ABSTRACT:Induction of cytochrome P450 3A4 (CYP3A4) is determined typically by employing primary culture of human hepatocytes and measuring CYP3A4 mRNA, protein and microsomal activity. Recently a pregnane X receptor (PXR) reporter gene assay was established to screen CYP3A4 inducers. To evaluate results from the PXR reporter gene assay with those from the aforementioned conventional assays, 14 drugs were evaluated for their ability to induce CYP3A4 and activate PXR. Sandwiched primary cultures of human hepatocytes from six donors were used and CYP3A4 activity was assessed by measuring microsomal testosterone 6␤-hydroxylase activity. Hepatic CYP3A4 mRNA and protein levels were also analyzed using branched DNA technology/Northern blotting and Western blotting, respectively. In general, PXR activation correlated with the induction potential observed in human hepatocyte cultures. Clotrimazole, phenobarbital, rifampin, and sulfinpyrazone highly activated PXR and increased CYP3A4 activity; carbamazepine, dexamethasone, dexamethasone-t-butylacetate, phenytoin, sulfadimidine, and taxol weakly activated PXR and induced CYP3A4 activity, and methotrexate and probenecid showed no marked activation in either system. Ritonavir and troleandomycin showed marked PXR activation but no increase (in the case of troleandomycin) or a significant decrease (in the case of ritonavir) in microsomal CYP3A4 activity. It is concluded that the PXR reporter gene assay is a reliable and complementary method to assess the CYP3A4 induction potential of drugs and other xenobiotics.

Influenza nucleoprotein (NP) plays multiple roles in the virus life cycle, including an essential function in viral replication as an integral component of the ribonucleoprotein complex, associating with viral RNA and polymerase within the viral core. The multifunctional nature of NP makes it an attractive target for antiviral intervention, and inhibitors targeting this protein have recently been reported. In a parallel effort, we discovered a structurally similar series of influenza replication inhibitors and show that they interfere with NP-dependent processes via formation of higherorder NP oligomers. Support for this unique mechanism is provided by site-directed mutagenesis studies, biophysical characterization of the oligomeric ligand:NP complex, and an X-ray cocrystal structure of an NP dimer of trimers (or hexamer) comprising three NP_A:NP_B dimeric subunits. Each NP_A:NP_B dimeric subunit contains two ligands that bridge two composite, protein-spanning binding sites in an antiparallel orientation to form a stable quaternary complex. Optimization of the initial screening hit produced an analog that protects mice from influenza-induced weight loss and mortality by reducing viral titers to undetectable levels throughout the course of treatment.antiinfluenza | oligomerization | polymerase inhibitor | protein-protein interaction | cooperative inhibition

The human transcription factor pregnane X receptor (hPXR) is a key regulator of enzyme expression, especially cytochrome P450 3A4 (CYP3A4). Due to the prominence of CYP3A4 in the elimination of many drugs, the development of high throughput in vitro models to predict the effect of drugs on CYP3A4 expression have increased. To better interpret and predict potential drug-drug interactions due to CYP3A4 enzyme induction, we evaluated 170 xenobiotics in a hPXR transactivation assay and compared these results to known clinical drug-drug interactions. Of the 170 xenobiotics tested, 54% of them demonstrated some level of hPXR transactivation. By taking into consideration cell culture conditions (solubility, cytotoxicity, appropriate drug concentration in media), as well as in vivo pharmacokinetics (therapeutic plasma C(max), distribution, route of administration, dosing regimen, liver exposure, potential to inhibit CYP3A4), the risk potential of CYP3A4 enzyme induction for most compounds reduced dramatically. By employing this overall interpretation strategy, the final percentage of compounds predicted to significantly induce CYP3A4 reduced to 5%, all of which are known to cause drug-drug interactions. Also, this is the first report that identifies several potent compounds that have the ability to transactivate hPXR that previously have not been identified, such as terbinafine, diclofenac, sildenafil, glimepiride, montelukast, and ticlopidine.

Epsins are endocytic proteins with a structured epsin N-terminal homology (ENTH) domain that binds phosphoinositides and a poorly structured C-terminal region that interacts with ubiquitin and endocytic machinery, including clathrin and endocytic scaffolding proteins. Yeast has two redundant genes encoding epsins, ENT1 and ENT2; deleting both genes is lethal. We demonstrate that the ENTH domain is both necessary and sufficient for viability of ent1⌬ent2⌬ cells. Mutational analysis of the ENTH domain revealed a surface patch that is essential for viability and that binds guanine nucleotide triphosphatase-activating proteins for Cdc42, a critical regulator of cell polarity in all eukaryotes. Furthermore, the epsins contribute to regulation of specific Cdc42 signaling pathways in yeast cells. These data support a model in which the epsins function as spatial and temporal coordinators of endocytosis and cell polarity.actin ͉ endocytosis ͉ polarity E ndocytosis is an essential mechanism for internalizing extracellular material and controlling the composition of the plasma membrane; this is critical for cellular homeostasis, including downregulation of signaling receptors and recycling of transmembrane proteins such as v-SNAREs that reside transiently at the plasma membrane (1). Many cytosolic proteins that contribute to the mechanisms and regulation of endocytosis have been identified, but assigning precise functions to each protein has been more challenging (2, 3). Some of these proteins may also participate in multiple steps or pathways (4, 5), either related to or independent from endocytosis, further complicating the elucidation of their function(s). Additionally, roles for the actin cytoskeleton in regulating or effecting specific stages of endocytosis are another active area of investigation (2). One goal is to identify multifunctional proteins that coordinate these various cellular processes.The epsin proteins are proposed to function as endocytic clathrin adaptors for ubiquitinated cargo (6, 7). They are found in all eukaryotes and have an N-terminal phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P 2 ]-binding epsin N-terminal homology (ENTH) domain, two ubiquitin interaction motifs, and several peptide ligands that bind components of the endocytic machinery (7). In addition to putative adaptor roles, it has been shown previously that mammalian epsin binds RalBP1͞RLIP76, a GTPase-activating protein (GAP) for Cdc42 and Rac1 (8). RalBP1 has been implicated in endocytosis, because it binds the plasma membrane clathrin adaptor AP-2 (8). The Cdc42 and Rac GTPases are key regulators of the actin cytoskeleton (9), thus suggesting that this complex links signaling, endocytosis, and actin cytoskeleton regulation.The budding yeast Saccharomyces cerevisiae has two epsins, Ent1 and Ent2; deleting either alone leads to no detectable phenotype, but a double deletion is lethal (10). Here we show that the ENTH domain of yeast epsin is necessary and sufficient for viability of ent1⌬ent2⌬ cells (⌬⌬). The essential function ...

No abstract