The Human Pregnane X Receptor (hPXR) is a nuclear receptor that regulates the expression of phase I and phase II drug-metabolizing enzymes, as well as that of drug transporters. Because this receptor plays a critical role in protecting tissues from potentially toxic endo- and xenobiotics, highly active agonists could represent novel therapeutic tools in treating several human diseases. Using an in vitro screening reporter system that allow to characterize hPXR activators and a first step of chemical modifications of an original agonist ligand (C2BA-4, 1-(2-chlorophenyl)-N-[1-(1-phenylethyl)-1H-benzimidazol-5-yl]methanesulfonamide), we identified compounds with a N-1H-benzimidazol-5-ylbenzenesulfonamide scaffold as a potent family of hPXR agonists. Further chemical modifications allowed us to identify enhanced activators, notably N-(1-benzyl-1H-benzimidazol-5-yl)-2,3,4,5,6-pentamethylbenzenesulfonamide (6n) with an EC(50) value in the subnanomolar range. Accordingly to their potent EC(50), these compounds induced an efficient protection of hPXR against proteolytic digestion by trypsin even at very low ligand concentrations and were able to induce the expression of the main target genes of hPXR, CYP3A4 and CYP2B6, in primary cultures of human hepatocytes.

The human pregnane X receptor ( hPXR) is a nuclear receptor that regulates the expression of phase I and II drug- metabolizing enzymes as well as that of drug transporters. In addition, this receptor plays a critical role in cholesterol homeostasis and in protecting tissues from potentially toxic endobiotics. hPXR is activated by a broad spectrum of low- affinity compounds including xenobiotics and endobiotics such as bile acids and their precursors. Crystallographic studies revealed a ligand binding domain ( LBD) with a large and conformable binding pocket that is likely to contribute to the ability of hPXR to respond to compounds of varying size and shape. Here, we describe an in silico method that allowed the identification of nine novel hPXR agonists. We further characterize the compound 1-( 2- chlorophenyl)- N-[ 1-( 1- phenylethyl)- 1H- benzimidazol5- yl] methanesulfonamide ( C2BA- 4), a methanesulfonamide that activates PXR specifically and more potently than does the reference compound 4-[ 2,2- bis( diethoxyphosphoryl) ethenyl]2,6- ditert- butyl- phenol ( SR12813) in our stable cell line expressing a Gal4- PXR and a GAL4 driven luciferase reporter gene. Furthermore treatment of primary human hepatocytes with C2BA- 4 results in a marked induction of the mRNA expression of hPXR target genes, such as cytochromes P450 3A4 and 2B6. Finally, C2BA- 4 is also able to induce hPXR- mediated in vivo luciferase expression in HGPXR stable bioluminescent cells implanted in mice. The study suggests new directions for the rational design of selective hPXR agonists and antagonists.

To study the structure–activity relationships (SAR) and the binding activity of pro-apoptotic Bak BH3 domain, we synthesised several 16mer peptide analogues corresponding to the region 72-GQVGRQLAIIGDDINR-87. Using different amino acids varying in length, steric and electronic properties, we investigated the role and the nature of physicochemical parameters of residues Val74, Leu78, Ile81 and Ile85, previously identified to be crucial for interactions. With this aim, we measured the affinity of these peptides on two anti-apoptotic proteins Bcl-xL and Bcl-2 by a polarization fluorescence competitive assay. We defined that the most potent peptide on Bcl-xL, which presents a 4.6-fold increase as compared to the parent peptide affinity, was obtained when Ile85 was mutated with a 4-chlorophenylalanine. Finally, assays of eight Bak peptide analogues on Bcl-2 allowed us to postulate that modulations at position 78 could afford peptides with a binding selectivity enhanced for Bcl-xL. These pharmacological and physicochemical parameter data should prove useful for the rational design of non-peptide ligands as potential antagonists of Bcl-2 protein interactions.