Inhibited Enzyme Activity Research Articles

In Vitro Inhibition of Xanthine Oxidase Purified from Arthritis Serum Patients by Nanocurcumin and Artemisinin Active Compounds.

Curcumin and artemisinin are commonly used in traditional East Asian medicine. In this study, we investigated the inhibitory effects of these active compounds on xanthine oxidase (XO) using allopurinol as a control. XO was purified from the serum of arthritis patients through ammonium sulfate precipitation (65%) and ion exchange chromatography on diethylaminoethyl (DEAE)-cellulose. The specific activity of the purified enzyme was 32.5 U/mg protein, resulting in a 7-fold purification with a yield of 66.8%. Molecular docking analysis revealed that curcumin had the strongest interaction energy with XO, with a binding energy of -9.28 kcal/mol. The amino acid residues Thr1077, Gln762, Phe914, Ala1078, Val1011, Glu1194, and Ala1079 were located closer to the binding site of curcumin than artemisinin, which had a binding energy of -7.2 kcal/mol. In vitro inhibition assays were performed using nanocurcumin and artemisinin at concentrations of 5, 10, 15, 20, and 25 µg/mL. Curcumin inhibited enzyme activity by 67-91%, while artemisinin had a lower inhibition ratio, which ranged from 40-70% compared to allopurinol as a control.

Inhibition of Pertussis Toxin by Human α-Defensins-1 and -5: Differential Mechanisms of Action

Whooping cough is a severe childhood disease, caused by the bacterium Bordetella pertussis, which releases pertussis toxin (PT) as a major virulence factor. Previously, we identified the human antimicrobial peptides α-defensin-1 and -5 as inhibitors of PT and demonstrated their capacity to inhibit the activity of the PT enzyme subunit PTS1. Here, the underlying mechanism of toxin inhibition was investigated in more detail, which is essential for developing the therapeutic potential of these peptides. Flow cytometry and immunocytochemistry revealed that α-defensin-5 strongly reduced PT binding to, and uptake into cells, whereas α-defensin-1 caused only a mild reduction. Conversely, α-defensin-1, but not α-defensin-5 was taken up into different cell lines and interacted with PTS1 inside cells, based on proximity ligation assay. In-silico modeling revealed specific interaction interfaces for α-defensin-1 with PTS1 and vice versa, unlike α-defensin-5. Dot blot experiments showed that α-defensin-1 binds to PTS1 and even stronger to its substrate protein Gαi in vitro. NADase activity of PTS1 in vitro was not inhibited by α-defensin-1 in the absence of Gαi. Taken together, these results suggest that α-defensin-1 inhibits PT mainly by inhibiting enzyme activity of PTS1, whereas α-defensin-5 mainly inhibits cellular uptake of PT. These findings will pave the way for optimization of α-defensins as novel therapeutics against whooping cough.

Characteristics and Application of a Novel Cold-Adapted and Salt-Tolerant Protease EK4-1 Produced by an Arctic Bacterium Mesonia algae K4-1.

Mesonia algae K4-1 from the Arctic secretes a novel cold-adapted and salt-tolerant protease EK4-1. It has the highest sequence similarity with Stearolysin, an M4 family protease from Geobacillus stearothermophilus, with only 45% sequence identity, and is a novel M4 family protease. Ek4-1 has a low optimal catalytic temperature (40 °C) and is stable at low temperatures. Moreover, EK4-1 is still active in 4 mol/L NaCl solution and is tolerant to surfactants, oxidizing agents and organic solvents; furthermore, it prefers the hydrolysis of peptide bonds at the P1' position as the hydrophobic residues, such as Leu, Phe and Val, and amino acids with a long side chain, such as Phe and Tyr. Mn2+and Mg2+ significantly promoted enzyme activity, while Fe3+, Co+, Zn2+ and Cu2+ significantly inhibited enzyme activity. Amino acid composition analysis showed that EK4-1 had more small-side-chain amino acids and fewer large-side-chain amino acids. Compared with a thermophilic protease Stearolysin, the cold-adapted protease EK4-1 contains more random coils (48.07%) and a larger active pocket (727.42 Å3). In addition, the acidic amino acid content of protease EK4-1 was higher than that of the basic amino acid, which might be related to the salt tolerance of protease. Compared with the homologous proteases EB62 and E423, the cold-adapted protease EK4-1 was more efficient in the proteolysis of grass carp skin, salmon skin and casein at a low temperature, and produced a large number of antioxidant peptides, with DPPH, ·OH and ROO· scavenging activities. Therefore, cold-adapted and salt-tolerant protease EK4-1 offers wide application prospects in the cosmetic and detergent industries.

Structural equation model was used to evaluate the effects of soil chemical environment, fertility and enzyme activity on eucalyptus biomass.

This paper aims to reveal the effects of multi-generational succession of eucalyptus on soil fertility, organic structure and biological properties. Soil samples were collected from eucalyptus plantations of different stand ages (5, 11, 17 and 21 years old) in a typical area in south Asia, soil organic fraction structure and content characteristics were investigated using Fourier transform infrared (FTIR), and structural equation modelling (SEM) was used to explore influences of soil fertility, enzyme activity and organic fraction on stand biomass. FTIR analysis showed that 11 infrared absorption peaks existed in the soils of this study area, attributed to silicates, aromatics, carbonate ions, sugars, esters, polysaccharides, aliphatic hydrocarbons and phenolic alcohols. Combined with the results of peak area integration, the content of esters, aromatics and phenolic alcohols was significantly higher in 17- and 21-year-old stand soils than in control soils. The results of SEM showed that organic components were negatively related (p < 0.01) to enzyme activity and biomass, with standardized coefficients of 0.53 and 0.49, respectively. In summary, multi-generation succession of eucalyptus trees can change the structure of soil organic functional group composition and promote the enrichment of aromatic and phenolic alcohol functional groups. Such changes can directly inhibit the increase in eucalyptus biomass and indirectly negatively affect biomass by inhibiting enzyme activity.

Computational aided design of a halotolerant CMP kinase for enzymatic synthesis of cytidine triphosphate.

The current biocatalytic method of industrial Cytidine triphosphate (CTP) production suffers from reaction rate loss. It is caused by gradually increasing acetate salt concentration, which inhibits enzyme activities and decreases the final yield. This work gave a possible solution to this problem through computational aided design of CMP kinase (CMPK), an enzyme in the CTP production system, to increase its stability in solution with high acetate salt concentration. Enlightened by the features of natural halophilic enzymes, the basic and neutral surface residues were replaced with acidic amino acids. This protein design strategy effectively increased the activity of CMPK in the working condition (acetate concentration over 1200mM). The halotolerant CMPK was applied in fed-batch production of CTP. The maximum titer was 201.4 ± 1.6mM, and the productivity was 12.6mML-1h-1, increased 26.4% and 27.8% from the process using wild-type CMPK, respectively.

Regulation of alanine racemase activity by carboxylates and the d-type substrate d-alanine.

Alanine racemases (ALRs) are essential for d-alanine (d-Ala) production in bacteria, and many ALRs have a conserved carbamylated lysine residue in the active site. Although short-chain carboxylates inhibit ALRs harbouring this lysine residue as substrate analogues, in an ALR variant with an alanine residue at this position, carboxylates behave as activators; however, this activation mechanism remains unclear. Here, we performed kinetic and structural analyses of U1ALR, an ALR from Latilactobacillus sakei UONUMA harbouring a glycine residue (Gly134) in the site of the carbamylated lysine residue. U1ALR was activated by various carboxylates and also by a G134K mutation, both of which caused a significant decrease in Km , indicating an increase in substrate affinity. The U1ALR crystal structure revealed the presence of an acetate molecule bound in a position and at an orientation resembling the conformation of the carbamylated lysine side chain observed in the structures of other ALRs. These results suggest a regulatory mechanism for U1ALR activity involving two carboxylate-binding sites: one with high affinity near Gly134, where an acetate molecule is observed in the crystal structure and carboxylate binding results in enzyme activation; the other is the substrate-binding site, where carboxylate binding inhibits enzyme activity. Furthermore, we observed no carboxylate/G134K-mediated activation in the presence of d-Ala at high concentrations, implying that d-Ala also exhibits low-affinity binding in the first carboxylate-binding site and prevents carboxylate/G134K-induced activation. Such regulation of enzyme activity by carboxylates and d-Ala may be ubiquitous in many ALRs from lactic acid bacteria sharing the same sequence characteristics.

Identification of anti-hyperuricemic components from Coix seed

Coix seed is a nutritious food with the effect of reducing uric acid. Few studies have been performed on the relevant active compounds. In this study, uric-acid-lowering active compounds were isolated and screened from Coix seed, and their structures were identified using NMR and mass spectrometry. The study showed that Coix seed effectively reduced serum uric acid and creatinine levels, suppressed liver xanthine oxidase activity, and improved kidney damage in mice due to hyperuricemia. Two major anti-hyperuricemic compounds were identified, namely, 1-phenyl-1,3,3-propanetriamine (compound 1) and dihydroferulic acid (compound 2), among which compound 1 was a new compound, and compound 2 was separated from Coix seed for the first time. Two compounds showed potent inhibitory activity in vitro against xanthine oxidase, with IC50 values of 230.48 and 373.09 μM, respectively. Furthermore, the molecular docking results indicated that the compounds could inhibit enzyme activity by interacting with residues within the active pocket of xanthine oxidase. This study provided a new alternative for the development of uric acid-lowering medications by identifying an anti-hyperuricemic alkaloid from Coix seed.

Unraveling the molecular mechanism of type II topoisomerase poisons with magnetic tweezers.

Topoisomerases are an essential class of enzymes that resolve topological problems on DNA (e.g., supercoils, knots, and links) that arise during transcription, replication, and recombination. As part of their reaction cycle, type II topoisomerases transiently cleave double-stranded DNA and form a covalent protein-DNA cleavage complex. Typically, these cleavage complexes are tightly regulated in vivo because they are highly vulnerable and can generate cytotoxic DNA breaks, yet some of the most efficacious and broad-spectrum antibacterials are topoisomerase poisons that exploit this vulnerability by disrupting cleavage complex regulation. Due to their biological and clinical importance, the effects of these poisons have been studied extensively with a variety of in vitro ensemble assays. These assays have shown that antibacterials increase overall levels of cleavage complexes and inhibit enzyme activity. However, the kinetics of individual poisoned cleavage complexes under physiologically relevant conditions is unknown. Therefore, we carried out single-molecule magnetic tweezer measurements of E. coli topo IV activity on supercoiled DNA. In the presence of topoisomerase poisons, we observed long pauses in enzyme activity that correspond to poison-stabilized cleavage complexes. We studied the effects of DNA tension, as well as supercoil handedness and extent, determining poison on-rates, cleavage-complex lifetimes, and topoisomerase activity with several common antibacterials under physiologically relevant conditions. Together, these results provide new mechanistic insight into how topoisomerase poisons disrupt cleavage complex regulation that is likely to be relevant to their action in vivo.

Visible-Light-Controlled Histone Deacetylase Inhibitors for Targeted Cancer Therapy.

The lack of selectivity of anticancer drugs limits current chemotherapy. Light-activatable drugs, whose activity can be precisely controlled with external light, could provide a more localized action of the drugs in the tumor, thus decreasing side effects and increasing efficacy. Herein, we introduce a series of photoswitchable azobenzene histone deacetylase inhibitors (HDACis) whose activity can be controlled by external visible light. Initial HDACis isomerized under ultraviolet light and were up to >50-fold more active under illumination than in the dark in enzyme assays. These were then optimized toward compounds responding to more permeable and less harmful green light by introducing o-halogen atoms into the azobenzene. Selected compounds decreased cell viability only under illumination in four different cancer cell lines. Overall, we present photoswitchable HDACis with optimized activation wavelengths, which inhibit enzyme activity and cell viability only upon illumination with visible light, contributing to the still limited toolbox of photoswitchable anticancer drugs.

Isoniazid improves cognitive performance, clears Aβ plaques, and protects dendritic synapses in APP/PS1 transgenic mice.

Alzheimer's disease (AD) is characterized by amyloid β (Aβ) aggregation and neuroinflammation. This study aimed to investigate the therapeutic effect of isoniazid (INH) against AD. The APP/PS1 transgenic mouse model of AD was adopted. The APP/PS1 mice received oral INH (45 mg/kg/d) for 14 days. The cognitive capability was assessed by the Morris Water Maze test. Amyloid plaques and Aβ levels were determined by immunohistochemistry and ELISA assay. The dendritic spines were analyzed by DiOlistic labeling. Immunofluorescence staining was used to observe the microglia and astrocytes. The Morris Water Maze test suggested that INH administration can effectively attenuate the reference memory deficit and improve the working memory of the APP/PS1 mice compared to the untreated mice (all p < 0.001). INH significantly decreased the Aβ plaques in the hippocampus and cortex and reduced the levels of Aβ1-40 and Aβ1-42 in the brain homogenates, cerebrospinal fluid, and serum (all p < 0.001). INH also inhibited enzyme activities of β-site amyloid precursor protein cleaving enzyme 1 (BACE1, p < 0.05) and monoamine oxidase B (Mao-b, p < 0.01). INH significantly increased the protrusion density in the hippocampus (p < 0.01). Immunofluorescence staining revealed that INH significantly reduced the number of activated microglia and astrocytes around the Aβ plaques (both p < 0.01). Isoniazid administration effectively improved cognitive performance, cleared Aβ plaques, protected dendritic synapses, and reduced innate immune cells around the Aβ plaques, suggesting that INH could be a potential drug for AD treatment.

High Molecular Weight α-Galactosidase from the Novel Strain Aspergillus sp. D-23 and Its Hydrolysis Performance

Aspergillus sp. D-23 was obtained by ultraviolet-diethyl sulfate (UV-DES) compound mutagenesis from Aspergillus sp. C18 that the α-galactosidase was purified from. According to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and non-denaturing polyacrylamide gel electrophoresis (Native-PAGE), the purified enzyme demonstrated apparent homogeneity. The monomeric α-galactosidase’s native molecular weight was 125 kDa. The optimal temperature of α-galactosidase was 65 °C, and 75% of the initial enzyme activity could be maintained between 45 and 55 °C. Its optimal pH was 5.0 with good pH stability. After incubating for 2 h at pH 3.0–8.0, it could retain more than 80% of its original activity. Different concentrations of metal ions had different effects on the α-galactosidase activity. High concentrations of Cu2+ could strongly inhibit enzyme activity and low concentrations of Fe2+ could promote enzyme activity. Additionally, as shown by thin layer chromatography and high-performance liquid chromatography, the enzyme also had good hydrolysis ability, which could efficiently hydrolyze melibiose and raffinose by more than 95%. Therefore, these excellent characteristics could make α-galactosidase a good candidate for the food and feed industries.

Pseudophosphorylation of Arabidopsis jasmonate biosynthesis enzyme lipoxygenase 2 via mutation of Ser600 inhibits enzyme activity

Jasmonates are oxylipin phytohormones critical for plant resistance against necrotrophic pathogens and chewing herbivores. An early step in their biosynthesis is catalyzed by non-heme iron lipoxygenases (LOX; EC 1.13.11.12). In Arabidopsis thaliana, phosphorylation of Ser600 of AtLOX2 was previously reported, but whether phosphorylation regulates AtLOX2 activity is unclear. Here, we characterize the kinetic properties of recombinant WT AtLOX2 (AtLOX2WT). AtLOX2WT displays positive cooperativity with α-linolenic acid (α-LeA, jasmonate precursor), linoleic acid (LA), and arachidonic acid (AA) as substrates. Enzyme velocity with endogenous substrates α-LeA and LA increased with pH. For α-LeA, this increase was accompanied by a decrease in substrate affinity at alkaline pH; thus, the catalytic efficiency for α-LeA was not affected over the pH range tested. Analysis of Ser600 phosphovariants demonstrated that pseudophosphorylation inhibits enzyme activity. AtLOX2 activity was not detected in phosphomimics Atlox2S600D and Atlox2S600M when α-LeA or AA were used as substrates. In contrast, phosphonull mutant Atlox2S600A exhibited strong activity with all three substrates, α-LeA, LA, and AA. Structural comparison between the AtLOX2 AlphaFold model and a complex between 8R-LOX and a 20C polyunsaturated fatty acid suggests a close proximity between AtLOX2 Ser600 and the carboxylic acid head group of the polyunsaturated fatty acid. This analysis indicates that Ser600 is located at a critical position within the AtLOX2 structure and highlights how Ser600 phosphorylation could affect AtLOX2 catalytic activity. Overall, we propose that AtLOX2 Ser600 phosphorylation represents a key mechanism for the regulation of AtLOX2 activity and, thus, the jasmonate biosynthesis pathway and plant resistance.

Java Plum (Syzygium cumini (L.) Skeels) Leaf Extract Lowers Serum Urea Levels in Lead-Acetate-Induced Rats

Lead is a hazardous metal to living things. Lead can cause oxidative stress in the body, inhibit enzyme activity, damage nucleic acids, and prevent DNA repair, resulting in cell death. Java plum (Syzygium cumini (L.) Skeels) leaves are rich with antioxidants. The research aimed to evaluate if Java plum leaf extract affected serum urea levels in lead acetate-induced rats. This study is an experimental study using 24 male rats with a randomized post-test only control group design. Rats were divided into three groups: the group that did not get any treatment, namely the negative control (NC); the group that was given lead acetate (40 mg/kg BW) was a positive control (PC); and the group that was given lead acetate (40 mg/kg BW) and Java plum leaf extract (150 mg/kg BW) was the treatment group (T), using an oral probe. The treatment was carried out for four weeks. After four weeks, the rats were killed, and their serum urea levels were examined using the urease-GLDH: enzymatic UV test method. The results showed the mean serum urea levels in the NC, PC, and T groups were 17.87±2.18, 22.79±2.52, and 18.12±2.19 mg/dl, respectively. There were significant differences in all groups (p-value &lt; 0.05) in the one-way Anova. Additionally, the post hoc Tukey HSD test demonstrated a statistically significant difference between the positive control and treatment groups as well as between the negative and positive control groups (p-value &lt; 0.05). The conclusion was that Java plum leaf extract was able to reduce serum urea levels under lead intoxication conditions.

Effects of chickpea protein fractions on α-amylase activity in digestion

This study concerns the effects of endogenous proteins on starch digestion kinetics. It investigates the effects in chickpeas of hydrolysates released from the endogenous proteins albumin, globulin and glutelin on the in vitro activity of porcine pancreatic α-amylase (PPA) and on starch digestion. Using a docking simulation, potential proteinaceous α-amylase inhibitors (α-AIs) belonging to the small peptides of albumin, globulin and glutelin are identified and their binding mechanisms with PPA are explored. It was found that cooking and pepsin hydrolysis led to severe degradation of protein fractions, accompanied by the enrichment of small (<10 kDa) peptides, among which peptides <3 kDa exhibited strong inhibitory activity (up to 50%). Most of the active α-AIs were therefore considered to be found in the fractions <3 kDa, and were further characterized by mass spectrometry combined with in silico analysis. Docking results showed that glutelin could produce the most α-AIs (45 peptides), followed by globulin (41) and albumin (12). Among these, 71 peptides were predicted to exhibit a competitive or uncompetitive type of inhibition on PPA, whereas 6 peptides performed a noncompetitive inhibition. The competitive and uncompetitive α-AIs inhibited enzyme activity mainly by binding to a flexible loop and three major catalytic residues in PPA, and the latter by interacting with the non-catalytic regions of PPA. Different inhibition types therefore can together to hinder the formation of enzyme-starch complexes, so that PPA activity is inhibited and starch digestibility could be reduced. • Chickpea proteins reduce starch digestion rate, which is nutritionally advantageous. • Cooking and pepsin hydrolysis produced albumin-, globulin- and glutelin-peptides. • Peptides<3 kDa had the highest α-amylase inhibitory activity. • 77 peptides were predicted to be α-amylase inhibitors. • Inhibitors exhibited competitive, uncompetitive and noncompetitive inhibition.

Effects of silver nanoparticles on denitrification and anammox in sediments of hypertrophic and mesotrophic lakes

The abundant production and wide usage of silver nanoparticles (Ag NPs) inevitably lead to their release into aquatic ecosystems. However, it is still unclear about how Ag NPs influence denitrification and anammox (DA) in freshwater sediments. To address this, the sediments of hypertrophic and mesotrophic lakes were exposed to 0.5 and 50 mg/L Ag NPs under anaerobic conditions for 7 days to explore the effects of Ag NPs on environmental variables, including redox potential (Eh), pH, organic matter (OM) and acid volatile sulfide (AVS), and the resulting influence on DA. Experimental results indicated that NO3−-N and NH4+-N levels were increased by the low (p > 0.05) and high doses of Ag NPs (p < 0.05) in comparison with the non-Ag control, revealing an inhibitive impact on DA. The level of total nitrogen (TN) was notably increased by the low and high doses of Ag NPs (p < 0.05), perhaps due to inhibited enzyme activity and corresponding encoding gene abundance, which were related to generating gaseous nitrogen such as N2O and N2. In addition, environmental factor Eh was significantly raised by Ag NPs (p < 0.05), further inhibiting DA. Moreover, the quantitative analysis unveiled that denitrifying and anammox bacteria in hypertrophic lakes evinced a stronger resistance to Ag NPs toxicity than those in mesotrophic lakes. Overall, our study revealed that short-term exposure to Ag NPs could inhibit DA in sediments. These findings provide an understanding enabling evaluation and prediction of the environmental risks of Ag NPs in freshwater lakes.

Recent Progress in the Study of Taste Characteristics and the Nutrition and Health Properties of Organic Acids in Foods.

Organic acids could improve the food flavor, maintain the nutritional value, and extend the shelf life of food. This review summarizes the detection methods and concentrations of organic acids in different foods, as well as their taste characteristics and nutritional properties. The composition of organic acids varies in different food. Fruits and vegetables often contain citric acid, creatine is a unique organic acid found in meat, fermented foods have a high content of acetic acid, and seasonings have a wide range of organic acids. Determination of the organic acid contents among different food matrices allows us to monitor the sensory properties, origin identification, and quality control of foods, and further provides a basis for food formulation design. The taste characteristics and the acid taste perception mechanisms of organic acids have made some progress, and binary taste interaction is the key method to decode multiple taste perception. Real food and solution models elucidated that the organic acid has an asymmetric interaction effect on the other four basic taste attributes. In addition, in terms of nutrition and health, organic acids can provide energy and metabolism regulation to protect the human immune and myocardial systems. Moreover, it also exhibited bacterial inhibition by disrupting the internal balance of bacteria and inhibiting enzyme activity. It is of great significance to clarify the synergistic dose-effect relationship between organic acids and other taste sensations and further promote the application of organic acids in food salt reduction.

Biological Characteristics of Recombinant Arthrobotrys oligospora Chitinase AO-801.

Chitinase AO-801 is a hydrolase secreted by Arthrobotrys oligospora during nematode feeding, while its role remained elusive. This study analyzed the molecular characteristics of recombinant chitinase of Arthrobotrys oligospora (reAO-801). AO-801 belongs to the typical glycoside hydrolase 18 family with conserved chitinase sequence and tertiary structure of (α/β)8 triose-phosphate isomerase (TIM) barrel. The molecular weight of reAO-801 was 42 kDa. reAO-801 effectively degraded colloidal and powdered chitin, egg lysate, and stage I larval lysate of Caenorhabditis elegans. The activity of reAO-801 reached its peak at 40˚C and pH values between 4-7. Enzyme activity was inhibited by Zn2+, Ca2+, and Fe3+, whereas Mg2+ and K+ potentiated its activity. In addition, urea, sodium dodecyl sulfate, and 2-mercaptoethanol significantly inhibited enzyme activity. reAO-801 showed complete nematicidal activity against C. elegans stage I larvae. reAO-801 broke down the C. elegans egg shells, causing them to die or die prematurely by hatching the eggs. It also invoked degradation of Haemonchus contortus eggs, resulting in apparent changes in the morphological structure. This study demonstrated the cytotoxic effect of reAO-801, which laid the foundation for further dissecting the mechanism of nematode infestation by A. oligospora.

Computational identification and experimental validation of anti-filarial lead molecules targeting metal binding/substrate channel residues of Cu/Zn SOD1 from Wuchereria bancrofti

Lymphatic filariasis (LF) is a neglected mosquito-borne parasitic disease, widely caused by Wuchereria bancrofti (Wb) in tropical and sub-tropical countries. During a blood meal, the filarial nematodes are transmitted to humans by the infected mosquito. To counter attack the invaded nematodes, the human immune system produces reactive oxygen species. However, the anti-oxidant enzymes of nematodes counteract the host oxidative cytotoxicity. Cu/Zn Superoxide dismutase (SOD1), a member of antioxidant enzymes and are widely used by the nematodes to sustain the host oxidative stress across its lifecycle, hence targeting SOD1 to develop suitable drug molecules would help to overcome the problems related to efficacy and activity of drugs upon different stages of nematodes. In order to find the potent inhibitors, a three-dimensional structure of Cu/Zn WbSOD1 was modelled and the structural stability was analysed through simulation studies. The structure-guided virtual screening approach has been used to identify lead molecules from the ChemBridge based on the docking score, ADMET properties and protein–ligand complex stability analysis. The identified compounds were observed to interact with the copper, metal binding residues (His48, His63, His80 and His120) and catalytically important residue Arg146, which play a crucial role in the disproportionation of incoming superoxide radicals of Cu/Zn WbSOD1. Further, in vitro validation of the selected leads in the filarial worm Setaria digitata exhibited higher inhibition and better IC50 compared to the standard drug ivermectin. Thus, the identified leads could potentially inhibit enzyme activity, which could subsequently act as drug candidates to control LF. Communicated by Ramaswamy H. Sarma

Phlorotannin Extracts from Ascophyllum nodosum Inhibited Proteases Activities and Structural Changes from Apostichopus japonicus

Fresh Apostichopus japonicas (A. japonicas) are easy to autolyze through the actions of endogenous proteases under certain circumstances, further inducing a decline in A. japonicas product quality and business value. To solve this serious problem in the A. japonicas industry, we investigated the inhibition of phlorotannin extracts (PhTE) on the activities and structural changes of crude endogenous proteinase (CEP) from A. japonicas. The results of enzyme activity and SDS-PAGE indicated that PhTE could inhibit enzyme activity in a dose dependent manner. Analysis of multiple spectroscopic methods, scanning electron microscopy, and differential scanning calorimetry indicated that PhTE promoted the cross-linking and aggregation of CEP and increased the particle size of the enzyme, thus changing the structure of the enzyme protein and decreasing the thermostability of protease. In conclusion, PhTE might be an effective inhibitor on endogenous proteinase in A. japonicas, which opened up a new way to prevent the A. japonicas autolysis.

Antifilarial efficacy of andrographolide: Ex vivo studies on bovine filarial parasite Setaria cervi

Lymphatic filariasis caused by filarial nematode is an important disease leading to considerable morbidity throughout tropical countries. Even after specific elimination programs, the disease continue to spread in endemic countries. Thus newer therapeutic interventions are urgently needed to control the spread. In the present study, we have seen the effect of andrographolide (andro), a diterpenoid lactone from the leaves of Andrographis paniculata on filarial parasite Setaria cervi. There was time and concentration dependent decrease in motility and viability leading to death of parasite after 6 h of the exposure of andro. Andro showed potential antifilarial activity with an IC50 value of 24.80 μM assessed through MTT assay. There was concentration dependent decrease in the antioxidant enzymes activity and increase in proapoptotic markers after 5 h exposure of andro. Further, molecular docking analysis revealed that andro binds with filarial glutathione-S-transferase at glutathione (GSH) binding site and inhibiting enzyme activity competitively. Andro induced oxidative stress mediated apoptosis in parasites as evidenced by increase in the intracellular reactive oxygen species (ROS) and apoptotic markers.Therefore this study suggested that andro could be further explored as a new antifilarial drug.