Structural basis of hepatitis B virus receptor binding.

Hepatitis B virus (HBV), a leading cause of developing hepatocellular carcinoma affecting more than 290 million people worldwide, is an enveloped DNA virus specifically infecting hepatocytes. Myristoylated preS1 domain of the HBV large surface protein binds to the host receptor sodium-taurocholate cotransporting polypeptide (NTCP), a hepatocellular bile acid transporter, to initiate viral entry. Here, we report the cryogenic-electron microscopy structure of the myristoylated preS1 (residues 2-48) peptide bound to human NTCP. The unexpectedly folded N-terminal half of the peptide embeds deeply into the outward-facing tunnel of NTCP, whereas the C-terminal half formed extensive contacts on the extracellular surface. Our findings reveal an unprecedented induced-fit mechanism for establishing high-affinity virus-host attachment and provide a blueprint for the rational design of anti-HBV drugs targeting virus entry.

Synthesis of Isomeric Tb3+ -Phthalocyanine Double-Decker Complexes Depending on the Difference in the Direction of Coordination Plane and Their Magnetic Properties.

Invited for the cover of this issue are Kentaro Tanaka at Nagoya University and co-workers. The image depicts three isomers of a terbium(III) phthalocyanine double-decker complex made from C4h symmetrically substituted phthalocyanines and their magnetic properties. Read the full text of the article at 10.1002/chem.202203272.

Synthesis of Isomeric Tb3+ -Phthalocyanine Double-Decker Complexes Depending on the Difference in the Direction of Coordination Plane and Their Magnetic Properties.

A C4h symmetrically substituted phthalocyanine, 1,8,15,22-tertrakis(2,4-dimethylpent-3-oxy)phthalocyanine (H2 TdMPPc), was used to synthesize Tb3+ -phthalocyanine double-decker complexes ([Tb(TdMPPc)2 ]s). Because H2 TdMPPc has C4h symmetry, S,S, R,R, and meso isomers of [Tb(TdMPPc)2 ] were obtained depending on the difference in the direction of the coordination plane of two C4h -type phthalocyanines with respect to a central Tb3+ ion. We investigated the physical properties of these [Tb(TdMPPc)2 ] isomers, including their single-ion magnetic properties, and found that the spin-reversal energy barrier (Ueff ) of the meso isomer was apparently higher than that of the enantiomers. Detailed crystal structural analyses indicated that the meso isomer has a more symmetrical structure than do the enantiomers, thereby suggesting that the higher Ueff of the meso isomer originated from the more highly symmetrical structure.

Design, Synthesis, and Evaluation of a Set of Carboxylic Acid and Phosphate Prodrugs Derived from HBV Capsid Protein Allosteric Modulator NVR 3-778.

Hepatitis B virus (HBV) capsid protein (Cp) is necessary for viral replication and the maintenance of viral persistence, having become an attractive target of anti-HBV drugs. To improve the water solubility of HBV capsid protein allosteric modulator (CpAM) NVR 3-778, a series of novel carboxylic acid and phosphate prodrugs were designed and synthesized using a prodrug strategy. In vitro HBV replication assay showed that these prodrugs maintained favorable antiviral potency (EC50 = 0.28−0.42 µM), which was comparable to that of NVR 3-778 (EC50 = 0.38 µM). More importantly, the cytotoxicity of prodrug N8 (CC50 > 256 µM) was significantly reduced compared to NVR 3-778 (CC50 = 13.65 ± 0.21 µM). In addition, the water solubility of prodrug N6 was hundreds of times better than that of NVR 3-778 in three phosphate buffers with various pH levels (2.0, 7.0, 7.4). In addition, N6 demonstrated excellent plasma and blood stability in vitro and good pharmacokinetic properties in rats. Finally, the hemisuccinate prodrug N6 significantly improved the candidate drug NVR 3-778's water solubility and increased metabolic stability while maintaining its antiviral efficacy.

Fungal Secondary Metabolite Exophillic Acid Selectively Inhibits the Entry of Hepatitis B and D Viruses.

Current anti-hepatitis B virus (HBV) drugs are suppressive but not curative for HBV infection, so there is considerable demand for the development of new anti-HBV agents. In this study, we found that fungus-derived exophillic acid inhibits HBV infection with a 50% maximal inhibitory concentration (IC50) of 1.1 µM and a 50% cytotoxic concentration (CC50) of >30 µM in primary human hepatocytes. Exophillic acid inhibited preS1-mediated viral attachment to cells but did not affect intracellular HBV replication. Exophillic acid appears to target the host cells to reduce their susceptibility to viral attachment rather than acting on the viral particles. We found that exophillic acid interacted with the HBV receptor, sodium taurocholate cotransporting polypeptide (NTCP). Exophillic acid impaired the uptake of bile acid, the original function of NTCP. Consistent with our hypothesis that it affects NTCP, exophillic acid inhibited infection with HBV and hepatitis D virus (HDV), but not that of hepatitis C virus. Moreover, exophillic acid showed a pan-genotypic anti-HBV effect. We thus identified the anti-HBV/HDV activity of exophillic acid and revealed its mode of action. Exophillic acid is expected to be a potential new lead compound for the development of antiviral agents.

Improving the Use of Deictic Verbs in Children with Autism Spectrum Disorder.

Background: Children with autism spectrum disorder (ASD) show difficulty in comprehension and production of the deictic verbs "come/go." Objective: To examine whether introducing conditions related to daily conversations into training would improve the use of deictic verbs. Methods: Six Japanese children with ASD participated. We set up multiple scenes where the questioner presented the sentence using "come/go" with/without deictic gestures, and children with ASD replied with "come/go." The conditions such as spatial relations between the two parties (face-to-face or side-by-side) and presentations of the gestures (moving one's arm toward or away from the body or moving one's upper body forward/backward) were introduced. Results: The appropriate use of deictic verbs during training and in daily life situations among children with ASD increased. Conclusions: Training children with ASD to look in the direction indicated by the questioner and to synchronize their bodies with the questioner's movements promotes their acquisition of deictic verbs.

Identification of Anti-Severe Acute Respiratory Syndrome-Related Coronavirus 2 (SARS-CoV-2) Oxysterol Derivatives In Vitro.

The development of effective antiviral drugs targeting the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is urgently needed to combat the coronavirus disease 2019 (COVID-19). We have previously studied the use of semi-synthetic derivatives of oxysterols, oxidized derivatives of cholesterol as drug candidates for the inhibition of cancer, fibrosis, and bone regeneration. In this study, we screened a panel of naturally occurring and semi-synthetic oxysterols for anti-SARS-CoV-2 activity using a cell culture infection assay. We show that the natural oxysterols, 7-ketocholesterol, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 27-hydroxycholesterol, substantially inhibited SARS-CoV-2 propagation in cultured cells. Among semi-synthetic oxysterols, Oxy210 and Oxy232 displayed more robust anti-SARS-CoV-2 activities, reducing viral replication more than 90% at 10 µM and 99% at 15 µM, respectively. When orally administered in mice, peak plasma concentrations of Oxy210 fell into a therapeutically relevant range (19 µM), based on the dose-dependent curve for antiviral activity in our cell-based assay. Mechanistic studies suggest that Oxy210 reduced replication of SARS-CoV-2 by disrupting the formation of double-membrane vesicles (DMVs); intracellular membrane compartments associated with viral replication. Our study warrants further evaluation of Oxy210 and Oxy232 as a safe and reliable oral medication, which could help protect vulnerable populations with increased risk of developing COVID-19.

σ-Bond Hydroboration of Cyclopropanes.

Hydroboration of alkenes is a classical reaction in organic synthesis in which alkenes react with boranes to give alkylboranes with subsequent oxidation resulting in alcohols. The double bond (π-bond) of alkenes can be readily reacted with boranes owing to its high reactivity. However, the single bond (σ-bond) of alkanes has never been reacted. To pursue the development of σ-bond cleavage, we selected cyclopropanes as model substrates since they present a relatively weak σ-bond. Herein, we describe an iridium-catalyzed hydroboration of cyclopropanes, resulting in ß-methyl alkylboronates. These unusually branched boronates can be derivatized by oxidation or cross-coupling chemistry, accessing "designer" products that are desired by practitioners of natural product synthesis and medicinal chemistry. Furthermore, mechanistic investigations and theoretical studies revealed the enabling role of the catalyst.

Serum uric acid and the incidence of CKD and hypertension.

BACKGROUND: Uric acid (UA) levels correlate positively with the prevalence of chronic kidney disease (CKD) and/or hypertension. We tested the hypothesis that UA may also have a link to a new incidence of CKD and hypertension. METHODS: Study design is a cohort study and the predictor is UA levels. Of the 15,470 screened cases, 8223 participants without CKD were eligible for the analysis of the incidence of CKD. Among these CKD candidates, 7569 participants were eligible for the analysis of the new development of hypertension. The observation period was 4 years. RESULTS: Relationship of UA with new cases of CKD. Higher UA levels had a closer association with the new development of CKD; 1.1 % (UA < 5 mg/dL), 1.5 % (5.0-5.9 mg/dL), 1.7 % (6.0-6.9 mg/dL), and 3.4 % (≧7 mg/dL), respectively (p < 0.001 by the Chi-square test). Cox proportional hazard analysis showed that the estimates of the CKD development were eGFR [Hazard Ratio (HR) 0.816, 95 % confidence intervals (CI) 0.791-0.840] and male gender (HR 0.562, 95 % CI 0.322-0.982). UA levels and new development of hypertension. Higher UA levels had a closer association with the new development of hypertension; 5.0 % (UA < 5 mg/dL), 8.9 % (5.0-5.9 mg/dL), 10.6 % (6.0-6.9 mg/dL), and 11.8 % (≧7 mg/dL), respectively (p < 0.001 by the Chi-square test). Cox proportional hazard analysis showed that the estimates of the hypertension development were BMI (HR 1.190, 95 % CI 1.155-1.226), age (HR 1.021, 95 % CI 1.010-1.032), HDL-cholesterol (HR 1.013, 95 % CI 1.007-1.019), male gender (HR 1.791, 95 % CI 1.338-2.395), UA level (HR 1.112, 95 % CI 1.024-1.207), and eGFR (HR 1008, 95 % CI 1.002-1.013). Furthermore, the logistic analysis showed that the odds ratio (OR) to estimate hypertension in the high UA group (UA ≧ 7 mg/dL; OR 1.33, 95 % CI 1.01-1.80) was greater than that in the low UA group (UA < 5 mg/dL). Kaplan-Meier analysis also confirmed the finding that the higher the UA levels the greater the hypertension development (p < 0.001 by the Log-rank test and Cox proportional hazard analysis). CONCLUSION: High UA levels are associated with the new development of hypertension, but not with the incidence of CKD.

Encapsulation of beraprost sodium in nanoparticles: analysis of sustained release properties, targeting abilities and pharmacological activities in animal models of pulmonary arterial hypertension.

Prostaglandin I2 (PGI2) and its analogues (such as beraprost sodium, BPS) are beneficial for the treatment of pulmonary arterial hypertension (PAH). The encapsulation of BPS in nanoparticles to provide sustained release and targeting abilities would improve both the therapeutic effect of BPS on PAH and the quality of life of patients treated with this drug. BPS was encapsulated into nanoparticles prepared from a poly(lactic acid) homopolymer and monomethoxy poly(ethyleneglycol)-poly(lactide) block copolymer. The accumulation of nanoparticles in damaged pulmonary arteries was examined using fluorescence-emitting rhodamine S-encapsulated nanoparticles. The monocrotaline-induced PAH rat model and the hypoxia-induced mouse model were used to examine the pharmacological activity of BPS-encapsulated nanoparticles. A nanoparticle, named BPS-NP, was selected among various types of BPS-encapsulated nanoparticles tested; this was based on the sustained release profile in vitro and blood clearance profile in vivo. Fluorescence-emitting rhodamine S-encapsulated nanoparticles were prepared in a similar manner to that of BPS-NP, and showed accumulation and prolonged residence in monocrotaline-damaged pulmonary peripheral arteries. Intravenous administration of BPS-NP (once per week, 20µg/kg) protected against monocrotaline-induced pulmonary arterial remodeling and right ventricular hypertrophy. The extent of this protection was similar to that observed with oral administration (once per day, 100µg/kg) of BPS alone. The once per week intravenous administration of BPS-NP (20µg/kg) also exhibited an ameliorative effect on hypoxia-induced pulmonary arterial remodeling and right ventricular hypertrophy. The beneficial effects of BPS-NP on PAH animal models seem to be mediated by its sustained release and tissue targeting profiles. BPS-NP may be useful for the treatment of PAH patients due to reduced dosages and frequency of BPS administration.

[Relationship between serum uric acid levels and muscle strength/volume: a new insight from a large-scale survey].

AIM: Serum uric acid (UA) concentration is regulated by its production in the liver and/or intestine and its rate of excretion from the kidneys. However, little is known about skeletal muscle involvement in determining the physiological UA level. The present trial explores whether muscle strength and/or muscle volume is associated with UA levels. MATERIAL & METHODS: Muscle strength was evaluated in terms of grasping power calculated as an average of right and left hand measurements in relation to other parameters in 14,333 subjects (median age; 41.2 years), who were recruited to the study. Skeletal muscle volume was calculated based on the bioimpedance method by subtracting estimated fat volume plus estimated bone weight from the total body weight. RESULTS: 1) Multiple regression analyses to explain the association with UA levels (dependent variable) revealed that BMI, BUN, triglyceride, muscle strength, AST, age and sex are independent variables. 2) Higher UA levels (assessed as 4 UA quartiles) are associated with higher muscle volume, muscle strength, BMI, DBP, and serum creatinine (Cr) concentration. 3) Greater DBP (assessed as 2 UA categories) was associated with higher BMI, muscle strength, muscle volume, UA levels and serum Cr concentration. 4) Regression coefficient "t" for muscle strength was the largest among the other parameters including serum Cr concentration in the UA level ranging from 5.5 to 6.5 mg/dL. CONCLUSION: There was an association between muscle strength/volume and UA levels in the near physiological UA range, suggesting that the circulating UA levels can be, at least in part, controlled by its production in the skeletal muscles.

Identification and characterization of genes involved in glutathione production in yeast.

Glutathione is a major peptide protecting cells against oxidative stress. To study the cellular processes affecting intracellular glutathione production, we screened Saccharomyces cerevisiae mutant collections and identified new eight yeast deletion mutants that produced more than 1.2-fold higher levels of intracellular glutathione: chc1, cst6, ddc1, def1, pep12, rts1, ubp6, and yih1. Furthermore, overexpression of the DEF1 and CYS4 genes led to a higher production of glutathione, similar to overexpression of GSH1. A multiplier effect on activation of glutathione synthesis was observed by a combination of overexpression of GSH1 and deletion of one of the eight genes. Metabolome analysis of the def1, pep12, and ubp6 deletion mutant, and DEF1-overexpressing strains showed that levels of intracellular methionine and oxidized glutathione were higher than in the control strains, suggesting that methionine biosynthesis was activated and the oxidative stress response was increased in these glutathione-overproductive strains. Moreover, overexpression of GSH1, CYS4, and DEF1 also increased glutathione production in Candida utilis. Taken together, these results will significantly contribute to more effective industrial production of glutathione using yeasts.

HSP70 inducers from Chinese herbs and their effect on melanin production.

Skin hyperpigmentation disorders as a result of abnormal melanin production induced by ultraviolet (UV) irradiation are both a clinical and a cosmetic problem. This melanin production is mediated by tyrosinase whose expression is positively regulated by microphthalmia-associated transcription factor (MITF). We recently found that expression of heat shock protein 70 (HSP70) inhibits melanin production. In this study, we searched for HSP70 inducers from Chinese herbs and selected an ethanol extract of Eupatorium lindleyanum (E. lindleyanum). Not only melanin production but also the activity and expression of tyrosinase were significantly suppressed in cells treated with E. lindleyanum extract as well as in HSP70-overexpressing cells. The expression of MITF was clearly suppressed in cells treated with E. lindleyanum extract but not in HSP70-overexpressing cells. These results suggest that E. lindleyanum extract suppresses the expression of tyrosinase and melanin production through both HSP70-dependent and HSP70-independent mechanisms.

Accelerated blood clearance phenomenon upon repeated injection of PEG-modified PLA-nanoparticles.

PURPOSE: We recently developed prostaglandin E(1) (PGE(1))-encapsulated nanoparticles, prepared with a poly(lactide) homopolymer (PLA, Mw = 17,500) and monomethoxy poly(ethyleneglycol)-PLA block copolymer (PEG-PLA) (NP-L20). In this study, we tested whether the accelerated blood clearance (ABC) phenomenon is observed with NP-L20 and other PEG-modified PLA-nanoparticles in rats. METHODS: The plasma levels of PGE(1) and anti-PEG IgM antibody were determined by EIA and ELISA, respectively. RESULTS: Second injections of NP-L20 were cleared much more rapidly from the circulation than first injections, showing that the ABC phenomenon was induced. This ABC phenomenon, and the accompanying induction of anti-PEG IgM antibody production, was optimal at a time interval of 7 days between the first and second injections. Compared to NP-L20, NP-L33s that were prepared with PLA (Mw = 28,100) and have a smaller particle size induced production of anti-PEG IgM antibody to a lesser extent. NP-L20 but not NP-L33s gave rise to the ABC phenomenon with a time interval of 14 days. NP-L33s showed a better sustained-release profile of PGE(1) than NP-L20. CONCLUSIONS: This study revealed that the ABC phenomenon is induced by PEG-modified PLA-nanoparticles. We consider that NP-L33s may be useful clinically for the sustained-release and targeted delivery of PGE(1).