Identification of small-molecule inhibitors against the interaction of RNA-binding protein PSF and its target RNA for cancer treatment.

Diverse cellular activities are modulated through a variety of RNAs, including long noncoding RNAs (lncRNAs), by binding to certain proteins. The inhibition of oncogenic proteins or RNAs is expected to suppress cancer cell proliferation. We have previously demonstrated that PSF interaction with its target RNAs, such as androgen-induced lncRNA CTBP1-AS, is critical for hormone therapy resistance in prostate and breast cancers. However, the action of protein-RNA interactions remains almost undruggable to date. High-throughput screening (HTS) has facilitated the discovery of drugs for protein-protein interactions. In the present study, we developed an in vitro alpha assay using Flag peptide-conjugated lncRNA, CTBP1-AS, and PSF. We then constructed an effective HTS screening system to explore small compounds that inhibit PSF-RNA interactions. Thirty-six compounds were identified and dose-dependently inhibited PSF-RNA interaction in vitro. Moreover, chemical optimization of these lead compounds and evaluation of cancer cell proliferation revealed two promising compounds, N-3 and C-65. These compounds induced apoptosis and inhibited cell growth in prostate and breast cancer cells. By inhibiting PSF-RNA interaction, N-3 and C-65 up-regulated signals that are repressed by PSF, such as the cell cycle signals by p53 and p27. Furthermore, using a mouse xenograft model for hormone therapy-resistant prostate cancer, we revealed that N-3 and C-65 can significantly suppress tumor growth and downstream target gene expression, such as the androgen receptor (AR). Thus, our findings highlight a therapeutic strategy through the development of inhibitors for RNA-binding events in advanced cancers.

A specific G9a inhibitor unveils BGLT3 lncRNA as a universal mediator of chemically induced fetal globin gene expression.

Sickle cell disease (SCD) is a heritable disorder caused by ß-globin gene mutations. Induction of fetal γ-globin is an established therapeutic strategy. Recently, epigenetic modulators, including G9a inhibitors, have been proposed as therapeutic agents. However, the molecular mechanisms whereby these small molecules reactivate γ-globin remain unclear. Here we report the development of a highly selective and non-genotoxic G9a inhibitor, RK-701. RK-701 treatment induces fetal globin expression both in human erythroid cells and in mice. Using RK-701, we find that BGLT3 long non-coding RNA plays an essential role in γ-globin induction. RK-701 selectively upregulates BGLT3 by inhibiting the recruitment of two major γ-globin repressors in complex with G9a onto the BGLT3 gene locus through CHD4, a component of the NuRD complex. Remarkably, BGLT3 is indispensable for γ-globin induction by not only RK-701 but also hydroxyurea and other inducers. The universal role of BGLT3 in γ-globin induction suggests its importance in SCD treatment.

A Novel Class of HIV-1 Inhibitors Targeting the Vpr-Induced G2-Arrest in Macrophages by New Yeast- and Cell-Based High-Throughput Screening.

The human immunodeficiency virus type 1 (HIV-1) accessory protein, Vpr, arrests the cell cycle of the G2 phase, and this Vpr-mediated G2 arrest is implicated in an efficient HIV-1 spread in monocyte-derived macrophages. Here, we screened new candidates for Vpr-targeting HIV-1 inhibitors by using fission yeast- and mammalian cell-based high-throughput screening. First, fission yeast strains expressing the HIV-1 Vpr protein were generated and then treated for 48 h with 20 µM of a synthetic library, including 140,000 chemical compounds. We identified 268 compounds that recovered the growth of Vpr-overexpressing yeast. The selected compounds were then tested in mammalian cells, and those displaying high cytotoxicity were excluded from further cell cycle analysis and imaging-based screening. A flow cytometry analysis confirmed that seven compounds recovered from the Vpr-induced G2 arrest. The cell toxicity and inhibitory effect of HIV-1 replication in human monocyte-derived macrophages (MDM) were examined, and three independent structural compounds, VTD227, VTD232, and VTD263, were able to inhibit HIV-1 replication in MDM. Furthermore, we showed that VTD227, but not VTD232 and VTD263, can directly bind to Vpr. Our results indicate that three new compounds and their derivatives represent new drugs targeting HIV-1 replication and can be potentially used in clinics to improve the current antiretroviral therapy.

Visit-to-visit blood pressure variability in children and adolescents with renal disease.

BACKGROUND: Increase in blood pressure (BP) variability (BPV) is associated with cardiovascular events, target organ damage, and arterial stiffness in adults. We previously reported that 24-h BPV may be associated with arterial stiffness and underlie white-coat hypertension (WCH). In this study, we examined whether visit-to-visit variability (VVV) could predict WCH and whether VVV correlated with eGFR, eGFR slope, and albuminuria/proteinuria in children and adolescents with renal diseases. METHODS: VVV was determined as average real variability of office BP measurements between visits, and 24-h BPV as the standard deviation of 24-h ambulatory BP. In 35 renal patients (25 boys and 10 girls, 7-18 years of age), divided into normotension (NT), WCH, and hypertension (HTN), the relationships between VVV and 24-h BPV and VVV in each BP category were studied. In separate 48 renal patients (24 boys and 24 girls, 2-18 years of age), the correlation between VVV and eGFR, eGFR slope, urine albumin or protein excretion was examined. RESULTS: Systolic VVV was significantly correlated with systolic office BP index. There was no correlation between VVV and 24-h BPV or 24-h pulse pressure. In addition, VVV was not different among NT, WCH, and HTN. Systolic VVV was significantly negatively correlated with eGFR but not with eGFR slope, albuminuria, or proteinuria. A cut-off value of systolic VVV for detecting eGFR < 60 ml/min per 1.73 m2 was 8.5. CONCLUSION: VVV could not predict WCH. Systolic VVV correlated with eGFR but not with eGFR slope, albuminuria/proteinuria. Increased VVV could be a marker of decreased eGFR.

Masked Isolated Nocturnal Hypertension in Children and Young Adults.

Isolated nocturnal hypertension (INH) is characterized by normal daytime blood pressure (BP) and elevated nighttime BP diagnosed by ambulatory BP monitoring. Masked isolated nocturnal hypertension (MINH) is a subtype of INH in which office BP is normal. We studied the frequency and characteristics of INH and MINH in children and young adults. One hundred and ninety-eight subjects seen by the pediatric nephrology service were studied retrospectively. Isolated nocturnal hypertension (INH) and MINH were diagnosed according to daytime and nighttime ABP and office BP in the case of the latter. One hundred and eighteen subjects (60%) had normotension, 6 (3%) had isolated daytime hypertension, 32 (16%) had INH, and 42 (21%) had day-night hypertension. Sixteen subjects had MINH (8.1%). The underlying diseases of MINH were as follows: no underlying disease 9 (56%), renal disease 6 (38%), and endocrine disease 1 (6%). There was no significant difference in the underlying disease, gender, age, and BMI between MINH and INH with elevated office BP. In conclusion, MINH is present in children and young adults. Since there were no specific features for MINH, screening with ambulatory or home BP monitoring during sleep may be appropriate.

White-Coat and Reverse White-Coat Effects Correlate with 24-h Pulse Pressure and Systolic Blood Pressure Variability in Children and Young Adults.

Masked hypertension (MH) and white-coat hypertension (WCH) are associated with organ damage. In the present study, we examined the correlation between the magnitude of white-coat effect (WCE) or reverse WCE (RWCE) and 24-h pulse pressure (PP), an indicator of target organ damage and arterial stiffness, in children and young adults. We also examined the relationship of WCE or RWCE and blood pressure (BP) variability, another predictor of clinical outcomes. One hundred and ninety-eight subjects were studied. According to the office BP and ambulatory BP, they were divided into normotension, WCH, MH, and hypertension. The magnitude of WCE or RWCE, along with male gender and 24-h systolic BP, was the determinant of 24-h PP. In subjects with 24-h PP ≥ 61 mmHg, the magnitude of WCE or RWCE, age, male ratio, height, weight, BMI, the percentage of secondary hypertension, that of MH, office systolic BP, and 24-h systolic BP were significantly greater. There was a progressive increase in 24-h PP from normotension, WCH, MH, to hypertension. BP variability in subjects with MH was numerically highest in both systolic and diastolic. Diastolic BP variability of WCH, MH, and hypertension was significantly higher than that of normotension. Finally, the magnitude of WCE or RWCE in systolic showed a significant correlation with systolic BP variability. In conclusion, the magnitude of WCE or RWCE correlates with 24-h PP and systolic BP variability, which may suggest increased arterial stiffness in WCH and MH.

Reactivity of cellulose reducing end in pyrolysis as studied by methyl glucoside-impregnation.

For better understanding of the roles of cellulose reducing ends during thermal degradation of cellulose and wood, cellulose samples impregnated with methyl-ß-D-glucopyranoside (GlcßOMe), a simple non-reducing sugar model, were pyrolyzed under N2 at relatively low temperatures of 200-280 °C. By the impregnation, cellulose was rather stabilized against discoloration and weight-loss through converting the reducing ends into the glycosides with GlcßOMe. Alternatively, polymerization and discoloration of GlcßOMe were accelerated in the presence of cellulose. A mechanism via reducing sugars as reactive intermediates formed through hydrolysis is proposed to explain these phenomena. These information would be useful to understand the interactions between cellulose and hemicellulose in wood cell wall as well as the role of the reducing ends in cellulose thermal degradation.

Spectomycin B1 as a novel SUMOylation inhibitor that directly binds to SUMO E2.

Conjugation of small ubiquitin-like modifier (SUMO) to protein (SUMOylation) regulates multiple biological systems by changing the functions and fates of a large number of proteins. Consequently, abnormalities in SUMOylation have been linked to multiple diseases, including breast cancer. Using an in situ cell-based screening system, we have identified spectomycin B1 and related natural products as novel SUMOylation inhibitors. Unlike known SUMOylation inhibitors such as ginkgolic acid, spectomycin B1 directly binds to E2 (Ubc9) and selectively blocks the formation of the E2-SUMO intermediate; that is, Ubc9 is the direct target of spectomycin B1. Importantly, either spectomycin B1 treatment or Ubc9 knockdown inhibited estrogen-dependent proliferation of MCF7 human breast-cancer cells. Our findings suggest that Ubc9 inhibitors such as spectomycin B1 have potential as therapeutic agents against hormone-dependent breast cancers.

Ambulatory blood pressure in prehypertensive children and adolescents.

BACKGROUND: Prehypertension is defined as blood pressure (BP) ≥ 90 th percentile, or ≥ 120/80 mmHg, but <95th percentile for age, sex, and height. Since the definition is made by conventional BP measurements and office BP can be quite variable, we studied whether prehypertension could be differentiated by ambulatory BP monitoring from normotension or hypertension (HTN) in children and adolescents. METHODS: One hundred and fifty-eight children (84 boys and 74 girls, aged 6-17 years, median 12) were studied. According to the office BP values, they were divided into normotension (80), prehypertension (20), and HTN (58). RESULTS: Systolic BP index and systolic daytime ambulatory BP (ABP) were significantly higher in prehypertensive patients than in normotensives and lower than hypertensives. When daytime ABP was used to diagnose HTN, four normotensive (5.0%), four prehypertensive (20.0%), and 27 hypertensive (46.6%) patients had HTN. Thus, in patients with prehypertension, the prevalence of masked HTN is significantly higher than in those with normotension. On the other hand, the prevalence of daytime ambulatory HTN is significantly lower, i.e., white-coat effect is more frequent, compared with hypertensive patients. CONCLUSION: Prehypertension lies between normotension and HTN in ABP values as well and is a good candidate for identifying masked HTN. Our data emphasize the importance of identifying prehypertension in children and adolescents.

Thermal glycosylation and degradation reactions occurring at the reducing ends of cellulose during low-temperature pyrolysis.

Thermal glycosylation and degradation reactions of cellulose (Avicel PH-101) were studied in the presence or absence of alcohols (glycerol, mannitol, 1,2,6-hexanetriol, 3-phenoxy-1,2-propanediol, and 1-tetradecanol) under N(2) at 60-280°C. In the presence of glycerol (heating time, 10 min), the reducing ends were converted into glycosides when the temperature of the glycerol was >140°C without the addition of any catalysts. A temperature of 140°C is close to that required for the initiation of thermal polymerization (glycosylation). Although the conversion was only around 20% in the range of 140-180°C, the reactivity increased above 200-240°C where the thermal expansion of cellulose crystals is reported to become significant. Finally, all reducing ends were converted into glycosides at 260°C. Such heterogeneous reactivity likely arose from the lower reactivities of the reducing ends in the crystalline region due to their lower accessibility to glycerol, although the reactivity in the non-crystalline region was similar to that of glucose. Alcohols that have a lower OH/C ratio did not react with the reducing ends, suggesting that the hydrophilicity of the alcohol was critical for the glycosylation reaction to proceed. The glycosylated cellulose samples were found to be significantly stabilized against pyrolytic coloration. The results of neat cellulose pyrolysis indicated that two competitive reactions, thermal glycosylation and degradation, formed a dark-colored substance at the reducing ends while the internal glucose units in the cellulose were comparatively stable.

Masked hypertension in children and young adults.

Masked hypertension, a high ambulatory blood pressure (ABP) in the presence of normal office blood pressure (BP), is recognized as a risk factor for cardiovascular complications in the adult population. We evaluated the prevalence of masked hypertension in pediatric patients. We studied 136 patients (59 boys and 77 girls, aged 6-25 years, mean 13.1+/-4.7 years). In all patients, office BP measurements with auscultatory technique were less than the 95th percentile for sex and age or <140/90 mmHg for those over 18 years. Masked hypertension was diagnosed when either systolic or diastolic daytime ABP values were equal to or greater than the 95th percentile for sex and height of reference values or > or =135 mmHg systolic or 85 mmHg diastolic BP for those over 15 years. Among 136 patients, 15 (11%) had masked hypertension. The prevalence of masked hypertension was higher in boys (19%) than in girls (5%), but not different between younger (< or =15 years) and older (>15 years) patients (11% vs. 12%). The diagnoses in the group with masked hypertension included 3 patients with diabetic nephropathy, 2 with obesity, and 2 with orthostatic dysregulation. In conclusion, masked hypertension is present in pediatric patients, and is more common in boys. Further study is needed to identify patients who may benefit from recognition of masked hypertension.

White coat effect and white coat hypertension in pediatric patients.

We evaluated the prevalence of white coat (WC) effect in pediatric age patients and that of white coat hypertension (WCH) in hypertensive pediatric patients. Two hundred and six patients (136 normotensive and 70 hypertensive patients, 107 boys and 99 girls, aged 6-25 years, mean 13.4, SD 4.7) were studied. Hypertension was diagnosed when systolic and/or diastolic blood pressure (BP) measurements with auscultatory technique were >or= the 95th percentile for sex and age. WC effect was defined as office BP minus daytime mean ambulatory BP (ABP). WCH was diagnosed in the hypertensive patients when daytime ABP values were < the 95th percentile for sex and height of reference values. There was a positive correlation between office BP and WC effect ( P<0.05). A WC effect of >or= 10 mmHg was observed more frequently in hypertensive patients (50%) than in normotensive patients (25%). Among 70 hypertensive patients, 33 (47%) had WCH. There was no significant difference in the prevalence of WCH in relation to age, gender, or the presence or absence of causes of hypertension. In conclusion, WC effect is frequently seen in pediatric patients, and is more common in subjects with higher office BP.