Phenotypic characteristics and immune response of Procypris merus following challenge with aquatic isolate of Klebsiella pneumoniae.

Currently, aquaculture is a relatively mature industry; however, disease problems are continuously threatening the industry and hindering its development to a certain extent. Klebsiella pneumoniae is one of the zoonotic bacteria widely present in different hosts and has caused some degree of harm to the aquaculture industry, posing a potential threat to the water environment and indirectly also affecting human food safety issues. In this study, K. pneumoniae was isolated from the aquaculture environment, named as ELD, and subjected to pathogenic and immunological related studies. The results of the study showed that the strain carries at least four virulence-related genes, magA, wabG, ureA and uge, and has developed resistance to at least seven antibacterial drugs, such as amoxicillin, doxycycline, rifampicin, and so on. Moreover, the strain is highly pathogenic and is capable of causing systemic clinical foci in Procypris merus. In addition, after infection with K. pneumoniae, the expression of IL-1ß, IL-8, HSP70 and C2 was upregulated in P. merus as a whole, whereas the expression of TNF-α did not change significantly in any of the tissues, which might be a kind of immune response of P. merus against K. pneumoniae infection. This study provides an important theoretical basis for the in-depth exploration of the pathogenic mechanism of K. pneumoniae in fish and the immune response that occurs after the disease is contracted in fish, as well as theoretical support for the development of effective preventive and therapeutic strategies against K. pneumoniae-infected aquatic animals in the future.

Changes in Chemical Composition of Flaxseed Oil during Thermal-Induced Oxidation and Resultant Effect on DSC Thermal Properties.

To investigate the changes in chemical composition of flaxseed oil during thermal-induced oxidation and the resultant effect on thermal properties, samples with different oxidation levels were obtained by being heated at 180 °C for two hours and four hours. The oxidation degree was evaluated using peroxide value (PV), extinction coefficient at 232 nm and 268 nm (K232 and K268), and total polar compounds (TPC). Using chromatography, the fatty acid profile and triacylglycerol (TAG) profile were examined. Differential scanning calorimetry (DSC) was used to determine the crystallization and melting profiles. Thermal-induced oxidation of flaxseed oil led to a significant increase (p < 0.05) in PV, K232, K268, and TPC, but the relative content of linolenic acid (Ln) and LnLnLn reduced dramatically (p < 0.05). TPC derived from lipid degradation affected both crystallization and melting profiles. Statistical correlations showed that the onset temperature (Ton) of the crystallization curve was highly correlated with K232, TPC, and the relative content of LnLnLn (p < 0.05), whereas the offset temperature (Toff) of the melting curve was highly correlated with the relative content of most fatty acids (p < 0.05). This finding provides a new way of rapid evaluation of oxidation level and changes of chemical composition for flaxseed oils using DSC.

Heterologous Expression and Characterization of a Novel Exo-Polygalacturonase from Aspergillus fumigatus Af293 and Its Application in Juice Extraction.

Exo-polygalacturonase (exo-PG) hydrolyzes pectin acids and liberates mono-galacturonate, which plays an important role in juice extraction, and has rarely been reported. Exo-PG (AfumExoPG28A) from Aspergillus fumigatus belongs to the glycoside hydrolase 28 family. In this study, its gene was cloned and the protein was expressed and secreted in Pichia pastoris with a maximal activity of 4.44 U/ml. The optimal temperature and pH of AfumExoPG28A were 55°C and 4.0, respectively. The enzyme exhibited activity over almost the entire acidic pH range (>20.0% activity at pH 2.5-6.5) and remained stable at pH 2.5-10.0 for 24 h. The Km and Vmax values of AfumExoPG28A were calculated by the substrate of polygalacturonic acid as 25.4 mg/ml and 23.6 U/mg, respectively. Addition of AfumExoPG28A (0.8 U/mg) increased the light transmittance and juice yield of plantain pulp by 11.7% and 9%, respectively. Combining AfumExoPG28A (0.8 U/mg) with an endo-PG (0.8 U/mg) from our laboratory, the enzymes increased the light transmittance and juice yield of plantain pulp by 45.7% and 10%, respectively. Thus, the enzyme's potential value in juice production was revealed by the remarkable acidic properties and catalytic activity in fruit pulp.

Design, synthesis and biological evaluation of potent NAD+-dependent DNA ligase inhibitors as potential antibacterial agents. Part 2: 4-amino-pyrido[2,3-d]pyrimidin-5(8H)-ones.

A series of 4-amino-pyrido[2,3-d]pyrimidin-5(8H)-ones were designed and synthesized as a novel class of inhibitors of NAD(+)-dependent DNA ligase that possess potency against Gram-positive bacteria.

Design, synthesis and biological evaluation of potent NAD+-dependent DNA ligase inhibitors as potential antibacterial agents. Part I: aminoalkoxypyrimidine carboxamides.

A series of 2,6-disubstituted aminoalkoxypyrimidine carboxamides (AAPCs) with potent inhibition of bacterial NAD(+)-dependent DNA ligase was discovered through the use of structure-guided design. Two subsites in the NAD(+)-binding pocket were explored to modulate enzyme inhibitory potency: a hydrophobic selectivity region was explored through a series of 2-alkoxy substituents while the sugar (ribose) binding region of NAD(+) was explored via 6-alkoxy substituents.

A Novel Endo-Polygalacturonase from Penicillium oxalicum: Gene Cloning, Heterologous Expression and Its Use in Acidic Fruit Juice Extraction.

An endo-polygalacturonase (endo-PGase) exhibiting excellent performance during acidic fruit juice production would be highly attractive to the fruit juice industry. However, candidate endo-PGases for this purpose have rarely been reported. In this study, we expressed a gene from Penicillium oxalicum in Pichia pastoris. The recombinant enzyme PoxaEnPG28C had an optimal enzyme activity at pH 4.5 and 45°C and was stable at pH 3.0-6.5 and < 45°C. The enzyme had a specific activity of 4,377.65 ± 55.37 U/mg towards polygalacturonic acid, and the Km and Vmax values of PoxaEnPG28C were calculated as 1.64 g/l and 6127.45 U/mg, respectively. PoxaEnPG28C increased the light transmittance of orange, lemon, strawberry and hawthorn juice by 13.9 ± 0.3%, 29.4 ± 3.8%, 95.7 ± 10.2% and 79.8 ± 1.7%, respectively; it reduced the viscosity of the same juices by 25.7 ± 1.6%, 52.0 ± 4.5%, 48.2 ± 0.7% and 80.5 ± 2.3%, respectively, and it increased the yield of the juices by 24.5 ± 0.7%, 12.7 ± 2.2%, 48.5 ± 4.2% and 104.5 ± 6.4%, respectively. Thus, PoxaEnPG28C could be considered an excellent candidate enzyme for acidic fruit juice production. Remarkably, fruit juice production using hawthorn as an material was reported for the first time.

Discovery of pyrazolthiazoles as novel and potent inhibitors of bacterial gyrase.

Bacterial DNA gyrase is an attractive target for the investigation of new antibacterial agents. Inhibitors of the GyrB subunit, which contains the ATP-binding site, are described in this communication. Novel, substituted 5-(1H-pyrazol-3-yl)thiazole compounds were identified as inhibitors of bacterial gyrase. Structure-guided optimization led to greater enzymatic potency and moderate antibacterial potency. Data are presented for the demonstration of selective enzyme inhibition of Escherichia coli GyrB over Staphylococcus aureus GyrB.

Novel thiol-based TACE inhibitors. Part 2: Rational design, synthesis, and SAR of thiol-containing aryl sulfones.

A series of potent thiol-containing aryl sulfone TACE inhibitors were designed and synthesized. The SAR and MMP selectivity of the series were investigated. In particular, compound 8b showed excellent in vitro potency against the isolated enzyme and good selectivity over MMP-2, -7, -8, -9, and -13. The X-ray structure of 8b in complex with TACE was also obtained.

Investigation of protein refolding using a fractional factorial screen: a study of reagent effects and interactions.

A recurring obstacle for structural genomics is the expression of insoluble, aggregated proteins. In these cases, the use of alternative salvage strategies, like in vitro refolding, is hindered by the lack of a universal refolding method. To overcome this obstacle, fractional factorial screens have been introduced as a systematic and rapid method to identify refolding conditions. However, methodical analyses of the effectiveness of refolding reagents on large sets of proteins remain limited. In this study, we address this void by designing a fractional factorial screen to rapidly explore the effect of 14 different reagents on the refolding of 33 structurally and functionally diverse proteins. The refolding data was analyzed using statistical methods to determine the effect of each refolding additive. The screen has been miniaturized for automation resulting in reduced protein requirements and increased throughput. Our results show that the choice of pH and reducing agent had the largest impact on protein refolding. Bis-mercaptoacetamide cyclohexane (BMC) and tris (2-carboxyethylphosphine) (TCEP) were superior reductants when compared to others in the screen. BMC was particularly effective in refolding disulfide-containing proteins, while TCEP was better for nondisulfide-containing proteins. From the screen, we successfully identified a positive synergistic interaction between nondetergent sulfobetaine 201 (NDSB 201) and BMC on Cdc25A refolding. The soluble protein resulting from this interaction crystallized and yielded a 2.2 Angstroms structure. Our method, which combines a fractional factorial screen with statistical analysis of the data, provides a powerful approach for the identification of optimal refolding reagents in a general refolding screen.

Phenotypic characterization of resistant Val36 variants of hepatitis C virus NS3-4A serine protease.

In patients chronically infected with hepatitis C virus (HCV) strains of genotype 1, rapid and dramatic antiviral activity has been observed with telaprevir (VX-950), a highly selective and potent inhibitor of the HCV NS3-4A serine protease. HCV variants with substitutions in the NS3 protease domain were observed in some patients during telaprevir dosing. In this study, purified protease domain proteins and reconstituted HCV subgenomic replicons were used for phenotypic characterization of many of these substitutions. V36A/M or T54A substitutions conferred less than eightfold resistance to telaprevir. Variants with double substitutions at Val36 plus Thr54 had approximately 20-fold resistance to telaprevir, and variants with double substitutions at Val36 plus Arg155 or Ala156 had >40-fold resistance to telaprevir. An X-ray structure of the HCV strain H protease domain containing the V36M substitution in a cocomplex with an NS4A cofactor peptide was solved at a 2.4-A resolution. Except for the side chain of Met36, the V36M variant structure is identical to that of the wild-type apoenzyme. The in vitro replication capacity of most variants was significantly lower than that of the wild-type replicon in cells, which is consistent with the impaired in vivo fitness estimated from telaprevir-dosed patients. Finally, the sensitivity of these replicon variants to alpha interferon or ribavirin remained unchanged compared to that of the wild-type.

Novel dual-targeting benzimidazole urea inhibitors of DNA gyrase and topoisomerase IV possessing potent antibacterial activity: intelligent design and evolution through the judicious use of structure-guided design and structure-activity relationships.

The discovery of new antibacterial agents with novel mechanisms of action is necessary to overcome the problem of bacterial resistance that affects all currently used classes of antibiotics. Bacterial DNA gyrase and topoisomerase IV are well-characterized clinically validated targets of the fluoroquinolone antibiotics which exert their antibacterial activity through inhibition of the catalytic subunits. Inhibition of these targets through interaction with their ATP sites has been less clinically successful. The discovery and characterization of a new class of low molecular weight, synthetic inhibitors of gyrase and topoisomerase IV that bind to the ATP sites are presented. The benzimidazole ureas are dual targeting inhibitors of both enzymes and possess potent antibacterial activity against a wide spectrum of relevant pathogens responsible for hospital- and community-acquired infections. The discovery and optimization of this novel class of antibacterials by the use of structure-guided design, modeling, and structure-activity relationships are described. Data are presented for enzyme inhibition, antibacterial activity, and in vivo efficacy by oral and intravenous administration in two rodent infection models.

Novel thiol-based TACE inhibitors: rational design, synthesis, and SAR of thiol-containing aryl sulfonamides.

A series of potent thiol-containing aryl sulfonamide TACE inhibitors was designed and synthesized. The SAR and MMP selectivity of the series were investigated. In particular, compound 4b has shown excellent in vitro potency against the isolated TACE enzyme and good selectivity over MMP-2, -7, -8, -9, and -13. The X-ray structure of 4b bound to TACE was obtained.

Phenotypic and structural analyses of hepatitis C virus NS3 protease Arg155 variants: sensitivity to telaprevir (VX-950) and interferon alpha.

Telaprevir (VX-950) is a highly selective, potent inhibitor of the hepatitis C virus (HCV) NS3.4A serine protease. It has demonstrated strong antiviral activity in patients chronically infected with genotype 1 HCV when dosed alone or in combination with peginterferon alfa-2a. Substitutions of Arg(155) of the HCV NS3 protease domain have been previously detected in HCV isolates from some patients during telaprevir dosing. In this study, Arg(155) was replaced with various residues in genotype 1a protease domain proteins and in genotype 1b HCV subgenomic replicons. Characterization of both the purified enzymes and reconstituted replicon cells demonstrated that substitutions of Arg(155) with these residues conferred low level resistance to telaprevir (<25-fold). An x-ray structure of genotype 1a HCV protease domain with the R155K mutation, in a complex with an NS4A co-factor peptide, was determined at a resolution of 2.5A. The crystal structure of the R155K protease is essentially identical to that of the wild-type apoenzyme (Protein Data Bank code 1A1R) except for the side chain of mutated residue 155. Telaprevir was docked into the x-ray structure of the R155K protease, and modeling analysis suggests that the P2 group of telaprevir loses several hydrophobic contacts with the Lys(155) side chain. It was demonstrated that replicon cells containing substitutions at NS3 protease residue 155 remain fully sensitive to interferon alpha or ribavirin. Finally, these variant replicons were shown to have reduced replication capacity compared with the wild-type HCV replicon in cells.

Inhibitors of hepatitis C virus NS3.4A protease. Effect of P4 capping groups on inhibitory potency and pharmacokinetics.

Reversible tetrapeptide-based compounds have been shown to effectively inhibit the hepatitis C virus NS3.4A protease. Inhibition of viral replicon RNA production in Huh-7 cells has also been demonstrated. We show herein that the inclusion of hydrogen bond donors on the P4 capping group of tetrapeptide-based inhibitors result in increased binding potency to the NS3.4A protease. The capping groups also impart significant effects on the pharmacokinetic profile of these inhibitors.

Preclinical profile of VX-950, a potent, selective, and orally bioavailable inhibitor of hepatitis C virus NS3-4A serine protease.

VX-950 is a potent, selective, peptidomimetic inhibitor of the hepatitis C virus (HCV) NS3-4A serine protease, and it demonstrated excellent antiviral activity both in genotype 1b HCV replicon cells (50% inhibitory concentration [IC50] = 354 nM) and in human fetal hepatocytes infected with genotype 1a HCV-positive patient sera (IC50 = 280 nM). VX-950 forms a covalent but reversible complex with the genotype 1a HCV NS3-4A protease in a slow-on, slow-off process with a steady-state inhibition constant (K(i)*) of 7 nM. Dissociation of the covalent enzyme-inhibitor complex of VX-950 and genotype 1a HCV protease has a half-life of almost an hour. A >4-log10 reduction in the HCV RNA levels was observed after a 2-week incubation of replicon cells with VX-950, with no rebound of viral RNA observed after withdrawal of the inhibitor. In several animal species, VX-950 exhibits a favorable pharmacokinetic profile with high exposure in the liver. In a recently developed HCV protease mouse model, VX-950 showed excellent inhibition of HCV NS3-4A protease activity in the liver. Therefore, the overall preclinical profile of VX-950 supports its candidacy as a novel oral therapy against hepatitis C.

In vitro studies of cross-resistance mutations against two hepatitis C virus serine protease inhibitors, VX-950 and BILN 2061.

VX-950 is a potent, small molecule, peptidomimetic inhibitor of the hepatitis C virus (HCV) NS3.4A serine protease and has recently been shown to possess antiviral activity in a phase I trial in patients chronically infected with genotype 1 HCV. In a previous study, we described in vitro resistance mutations against either VX-950 or another HCV NS3.4A protease inhibitor, BILN 2061. Single amino acid substitutions that conferred drug resistance (distinct for either inhibitor) were identified in the HCV NS3 serine protease domain. The dominant VX-950-resistant mutant (A156S) remains sensitive to BILN 2061. The major BILN 2061-resistant mutants (D168V and D168A) are fully susceptible to VX-950. Modeling analysis suggested that there are different mechanisms of resistance for these mutations induced by VX-950 or BILN 2061. In this study, we identified mutants that are cross-resistant to both HCV protease inhibitors. The cross-resistance conferred by substitution of Ala(156) with either Val or Thr was confirmed by characterization of the purified enzymes and reconstituted replicon cells containing the single amino acid substitution A156V or A156T. Both cross-resistance mutations (A156V and A156T) displayed significantly diminished fitness (or replication capacity) in a transient replicon cell system.

Crystal structures of Escherichia coli topoisomerase IV ParE subunit (24 and 43 kilodaltons): a single residue dictates differences in novobiocin potency against topoisomerase IV and DNA gyrase.

Topoisomerase IV and DNA gyrase are related bacterial type II topoisomerases that utilize the free energy from ATP hydrolysis to catalyze topological changes in the bacterial genome. The essential function of DNA gyrase is the introduction of negative DNA supercoils into the genome, whereas the essential function of topoisomerase IV is to decatenate daughter chromosomes following replication. Here, we report the crystal structures of a 43-kDa N-terminal fragment of Escherichia coli topoisomerase IV ParE subunit complexed with adenylyl-imidodiphosphate at 2.0-A resolution and a 24-kDa N-terminal fragment of the ParE subunit complexed with novobiocin at 2.1-A resolution. The solved ParE structures are strikingly similar to the known gyrase B (GyrB) subunit structures. We also identified single-position equivalent amino acid residues in ParE (M74) and in GyrB (I78) that, when exchanged, increased the potency of novobiocin against topoisomerase IV by nearly 20-fold (to 12 nM). The corresponding exchange in gyrase (I78 M) yielded a 20-fold decrease in the potency of novobiocin (to 1.0 micro M). These data offer an explanation for the observation that novobiocin is significantly less potent against topoisomerase IV than against DNA gyrase. Additionally, the enzyme kinetic parameters were affected. In gyrase, the ATP K(m) increased approximately 5-fold and the V(max) decreased approximately 30%. In contrast, the topoisomerase IV ATP K(m) decreased by a factor of 6, and the V(max) increased approximately 2-fold from the wild-type values. These data demonstrate that the ParE M74 and GyrB I78 side chains impart opposite effects on the enzyme's substrate affinity and catalytic efficiency.

In vitro resistance studies of hepatitis C virus serine protease inhibitors, VX-950 and BILN 2061: structural analysis indicates different resistance mechanisms.

We have used a structure-based drug design approach to identify small molecule inhibitors of the hepatitis C virus (HCV) NS3.4A protease as potential candidates for new anti-HCV therapies. VX-950 is a potent NS3.4A protease inhibitor that was recently selected as a clinical development candidate for hepatitis C treatment. In this report, we describe in vitro resistance studies using a subgenomic replicon system to compare VX-950 with another HCV NS3.4A protease inhibitor, BILN 2061, for which the Phase I clinical trial results were reported recently. Distinct drug-resistant substitutions of a single amino acid were identified in the HCV NS3 serine protease domain for both inhibitors. The resistance conferred by these mutations was confirmed by characterization of the mutant enzymes and replicon cells that contain the single amino acid substitutions. The major BILN 2061-resistant mutations at Asp(168) are fully susceptible to VX-950, and the dominant resistant mutation against VX-950 at Ala(156) remains sensitive to BILN 2061. Modeling analysis suggests that there are different mechanisms of resistance to VX-950 and BILN 2061.

Inhibitors of hepatitis C virus NS3.4A protease 2. Warhead SAR and optimization.

The alpha-ketoamide warhead (e.g., 15) was found to be a practical replacement for aliphatic aldehydes in a series of HCV NS3.4A protease inhibitors. Structure-activity relationships and prime side optimization are discussed.

Inhibitors of hepatitis C virus NS3.4A protease 1. Non-charged tetrapeptide variants.

Tetrapeptide-based peptidomimetic compounds have been shown to effectively inhibit the hepatitis C virus NS3.4A protease without the need of a charged functionality. An aldehyde is used as a prototype reversible electrophilic warhead. The SAR of the P1 and P2 inhibitor positions is discussed.