heterodimerization, coactivator recruitment and ligand binding. These findings were further supported by molecular dynamics simulations, which offered an explanation for the behavior of this isoform on the molecular level. FXRα5 exhibited low uniform expression levels in nearly all human tissues. Our systematic analysis of FXR splice variants in human hepatocytes resulted in the identification of four novel FXR isoforms, which all proved to be functionally deficient, but one novel variant, FXRα5, also displayed dominant negative activity. The possible associations with and roles of these novel isoforms in human liver diseases require further investigation.

Oligonucleotide-based pharmaceuticals: Non-clinical and clinical safety signals and non-clinical testing strategies.

Antisense oligonucleotides, short interfering RNAs (siRNAs) and aptamers are oligonucleotide-based pharmaceuticals with a promising role in targeted therapies. Currently, five oligonucleotide-based pharmaceuticals have achieved marketing authorization in Europe or USA and many more are undergoing clinical testing. However, several safety concerns have been raised in non-clinical and clinical studies. Oligonucleotides share properties with both chemical and biological pharmaceuticals and therefore they pose challenges also from the regulatory point of view. We have analyzed the safety data of oligonucleotides and evaluated the applicability of current non-clinical toxicological guidelines for assessing the safety of oligonucleotide-based pharmaceuticals. Oligonucleotide-based pharmaceuticals display a similar toxicological profile, exerting adverse effects on liver and kidney, evoking hematological alterations, as well as causing immunostimulation and prolonging the coagulation time. It is possible to extrapolate some of these effects from non-clinical studies to humans. However, evaluation strategies for genotoxicity testing of "non-natural" oligonucleotides should be revised. Additionally, the selective use of surrogates and prediction of clinical endpoints for non-clinically observed immunostimulation is complicated by its multiple potential manifestations, demanding improvements in the testing strategies. Utilizing more relevant and mechanistic-based approaches and taking better account of species differences, could possibly improve the prediction of relevant immunological/proinflammatory effects in humans.

Target Hopping from Protein Kinases to PXR: Identification of Small-Molecule Protein Kinase Inhibitors as Selective Modulators of Pregnane X Receptor from TüKIC Library.

Small-molecule protein kinase inhibitors are used for the treatment of cancer, but off-target effects hinder their clinical use. Especially off-target activation of the pregnane X receptor (PXR) has to be considered, as it not only governs drug metabolism and elimination, but also can promote tumor growth and cancer drug resistance. Consequently, PXR antagonism has been proposed for improving cancer drug therapy. Here we aimed to identify small-molecule kinase inhibitors of the Tübingen Kinase Inhibitor Collection (TüKIC) compound library that would act also as PXR antagonists. By a combination of in silico screen and confirmatory cellular reporter gene assays, we identified four novel PXR antagonists and a structurally related agonist with a common phenylaminobenzosuberone scaffold. Further characterization using biochemical ligand binding and cellular protein interaction assays classified the novel compounds as mixed competitive/noncompetitive, passive antagonists, which bind PXR directly and disrupt its interaction with coregulatory proteins. Expression analysis of prototypical PXR target genes ABCB1 and CYP3A4 in LS174T colorectal cancer cells and HepaRG hepatocytes revealed novel antagonists as selective receptor modulators, which showed gene- and tissue-specific effects. These results demonstrate the possibility of dual PXR and protein kinase inhibitors, which might represent added value in cancer therapy.

Discrepancy in interactions and conformational dynamics of pregnane X receptor (PXR) bound to an agonist and a novel competitive antagonist.

Pregnane X receptor (PXR) is a nuclear receptor with an essential role in regulating drug metabolism genes. While the mechanism of action for ligand-mediated PXR agonism is well-examined, its ligand-mediated inhibition or antagonism is poorly understood. Here we employ microsecond timescale all-atom molecular dynamics (MD) simulations to investigate how our newly identified dual kinase and PXR inhibitor, compound 100, acts as a competitive PXR antagonist and not as a full agonist. We study the PXR ligand binding domain conformational changes associated with compound 100 and compare the results to the full agonist SR12813, in presence and absence of the coactivator. Furthermore, we complement our research by experimentally disclosing the effect of eight key-residue mutations on PXR activation. Finally, simulations of P2X4 inhibitor (BAY-1797) in complex with PXR, which shares an identical structural moiety with compound 100, provide further insights to ligand-induced PXR behaviour. Our MD data suggests ligand-specific influence on conformations of different PXR-LBD regions, including α6 region, αAF-2, α1-α2', ß1'-α3 and ß1-ß1' loop. Our results provide important insights on conformational behaviour of PXR and offers guidance how to alleviate PXR agonism or to promote PXR antagonism.