Synergistic activation of human pregnane X receptor by binary cocktails of pharmaceutical and environmental compounds.

Humans are chronically exposed to multiple exogenous substances, including environmental pollutants, drugs and dietary components. Many of these compounds are suspected to impact human health, and their combination in complex mixtures could exacerbate their harmful effects. Here we demonstrate that a pharmaceutical oestrogen and a persistent organochlorine pesticide, both exhibiting low efficacy when studied separately, cooperatively bind to the pregnane X receptor, leading to synergistic activation. Biophysical analysis shows that each ligand enhances the binding affinity of the other, so the binary mixture induces a substantial biological response at doses at which each chemical individually is inactive. High-resolution crystal structures reveal the structural basis for the observed cooperativity. Our results suggest that the formation of 'supramolecular ligands' within the ligand-binding pocket of nuclear receptors contributes to the synergistic toxic effect of chemical mixtures, which may have broad implications for the fields of endocrine disruption, toxicology and chemical risk assessment.

One Question, Multiple Answers: Biochemical and Biophysical Screening Methods Retrieve Deviating Fragment Hit Lists.

Fragment-based lead discovery is gaining momentum in drug development. Typically, a hierarchical cascade of several screening techniques is consulted to identify fragment hits which are then analyzed by crystallography. Because crystal structures with bound fragments are essential for the subsequent hit-to-lead-to-drug optimization, the screening process should distinguish reliably between binders and non-binders. We therefore investigated whether different screening methods would reveal similar collections of putative binders. First we used a biochemical assay to identify fragments that bind to endothiapepsin, a surrogate for disease-relevant aspartic proteases. In a comprehensive screening approach, we then evaluated our 361-entry library by using a reporter-displacement assay, saturation-transfer difference NMR, native mass spectrometry, thermophoresis, and a thermal shift assay. While the combined results of these screening methods retrieve 10 of the 11 crystal structures originally predicted by the biochemical assay, the mutual overlap of individual hit lists is surprisingly low, highlighting that each technique operates on different biophysical principles and conditions.

Small molecules inhibit the interaction of Nrf2 and the Keap1 Kelch domain through a non-covalent mechanism.

Keap1 binds to the Nrf2 transcription factor to promote its degradation, resulting in the loss of gene products that protect against oxidative stress. While cell-active small molecules have been identified that modify cysteines in Keap1 and effect the Nrf2 dependent pathway, few act through a non-covalent mechanism. We have identified and characterized several small molecule compounds that specifically bind to the Keap1 Kelch-DC domain as measured by NMR, native mass spectrometry and X-ray crystallography. One compound upregulates Nrf2 response genes measured by a luciferase cell reporter assay. The non-covalent inhibition strategy presents a reasonable course of action to avoid toxic side-effects due to non-specific cysteine modification.

Activation of RXR-PPAR heterodimers by organotin environmental endocrine disruptors.

The nuclear receptor retinoid X receptor-alpha (RXR-alpha)-peroxisome proliferator-activated receptor-gamma (PPAR-gamma) heterodimer was recently reported to have a crucial function in mediating the deleterious effects of organotin compounds, which are ubiquitous environmental contaminants. However, because organotins are unrelated to known RXR-alpha and PPAR-gamma ligands, the mechanism by which these compounds bind to and activate the RXR-alpha-PPAR-gamma heterodimer at nanomolar concentrations has remained elusive. Here, we show that tributyltin (TBT) activates all three RXR-PPAR-alpha, -gamma, -delta heterodimers, primarily through its interaction with RXR. In addition, the 1.9 A resolution structure of the RXR-alpha ligand-binding domain in complex with TBT shows a covalent bond between the tin atom and residue Cys 432 of helix H11. This interaction largely accounts for the high binding affinity of TBT, which only partly occupies the RXR-alpha ligand-binding pocket. Our data allow an understanding of the binding and activation properties of the various organotins and suggest a mechanism by which these tin compounds could affect other nuclear receptor signalling pathways.

Amphioxus postembryonic development reveals the homology of chordate metamorphosis.

Most studies in evolution are centered on how homologous genes, structures, and/or processes appeared and diverged. Although historical homology is well defined as a concept, in practice its establishment can be problematic, especially for some morphological traits or developmental processes. Metamorphosis in chordates is such an enigmatic character. Defined as a spectacular postembryonic larva-to-adult transition, it shows a wide morphological diversity between the different chordate lineages, suggesting that it might have appeared several times independently. In vertebrates, metamorphosis is triggered by binding of the thyroid hormones (THs) T(4) and T(3) to thyroid-hormone receptors (TRs). Here we show that a TH derivative, triiodothyroacetic acid (TRIAC), induces metamorphosis in the cephalochordate amphioxus. The amphioxus TR (amphiTR) mediates spontaneous and TRIAC-induced metamorphosis because it strongly binds to TRIAC, and a specific TR antagonist, NH3, inhibits both spontaneous and TRIAC-induced metamorphosis. Moreover, as in amphibians, amphiTR expression levels increase around metamorphosis and are enhanced by THs. Therefore, TH-regulated metamorphosis, mediated by TR, is an ancestral feature of all chordates. This conservation of a regulatory network supports the homology of metamorphosis in the chordate lineage.