Structure-Activity Relationships and Discovery of (S)-6-Isopropyl-2-methoxy-3-(3-methoxypropoxy)-10-oxo-5,10-dihydro-6H-pyrido[1,2-h][1,7]naphthyridine-9-carboxylic Acid (AB-452), a Novel Orally Available HBV RNA Destabilizer.

Approved therapies for hepatitis B virus (HBV) treatment include nucleos(t)ides and interferon alpha (IFN-α) which effectively suppress viral replication, but they rarely lead to cure. Expression of viral proteins, especially surface antigen of the hepatitis B virus (HBsAg) from covalently closed circular DNA (cccDNA) and the integrated genome, is believed to contribute to the persistence of HBV. This work focuses on therapies that target the expression of HBV proteins, in particular HBsAg, which differs from current treatments. Here we describe the identification of AB-452, a dihydroquinolizinone (DHQ) analogue. AB-452 is a potent HBV RNA destabilizer by inhibiting PAPD5/7 proteins in vitro with good in vivo efficacy in a chronic HBV mouse model. AB-452 showed acceptable tolerability in 28-day rat and dog toxicity studies, and a high degree of oral exposure in multiple species. Based on its in vitro and in vivo profiles, AB-452 was identified as a clinical development candidate.

Apoptotic cell-derived exosomes: messages from dying cells.

Apoptosis, a type of programmed cell death that plays a key role in both healthy and pathological conditions, releases extracellular vesicles such as apoptotic bodies and microvesicles, but exosome release due to apoptosis is not yet commonly accepted. Here, the reports demonstrating the presence of apoptotic exosomes and their roles in inflammation and immune responses are summarized, together with a general summary of apoptosis and extracellular vesicles. In conclusion, apoptosis is not just a 'silent' type of cell death but an active form of communication from dying cells to live cells through exosomes.

Sustainable electricity generation and ammonium removal by microbial fuel cell with a microalgae assisted cathode at various environmental conditions.

Power generation and nutrient removal by a microbial fuel cell assisted with an algae growth in cathode chamber were evaluated along with continuous measurements of dissolved oxygen (DO) at various light and temperature conditions. Under light/dark regime of 18/6 hrs, a sustainable voltage of 0.31 V was observed with sufficient DO in the dark period. However, maximum NH4+-N removal rate (22.7 mg/L.d) and removal percentage (95.5%) was observed with 12/12 light/dark regime. Voltage and nitrogen removal increased with higher photon flux density up to 92 µE m-2 s-1, and the highest 0.35 V voltage and NH4+-N removal rate of 26.5 mg/L.d were obtained. A higher temperature condition was favorable to improve the performance of algae cathode MFCs, but no substantial influence was observed relatively (19-35 °C). This study suggests that algae-cathode MFCs could continuously generate sustainable power with nutrient removal at optimum light and temperature conditions.

Performance of an air-cathode microbial fuel cell under varied relative humidity conditions in the cathode chamber.

The performance of an air-cathode microbial fuel cell (MFC) with a cap arrangement was significantly affected by humidity conditions in the cathode. An MFC at a relative humidity (RH) of 88% produced a highest cell voltage of 0.42 V (600 Ω) compared to other operations at 50% (0.34 V) and 30% (0.29 V) RHs. During polarization analysis, MFC operation at 88% RH produced a maximum power density of 0.377 W/m2 (a current density of 1.5 A/m2), which was 1.8 and 2.9 times higher than with 50% and 30% RHs, respectively. Cyclic voltammogram analysis revealed a higher reduction current of - 0.073 A with 88% RH. Furthermore, no increase in dissolved oxygen concentration in the anode chamber was observed with 88% RH. This result suggests that control of humidity conditions in cathode chamber could maximize power generation from an air-cathode MFC.

Inhibition of HBV replication by N-hydroxyisoquinolinedione and N-hydroxypyridinedione ribonuclease H inhibitors.

We recently developed a screening system capable of identifying and evaluating inhibitors of the Hepatitis B virus (HBV) ribonuclease H (RNaseH), which is the only HBV enzyme not targeted by current anti-HBV therapies. Inhibiting the HBV RNaseH blocks synthesis of the positive-polarity DNA strand, causing early termination of negative-polarity DNA synthesis and accumulation of RNA:DNA heteroduplexes. We previously reported inhibition of HBV replication by N-hydroxyisoquinolinediones (HID) and N-hydroxypyridinediones (HPD) in human hepatoma cells. Here, we report results from our ongoing efforts to develop more potent anti-HBV RNaseH inhibitors in the HID/HPD compound classes. We synthesized and screened additional HIDs and HPDs for preferential suppression of positive-polarity DNA in cells replicating HBV. Three of seven new HIDs inhibited HBV replication, however, the therapeutic indexes (TI = CC50/EC50) did not improve over what we previously reported. All nine of the HPDs inhibited HBV replication with EC50s ranging from 110 nM to 4 µM. Cellular cytotoxicity was evaluated by four assays and CC50s ranged from 15 to >100 µM. The best compounds have a calculated TI of >300, which is a 16-fold improvement over the primary HPD hit. These studies indicate that the HPD compound class holds potential for antiviral discovery.

Application of high-salinity stress for enhancing the lipid productivity of Chlorella sorokiniana HS1 in a two-phase process.

Increased lipid accumulation of algal cells as a response to environmental stress factors attracted much attention of researchers to incorporate this stress response into industrial algal cultivation process with the aim of enhancing algal lipid productivity. This study applies high-salinity stress condition to a two-phase process in which microalgal cells are initially grown in freshwater medium until late exponential phase and subsequently subjected to high-salinity condition that induces excessive lipid accumulation. Our initial experiment revealed that the concentrated culture of Chlorella sorokiniana HS1 exhibited the intense fluorescence of Nile red at the NaCl concentration of 60 g/L along with 1 g/L of supplemental bicarbonate after 48 h of induction period without significantly compromising cultural integrity. These conditions were further verified with the algal culture grown for 7 days in a 1 L bottle reactor that reached late exponential phase; a 12% increment in the lipid content of harvested biomass was observed upon inducing high lipid accumulation in the concentrated algal culture at the density of 5.0 g DW/L. Although an increase in the sum of carbohydrate and lipid contents of harvested biomass indicated that the external carbon source supplemented during the induction period increased overall carbon assimilation, a decrease in carbohydrate content suggested the potential reallocation of cellular carbon that promoted lipid droplet formation under high-salinity stress. These results thus emphasize that the two-phase process can be successfully implemented to enhance algal lipid productivity by incorporating high-salinity stress conditions into the pre-concentrated sedimentation ponds of industrial algal production system.

The performance and long-term stability of low-cost separators in single-chamber bottle-type microbial fuel cells.

This study evaluates long-term stability of low-cost separators in single-chamber bottle-type microbial fuel cells with domestic wastewater. Low-cost separators tested in this study were nonwoven fabrics (NWF) of polypropylene (PP80, PP100), textile fabrics of polyphenylene sulfide (PPS), sulfonated polyphenylene sulfide (SPPS), and cellulose esters. NWF PP80 separator generated the highest power density of 280 mW/m2, which was higher than with ion-exchange membranes (cation exchange membrane; CEM = 271 mW/m2, cation exchange membrane; CMI = 196 mW/m2, Nafion = 260 mW/m2). MFC operations with other size-selective separators such as SPPS, PPS, and cellulose esters exhibited power densities of 261, 231, and 250 mW/m2, respectively. During a 280-day operation, initial power density of PP80 (278 mW/m2) was decreased to 257 mW/m2, but this decrease was smaller than with others (Nafion: 265-230 mW/m2; PP100: 220-126 mW/m2). The anode potential of around -430 mV did not change much with all separators in the long-term operation, but the initial cathode potential gradually decreased. Fouling analysis suggested that the presence of carbonaceous substance on Nafion and PP80 after 280 days of operation and Nafion was subject to be more biofouling.

Enhanced performance of an air-cathode microbial fuel cell with oxygen supply from an externally connected algal bioreactor.

An algae bioreactor (ABR) was externally connected to air-cathode microbial fuel cells (MFCs) to increase power generation by supplying a high amount of oxygen to cathode electrode. The MFC with oxygen fed from ABR produced maximum cell voltage and cathode potential at a fixed loading of 459 mV and 10 mV, respectively. During polarization analysis, the MFC displayed a maximum power density of 0.63 W/m(2) (at 2.06 A/m(2)) using 39.2% O2 from ABR, which was approximately 30% higher compared with use of atmospheric air (0.44 W/m(2), 20.8% O2,). The cyclic voltammogram analysis exhibited a higher reduction current of -137 mA with 46.5% O2 compared to atmospheric air (-115 mA). Oxygen supply by algae bioreactor to air-cathode MFC could also maintain better MFC performance in long term operation by minimizing cathode potential drop over time.

Determination of Microbial Growth by Protein Assay in an Air-Cathode Single Chamber Microbial Fuel Cell.

Microbial fuel cells (MFCs) have gathered attention as a novel bioenergy technology to simultaneously treat wastewater with less sludge production than the conventional activated sludge system. In two different operations of the MFC and aerobic process, microbial growth was determined by the protein assay method and their biomass yields using real wastewater were compared. The biomass yield on the anode electrode of the MFC was 0.02 g-COD-cell/g- COD-substrate and the anolyte planktonic biomass was 0.14 g-COD-cell/g-COD-substrate. An MFC without anode electrode resulted in the biomass yield of 0.07 ± 0.03 g-COD-cell/g-COD-substrate, suggesting that oxygen diffusion from the cathode possibly supported the microbial growth. In a comparative test, the biomass yield under aerobic environment was 0.46 ± 0.07 g-COD-cell/g-COD-substrate, which was about 3 times higher than the total biomass value in the MFC operation.

Evaluation of microbial fuel cell operation using algae as an oxygen supplier: carbon paper cathode vs. carbon brush cathode.

Microbial fuel cell (MFC) and its cathode performances were compared with use of carbon fiber brush and plain carbon paper cathode electrodes in algae aeration. The MFC having carbon fiber brush cathode exhibited a voltage of 0.21 ± 0.01 V (1,000 Ω) with a cathode potential of around -0.14 ± 0.01 V in algal aeration, whereas MFC with plain carbon paper cathode resulted in a voltage of 0.06 ± 0.005 V with a cathode potential of -0.39 ± 0.01 V. During polarizations, MFC equipped with carbon fiber brush cathode showed a maximum power density of 30 mW/m(2), whereas the MFC equipped with plain carbon paper showed a power density of 4.6 mW/m(2). In algae aeration, the internal resistance with carbon fiber brush cathode was 804 Ω and with plain carbon paper it was 1,210 Ω. The peak currents of MFC operation with carbon fiber brush and plain carbon paper cathodes were -31 mA and -850 µA, respectively.

Discovery of a novel class of potent HCV NS4B inhibitors: SAR studies on piperazinone derivatives.

HTS screening identified compound 2a (piperazinone derivative) as a low micromolar HCV genotype 1 (GT-1) inhibitor. Resistance mapping studies suggested that this piperazinone chemotype targets the HCV nonstructural protein NS4B. Extensive SAR studies were performed around 2a and the amide function and the C-3/C-6 cis stereochemistry of the piperazinone core were essential for HCV activity. A 10-fold increase in GT-1 potency was observed when the chiral phenylcyclopropyl amide side chain of 2a was replaced with p-fluorophenylisoxazole-carbonyl moiety (67). Replacing the C-6 nonpolar hydrophobic moiety of 67 with a phenyl moiety (95) did not diminish the GT-1 potency. A heterocyclic thiophene moiety (103) and an isoxazole moiety (108) were incorporated as isosteric replacements for the C-6 phenyl moiety (95), resulting in significant improvement in GT-1b and 1a potency. However, the piperazonone class of compounds lacks GT-2 activity and, consequently, were not pursued further into development.

Structure-activity relationship (SAR) optimization of 6-(indol-2-yl)pyridine-3-sulfonamides: identification of potent, selective, and orally bioavailable small molecules targeting hepatitis C (HCV) NS4B.

A novel, potent, and orally bioavailable inhibitor of hepatitis C RNA replication targeting NS4B, compound 4t (PTC725), has been identified through chemical optimization of the 6-(indol-2-yl)pyridine-3-sulfonamide 2 to improve DMPK and safety properties. The focus of the SAR investigations has been to identify the optimal combination of substituents at the indole N-1, C-5, and C-6 positions and the sulfonamide group to limit the potential for in vivo oxidative metabolism and to achieve an acceptable pharmacokinetic profile. Compound 4t has excellent potency against the HCV 1b replicon, with an EC50 = 2 nM and a selectivity index of >5000 with respect to cellular GAPDH. Compound 4t has an overall favorable pharmacokinetic profile with oral bioavailability values of 62%, 78%, and 18% in rats, dogs, and monkeys, respectively, as well as favorable tissue distribution properties with a liver to plasma exposure ratio of 25 in rats.

Increased power generation from primary sludge by a submersible microbial fuel cell and optimum operational conditions.

Microbial fuel cells (MFCs) have received attention as a promising renewable energy technology for waste treatment and energy recovery. We tested a submersible MFC with an innovative design capable of generating a stable voltage of 0.250 ± 0.008 V (with a fixed 470 Ω resistor) directly from primary sludge. In a polarization test, the maximum power density was 0.18 W/m(2) at a current density of 0.8 A/m(2) with an external resistor of 300 Ω. The anodic solution of the primary sludge needs to be adjusted to a pH 7 for high power generation. The modified primary sludge with an added phosphate buffer prolonged the current generation and increased the power density by 7 and 1.5 times, respectively, in comparison with raw primary sludge. These findings suggest that energy recovery from primary sludge can be maximized using an advanced MFC system with optimum conditions.

HCV NS5A replication complex inhibitors. Part 2: investigation of stilbene prolinamides.

In a previous disclosure,(1) we reported the dimerization of an iminothiazolidinone to form 1, a contributor to the observed inhibition of HCV genotype 1b replicon activity. The dimer was isolated via bioassay-guided fractionation experiments and shown to be a potent inhibitor of genotype 1b HCV replication for which resistance mapped to the NS5A protein. The elements responsible for governing HCV inhibitory activity were successfully captured in the structurally simplified stilbene prolinamide 2. We describe herein the early SAR and profiling associated with stilbene prolinamides that culminated in the identification of analogs with PK properties sufficient to warrant continued commitment to this chemotype. These studies represent the key initial steps toward the discovery of daclatasvir (BMS-790052), a compound that has demonstrated clinical proof-of-concept for inhibiting the NS5A replication complex in the treatment of HCV infection.

Solid-phase synthesis of a library based on biphenyl-containing trypsin-like serine protease inhibitors.

The solid-phase synthesis of a library based on an unusual biphenyl-containing trypsin-like serine protease inhibitor is described. Key to this effort was the synthesis of a highly functionalized aryl boronic acid reagent which required the development of a novel and efficient method to convert a triflate to a pinacolboronate in large scale.

Design and synthesis of a G-protein-coupled receptor antagonist library of aryloxyalkanolamines using a polymer-supported acyclic acetal linker.

A G-Protein-coupled receptor-targeted library of aryloxypropanolamines and aryloxybutanolamines was efficiently executed using a novel, polymer-supported acyclic acetal linker, producing compounds in good yields and purities.

Synthesis and applications of a novel polymer-supported EEDQ reagent.

A simple synthetic protocol for a novel polymer-supported EEDQ reagent is reported. This reagent promotes solution-phase amide bond formation without any additives or base, as well as the selective coupling of aliphatic amines with acids in the presence of aromatic amines.