Vertebrate Ssu72 regulates and coordinates 3'-end formation of RNAs transcribed by RNA polymerase II.

In eukaryotes, the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is composed of tandem repeats of the heptapeptide YSPTSPS, which is subjected to reversible phosphorylation at Ser2, Ser5, and Ser7 during the transcription cycle. Dynamic changes in CTD phosphorylation patterns, established by the activities of multiple kinases and phosphatases, are responsible for stage-specific recruitment of various factors involved in RNA processing, histone modification, and transcription elongation/termination. Yeast Ssu72, a CTD phosphatase specific for Ser5 and Ser7, functions in 3'-end processing of pre-mRNAs and in transcription termination of small non-coding RNAs such as snoRNAs and snRNAs. Vertebrate Ssu72 exhibits Ser5- and Ser7-specific CTD phosphatase activity in vitro, but its roles in gene expression and CTD dephosphorylation in vivo remain to be elucidated. To investigate the functions of vertebrate Ssu72 in gene expression, we established chicken DT40 B-cell lines in which Ssu72 expression was conditionally inactivated. Ssu72 depletion in DT40 cells caused defects in 3'-end formation of U2 and U4 snRNAs and GAPDH mRNA. Surprisingly, however, Ssu72 inactivation increased the efficiency of 3'-end formation of non-polyadenylated replication-dependent histone mRNA. Chromatin immunoprecipitation analyses revealed that Ssu72 depletion caused a significant increase in both Ser5 and Ser7 phosphorylation of the Pol II CTD on all genes in which 3'-end formation was affected. These results suggest that vertebrate Ssu72 plays positive roles in 3'-end formation of snRNAs and polyadenylated mRNAs, but negative roles in 3'-end formation of histone mRNAs, through dephosphorylation of both Ser5 and Ser7 of the CTD.

Coformer screening using thermal analysis based on binary phase diagrams.

PURPOSE: The advent of cocrystals has demonstrated a growing need for efficient and comprehensive coformer screening in search of better development forms, including salt forms. Here, we investigated a coformer screening system for salts and cocrystals based on binary phase diagrams using thermal analysis and examined the effectiveness of the method. METHODS: Indomethacin and tenoxicam were used as models of active pharmaceutical ingredients (APIs). Physical mixtures of an API and 42 kinds of coformers were analyzed using Differential Scanning Calorimetry (DSC) and X-ray DSC. We also conducted coformer screening using a conventional slurry method and compared these results with those from the thermal analysis method and previous studies. RESULTS: Compared with the slurry method, the thermal analysis method was a high-performance screening system, particularly for APIs with low solubility and/or propensity to form solvates. However, this method faced hurdles for screening coformers combined with an API in the presence of kinetic hindrance for salt or cocrystal formation during heating or if there is degradation near the metastable eutectic temperature. CONCLUSIONS: The thermal analysis and slurry methods are considered complementary to each other for coformer screening. Feasibility of the thermal analysis method in drug discovery practice is ensured given its small scale and high throughput.

High-throughput evaluation method for drug association with pregnane X receptor (PXR) using differential scanning fluorometry.

The pregnane xenobiotic receptor (PXR) is a key transcriptional regulator of cytochrome P450 (CYP) 3A, a crucial enzyme in the metabolism and detoxification of xenobiotics and endobiotics. PXR is activated by a wide variety of chemicals and serves as a master regulator of detoxification in mammals. Here, we report a fast evaluation method for PXR-drug interactions using differential scanning fluorometry (DSF). DSF analysis revealed that PXR associates with a fluorescence dye in the native state as well as in the unfolded state, which prevented precise evaluation of any shift in the transition midpoint (ΔT (m)) due to association with a drug. Hence, we defined a new parameter, (dF/dT)(50), where F is fluorescence intensity and T is temperature, to describe the ligand concentration. (dF/dT)(50) exhibited better correlation with EC(50) (r(2) = 0.84) than with ΔT m (r(2) = 0.71). The correlation of ΔT m measured using differential scanning calorimetry (DSC) with EC(50) (r(2) = 0.86) was similar to the above (dF/dT)(50) correlation. Therefore, the use of (dF/dT)(50) enables DSF to be used for the rapid evaluation of PXR-drug interactions and could provide prescreening to narrow down the collection of candidate ligands that most likely result in transcriptional activation of CYP3A4.

Detection of cocrystal formation based on binary phase diagrams using thermal analysis.

PURPOSE: Although a number of studies have reported that cocrystals can form by heating a physical mixture of two components, details surrounding heat-induced cocrystal formation remain unclear. Here, we attempted to clarify the thermal behavior of a physical mixture and cocrystal formation in reference to a binary phase diagram. METHODS: Physical mixtures prepared using an agate mortar were heated at rates of 2, 5, 10, and 30 °C/min using differential scanning calorimetry (DSC). Some mixtures were further analyzed using X-ray DSC and polarization microscopy. RESULTS: When a physical mixture consisting of two components which was capable of cocrystal formation was heated using DSC, an exothermic peak associated with cocrystal formation was detected immediately after an endothermic peak. In some combinations, several endothermic peaks were detected and associated with metastable eutectic melting, eutectic melting, and cocrystal melting. In contrast, when a physical mixture of two components which is incapable of cocrystal formation was heated using DSC, only a single endothermic peak associated with eutectic melting was detected. CONCLUSION: These experimental observations demonstrated how the thermal events were attributed to phase transitions occurring in a binary mixture and clarified the relationship between exothermic peaks and cocrystal formation.

Identification of 4-quinolone derivatives as inhibitors of reactive oxygen species production from human umbilical vein endothelial cells.

Oxidative stress is widely recognized as being associated with a number of disorders, including metabolic dysfunction and atherosclerosis. A series of substituted 4-quinolone derivatives were prepared and evaluated as inhibitors of reactive oxygen species (ROS) production from human umbilical vein endothelial cells (HUVECs). One compound in particular, 2-({[4-(3-hydroxy-3-methylbutoxy)pyridin-2-yl]oxy}methyl)-3-methylquinolin-4(1H)-one (25b), inhibited ROS production from HUVECs with an IC(50) of 140 nM. This compound also exhibited low CYP2D6 inhibitory activity, high aqueous solubility, and good in vitro metabolic stability. An in vivo pharmacokinetic study of this compound in SD rats revealed high oral bioavailability and a long plasma half-life.

Prolyl isomerase Pin1 shares functional similarity with phosphorylated CTD interacting factor PCIF1 in vertebrate cells.

The carboxy-terminal domain (CTD) of the RNA polymerase II (Pol II) largest subunit undergoes reversible phosphorylation during transcription cycle. The phosphorylated CTD plays critical roles in coordinating transcription with chromatin modification and RNA processing by serving as a scaffold to recruit various proteins. Recently, we identified a novel human WW domain-containing protein PCIF1 as a phosphorylated CTD-interacting factor and demonstrated that PCIF1 negatively modulates Pol II activity in vivo. In the present study, to explore cellular functions of PCIF1, we generated PCIF1-deficient chicken DT40 cell lines. We observed significant up-regulation of WW domain-containing prolyl isomerase Pin1 in two independently established PCIF1-deficient mutant clones. As reconstitution of PCIF1 in the mutants did not reduce Pin1 expression, PCIF1 may not be a negative regulator of Pin1 expression. We assume that Pin1 over-expression might suppress defects caused by PCIF1 deficiency in DT40 cells. We furthermore compared PCIF1 and Pin1 for their functional properties and found that these two proteins exhibit most similar target specificity among other CTD-binding WW proteins, overlapping subcellular localization and comparative inhibitory effects on transcriptional activation by Pol II in human cultured cells. These results suggest that Pin1 may have overlapping cellular function with PCIF1 in vertebrate cells.