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.

4-Nonylphenol (4-NP), a para-substituted phenolic compound mixture comprising a straight or branched carbon chain, is a widespread ubiquitous environmental pollutant and food contaminant. 4-NP, particularly the branched form, has been identified as an endocrine disruptor with potent activities on estrogen nuclear receptors. Constitutive Androstane Receptor (CAR, NR1I3) is another crucial nuclear receptor that regulates hepatic lipid, glucose, and steroid metabolism and is involved in endocrine disruption mechanism. The nonylphenol mixture has been described as an extremely potent activator of both human and rodent CAR. However, detailed mechanistic aspects of CAR activation by NP are enigmatic and it is not known if 4-NP can directly interact with the CAR ligand binding domain (LBD). Here we examined interactions of individual branched (22NP, 33NP, and 353NP) and linear 4-NPs with CAR and its variants using molecular dynamics (MD) simulations, cellular experiments with various CAR constructs or CAR ligand binding domain (LBD) mutants, or in differentiated HepaRG hepatocyte cellular model. Our results demonstrate that branched 4-NPs display more stable poses to activate both wild-type (wt)CAR1 as well as CAR3 variant LBDs in MD simulations. Consistently, branched 4-NP activated CAR3 and wtCAR1 LBD more efficiently than linear 4-NP or tert-nonylphenol (22NP). Furthermore, in HepaRG cells, we observed that all NP significantly up-regulated CYP2B6, a relevant hallmark for CAR activation. This is the first study to provide detailed insights into the direct interaction between individual 4-NPs and the human CAR-LBD, as well as its dominant variant CAR3 The work could contribute to the safer use of individual NPs in many areas of industry.

Human-targeted drugs are increasingly recognized to affect bacterial physiology, yet their impact beyond growth inhibition, and the underlying molecular mechanisms, remain poorly understood. Here, we systematically mapped how chemically diverse host-associated compounds influence virulence and intrinsic antibiotic resistance in Salmonella enterica serovar Typhimurium. Using a high-throughput reporter approach, we quantified transcriptional response of 26 key stress promoters in Salmonella against 2,415 compounds, including FDA-approved drugs, food-derived molecules, and human metabolites. We found that non-antibiotic compounds induce transcriptional responses distinct from those elicited by antibiotics, uncovering a regulatory shift from general stress responses to compound-specific regulation driven by ligand-responsive MarR- and TetR-family regulators. In particular, more than 200 non-antibiotic drugs modulate virulence or intrinsic resistance pathways through the central regulators SlyA or RamR, respectively. By integrating our large-scale dataset with machine learning, we further defined chemical features associated with ramA transcriptional activation in Salmonella , and predicted ∼200 additional candidate inducers of intrinsic antibiotic resistance in this pathogen. Together, our results uncovered a large and previously unrecognized chemical landscape that reshapes bacterial stress responses through ligand-responsive regulatory mechanisms, with implications for how host-associated compounds influence pathogen virulence and antibiotic resistance. Highlights: Mapping the transcriptional response of key Salmonella stress pathways across a large chemical space shows that antibiotics and non-antibiotic compounds elicit distinct stress responses in Salmonella The activity of the virulence regulator SlyA is modulated by non-antibiotic compounds SlyA-inhibitors increase sensitivity to polymyxin B and decrease Salmonella virulence in cell culture Human-targeted drugs increase intrinsic antibiotic resistance via RamR-RamA in Salmonella Machine learning approach enables large-scale, out-of-sample prediction of human-targeted drugs inducing ramA expression in Salmonella

The pregnane X receptor (PXR) displays exceptional ligand promiscuity, yet the molecular basis of its antagonism remains poorly defined. PXR inhibition has traditionally been attributed to the displacement of coactivators from the activation function‐2 (AF‐2) surface. Here, we show that PXR antagonism is mechanistically heterogeneous, with distinct inhibitory mechanisms determined by ligand structure. Comparative analysis of imidazo[1,2‐a]pyridine, MI891, and SPA70 antagonists reveals multiple non‐canonical inhibitory mechanisms that decouple coregulator recruitment from transcriptional output. Notably, SPA70 preserves compatibility with coactivator recruitment while remaining transcriptionally inactive, consistent with concurrent corepressor engagement and a distinct coactivator‐compatible antagonist state. In contrast, MI891 stabilizes a PXR conformation that favors NCoR1 and SMRT binding and impairs nuclear translocation. MD simulations and mutagenesis reveal ligand‐specific perturbations of α12 and the α10/α11 region that underlie these effects. Together, these findings establish a ligand‐encoded framework for PXR antagonism that extends beyond the classical on/off switch model.

The pregnane X receptor (PXR) displays exceptional ligand promiscuity, yet the molecular basis of its antagonism remains poorly defined. PXR inhibition has traditionally been attributed to the displacement of coactivators from the activation function‐2 (AF‐2) surface. Here, we show that PXR antagonism is mechanistically heterogeneous, with distinct inhibitory mechanisms determined by ligand structure. Comparative analysis of imidazo[1,2‐a]pyridine, MI891, and SPA70 antagonists reveals multiple non‐canonical inhibitory mechanisms that decouple coregulator recruitment from transcriptional output. Notably, SPA70 preserves compatibility with coactivator recruitment while remaining transcriptionally inactive, consistent with concurrent corepressor engagement and a distinct coactivator‐compatible antagonist state. In contrast, MI891 stabilizes a PXR conformation that favors NCoR1 and SMRT binding and impairs nuclear translocation. MD simulations and mutagenesis reveal ligand‐specific perturbations of α12 and the α10/α11 region that underlie these effects. Together, these findings establish a ligand‐encoded framework for PXR antagonism that extends beyond the classical on/off switch model.