Structure-guided design of N-phenyl tertiary amines as transrepression-selective liver X receptor modulators with anti-inflammatory activity.

A cocrystal structure of T1317 (3) bound to hLXRbeta was utilized in the design of a series of substituted N-phenyl tertiary amines. Profiling in binding and functional assays led to the identification of LXR modulator GSK9772 ( 20) as a high-affinity LXRbeta ligand (IC 50 = 30 nM) that shows separation of anti-inflammatory and lipogenic activities in human macrophage and liver cell lines, respectively. A cocrystal structure of the structurally related analog 19 bound to LXRbeta reveals regions within the receptor that can affect receptor modulation through ligand modification. Mechanistic studies demonstrate that 20 is greater than 10-fold selective for LXR-mediated transrepression of proinflammatory gene expression versus transactivation of lipogenic signaling pathways, thus providing an opportunity for the identification of LXR modulators with improved therapeutic indexes.

Plant sterols and stanols: effects on mixed micellar composition and LXR (target gene) activation.

Plant stanols and sterols of the 4-desmethyl family (e.g., sitostanol and sitosterol) effectively decrease LDL cholesterol concentrations, whereas 4,4-dimethylsterols (alpha-amyrin and lupeol) do not. Serum carotenoid concentrations, however, are decreased by both plant sterol families. The exact mechanisms underlying these effects are not known, although effects on micellar composition have been suggested. With a liver X receptor (LXR) coactivator peptide recruitment assay, we showed that plant sterols and stanols from the 4-desmethylsterol family activated both LXRalpha and LXRbeta, whereas 4,4-dimethyl plant sterols did not. In fully differentiated Caco-2 cells, the functionality of this effect was shown by the increased expression of ABCA1, one of the known LXR target genes expressed by Caco-2 cells in measurable amounts. The LXR-activating potential of the various plant sterols/stanols correlated positively with ABCA1 mRNA expression. Reductions in serum hydrocarbon carotenoids could be explained by the effects of the 4-desmethyl family and 4,4-dimethylsterols on micellar carotenoid incorporation. Our findings indicate that the decreased intestinal absorption of cholesterol and carotenoids by plant sterols and stanols is caused by two distinct mechanisms.

Discovery of substituted maleimides as liver X receptor agonists and determination of a ligand-bound crystal structure.

Substituted 3-(phenylamino)-1H-pyrrole-2,5-diones were identified from a high throughput screen as inducers of human ATP binding cassette transporter A1 expression. Mechanism of action studies led to the identification of GSK3987 as an LXR ligand. GSK3987 recruits the steroid receptor coactivator-1 to human LXRalpha and LXRbeta with EC(50)s of 40 nM, profiles as an LXR agonist in functional assays, and activates LXR though a mechanism that is similar to first generation LXR agonists.

Cyclin D and cdk4 are required for normal development beyond the blastula stage in sea urchin embryos.

cdk4 mRNA and protein are constitutively expressed in sea urchin eggs and throughout embryonic development. In contrast, cyclin D mRNA is barely detectable in eggs and early embryos, when the cell cycles consist of alternating S and M phases. Cyclin D mRNA increases dramatically in embryos at the early blastula stage and remains at a constant level throughout embryogenesis. An increase in cdk4 kinase activity occurs concomitantly with the increase in cyclin D mRNA. Ectopic expression of cyclin D mRNA in eggs arrests development before the 16-cell stage and causes eventual embryonic death, suggesting that activation of cyclin D/cdk4 in cleavage cell cycles is lethal to the embryo. In contrast, blocking cyclin D or cdk4 expression with morpholino antisense oligonucleotides results in normal development of early gastrula-stage embryos but abnormal, asymmetric larvae. These results suggest that in sea urchins, cyclin D and cdk4 are required for normal development and perhaps the patterning of the developing embryo, but may not be directly involved in regulating entry into the cell cycle.