The nuclear receptor PXR (pregnane X receptor) is a broadspecificity sensor that recognizes a wide variety of synthetic drugs and xenobiotic agents. On activation by these compounds, PXR coordinately induces a network of transporters, cytochrome P450 enzymes, and other genes that effectively clear xenobiotics from the liver and intestine. Like PXR, the majority of its target genes also possess a broad specificity for exogenous compounds. Thus, PXR is both a sensor and effector in a well integrated and generalized pathway for chemical immunity. Although it is clear that PXR responds to numerous foreign compounds, it is unclear whether it possesses an endogenous ligand. To address this issue, we noted that there is substantial overlap in the substrate specificities of PXR and its critical CYP3A target gene. This prompted us to ask whether endogenous CYP3A substrates also serve as PXR ligands. We demonstrate that 5-cholestane-3␣,7␣,12␣-triol (triol), a cholesterol-derived CYP3A substrate, is a potent PXR agonist that effectively induces cyp3a expression in mice. This defines a critical salvage pathway that can be autoinduced to minimize triol accumulation. In contrast, triol can accumulate to very high levels in humans, and unlike mice, these people develop the severe clinical manifestations of cerebrotendinous xanthomatosis. The reason for these dramatic species differences has remained unclear. We now demonstrate that triol fails to activate human PXR or induce the CYP3A-salvage pathway. This explains why humans are more susceptible to sterol accumulation and suggests that synthetic ligands for human PXR could be used to treat cerebrotendinous xanthomatosis and other disorders of cholesterol excess.
Employing a capillary-scale NMR probe enables the miniaturisation of structure determination and de-replication of purified natural products from plants using only 5-100 microg of material. Approximately 5 microg are required to perform one-dimensional proton and two-dimensional homonuclear (COSY and NOESY) NMR experiments; some 30 microg are needed to acquire HMQC- or HSQC-NMR spectra; ca. 75-100 microg are necessary to measure HMBC-NMR spectra; and around 200 microg of a compound are needed to perform 13C- and DEPT-NMR experiments. In order to illustrate the integration of the outputs from high-throughput natural product chemistry methods with the capabilities of the state-of-the-art CapNMR technology, the preparation of a natural product library from the extract of Penstemon centranthifolius, and the subsequent isolation, purification and structure determination of six known iridoid glycosides with 25-300 microg of material are presented.
One new (1) and four known (2-5) ursene triterpenes with potent inhibition of the formation of the bacterial biofilm Pseudomonas aeruginosa PA01 were obtained from Diospyros dendo using a high-throughput natural products chemistry procedure. These compounds were isolated as mass-limited samples. The miniaturization of the structure elucidation and dereplication was performed primarily utilizing a capillary-scale NMR probe.
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