The Human Paraoxonase 2: An Optimized Procedure for Refolding and Stabilization Facilitates Enzyme Analyses and a Proteomics Approach.

The human paraoxonase 2 (PON2) is the oldest member of a small family of arylesterase and lactonase enzymes, representing the first line of defense against bacterial infections and having a major role in ROS-associated diseases such as cancer, cardiovascular diseases, neurodegeneration, and diabetes. Specific Post-Translational Modifications (PTMs) clustering nearby two residues corresponding to pon2 polymorphic sites and their impact on the catalytic activity are not yet fully understood. Thus, the goal of the present study was to develop an improved PON2 purification protocol to obtain a higher amount of protein suitable for in-depth biochemical studies and biotechnological applications. To this end, we also tested several compounds to stabilize the active monomeric form of the enzyme. Storing the enzyme at 4 °C with 30 mM Threalose had the best impact on the activity, which was preserved for at least 30 days. The catalytic parameters against the substrate 3-Oxo-dodecanoyl-Homoserine Lactone (3oxoC12-HSL) and the enzyme ability to interfere with the biofilm formation of Pseudomonas aeruginosa (PAO1) were determined, showing that the obtained enzyme is well suited for downstream applications. Finally, we used the purified rPON2 to detect, by the direct molecular fishing (DMF) method, new putative PON2 interactors from soluble extracts of HeLa cells.

Active site engineering of Zn-doped mesoporous ceria toward highly efficient organophosphorus hydrolase-mimicking nanozyme.

Hydrolase-mimicking nanozymes have received increasing attention in recent years, but the effective rational design and development of these materials has not been realized, as they are not at present considered a critical research target. Herein, we report that Zn-doped mesoporous ceria (Zn-m-ceria) engineered to have an abundance of two different active sites with different functions-one that allows both co-adsorption binding of organophosphate (OP) and water and another that serves as a general base-has significant organophosphorus hydrolase (OPH)-like catalytic activity. Specifically, Zn-m-ceria exhibits a catalytic efficiency over 75- and 25-fold higher than those of m-ceria and natural OPH, respectively. First-principles calculations reveal the importance of Zn for the OPH-mimicking activity of the material, promoting substrate adsorption and proton-binding. The OPH-like Zn-m-ceria catalyst is successfully applied to detect a model OP, methyl paraoxon, in spiked tap water samples with excellent sensitivity, stability, and detection precision. We expect that these findings will promote research based on the rational engineering of the active site of nanozymes and efficient strategies for obtaining a diverse range of catalysts that mimic natural enzymes, and hence the utilization in real-world applications of enzyme-mimicking catalysts with properties superior to their natural analogs should follow.

Structural Characteristics of PON1 with Leu55Met and Gln192Arg Variants Influencing Oxidative-Stress-Related Diseases: An Integrated Molecular Modeling and Dynamics Study.

Background and Objectives: PON1 is a multi-functional antioxidant protein that hydrolyzes a variety of endogenous and exogenous substrates in the human system. Growing evidence suggests that the Leu55Met and Gln192Arg substitutions alter PON1 activity and are linked with a variety of oxidative-stress-related diseases. Materials and Methods: We implemented structural modeling and molecular dynamics (MD) simulation along with essential dynamics of PON1 and molecular docking with their endogenous (n = 4) and exogenous (n = 6) substrates to gain insights into conformational changes and binding affinity in order to characterize the specific functional ramifications of PON1 variants. Results: The Leu55Met variation had a higher root mean square deviation (0.249 nm) than the wild type (0.216 nm) and Gln192Arg (0.202 nm), implying increased protein flexibility. Furthermore, the essential dynamics analysis confirms the structural change in PON1 with Leu55Met vs. Gln192Arg and wild type. Additionally, PON1 with Leu55Met causes local conformational alterations at the substrate binding site, leading to changes in binding affinity with their substrates. Conclusions: Our findings highlight the structural consequences of the variants, which would increase understanding of the role of PON1 in the pathogenesis of oxidative-stress-related diseases, as well as the management of endogenous and exogenous chemicals in the treatment of diseases.

Substrate-dependent inactivation of recombinant paraoxonase 1 during catalytic dihydrocoumarin turnover and the protective properties of surfactants.

Human paraoxonase-1 (PON1) is the most studied member of the paraoxonases (PONs) family and catalyzes the hydrolysis of various substrates (lactones, aryl esters, and paraoxon). Numerous studies link PON1 to oxidative stress-related diseases such as cardiovascular disease, diabetes, HIV infection, autism, Parkinson's, and Alzheimer's, where the kinetic behavior of an enzyme is characterized by initial rates or by modern methods that obtain enzyme kinetic parameters by fitting the computed curves over the entire time-courses of product formation (progress curves). In the analysis of progress curves, the behavior of PON1 during hydrolytically catalyzed turnover cycles is unknown. Hence, progress curves for enzyme-catalyzed hydrolysis of the lactone substrate dihydrocoumarin (DHC) by recombinant PON1 (rePON1) were analyzed to investigate the effect of catalytic DHC turnover on the stability of rePON1. Although rePON1 was significantly inactivated during the catalytic DHC turnover, its activity was not lost due to the product inhibition or spontaneous inactivation of rePON1 in the sample buffers. Examination of the progress curves of DHC hydrolysis by rePON1 led to the conclusion that rePON1 inactivates itself during catalytic DHC turnover hydrolysis. Moreover, human serum albumin or surfactants protected rePON1 from inactivation during this catalytic process, which is significant because the activity of PON1 in clinical samples is measured in the presence of albumin.

Evaluation of in vitro effect, molecular docking, and molecular dynamics simulations of some dihydropyridine-class calcium channel blockers on human serum paraoxonase 1 (hPON1) enzyme activity.

Paraoxonase 1 (PON1) was purified 148.80-fold in 37.92% yield by hydrophobic interaction chromatography technique. The purity of PON1 was checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with a single band of 43 kDa. The in vitro effects of nine different calcium channel blockers on PON1 activity were evaluated. All drugs strongly decreased PON1 activity, and IC50 levels were between 13.987 ± 0.59 and 238.104 ± 2.14 µM, Ki values between 8.58 ± 0.36 and 111 ± 1.27 µM. The drugs with the strongest inhibitory effect were nisoldipine with 13.987 ± 0.59 µM and nicardipine with 20.158 ± 0.43 µM. The mechanism of action for the inhibition of the enzyme by nisoldipine and nicardipine was investigated through molecular docking. The stability of enzyme-ligand complexes obtained from the docking was explored through molecular dynamics simulation. The binding affinity of the ligands toward the enzyme was also investigated through MMPBSA (molecular mechanics Poisson-Boltzmann surface area method). The computational analysis demonstrated these compounds could inhibit the enzyme. Nisoldipine had the strongest binding, and its complex was the most stable one. Furthermore, nicardipine was found to have the highest affinity toward the enzyme.

Self-Assembling Enzymatic Nanocomplexes with Polypeptides and Low-Weight Organic Compounds: Preparation, Characterization, and Application of New Antibacterials.

The self-assembling of nanosized materials is a promising field for research and development. Multiple approaches are applied to obtain inorganic, organic and composite nanomaterials with different functionality. In the present work, self-assembling nanocomplexes (NCs) were prepared on the basis of enzymes and polypeptides followed by the investigation of the influence of low-molecular weight biologically active compounds on the properties of the NCs. For that, the initially possible formation of catalytically active self-assembling NCs of four hydrolytic enzymes with nine effectors was screened via molecular modeling. It allowed the selection of two enzymes (hexahistidine-tagged organophosphorus hydrolase and penicillin acylase) and two compounds (emodin and naringenin) having biological activity. Further, such NCs based on surface-modified enzymes were characterized by a batch of physical and biochemical methods. At least three NCs containing emodin and enzyme (His6-OPH and/or penicillin acylase) have been shown to significantly improve the antibacterial activity of colistin and, to a lesser extent, polymyxin B towards both Gram-positive bacteria (Bacillus subtilis) and Gram-negative bacteria (Escherichia coli).

Insights in detection and analysis of organophosphates using organophosphorus acid anhydrolases (OPAA) enzyme-based biosensors.

The human population is dependent on agriculture for its food requirements and survival. Several insecticides and pesticides have found their use for improvements in agricultural yields. Organophosphates (OP) are one of the many compounds used as insecticides and pesticides. OPs have also been used to develop G and V-series chemicals which act as highly toxic nerve agents that can severely influence the normal function of the nervous system in all living beings. Thus, OP compounds utilized as insecticides/pesticides and nerve agents are hazardous to the environment, lethal for humans and other non-target animals. To avoid their toxicity, approaches to detect and neutralize them have become essential. A variety of analytical procedures such as electrochemical processes and chromatography methods, namely liquid and gas chromatography, have been employed to detect OPs. Though these techniques are sensitive and highly accurate they suffer from drawbacks, for instance: their bulky nature and expensive instrumentation, the difficulty of operation, long detection times, and they can yield unpredictable results with variable sample complexities. With the advent of several types of biosensors, the assay of OP compounds has become simpler, faster, cost-effective with improved sensitivity, and provides the capability for onsite detection. OP biosensor assays typically utilize several enzymes with the capability to hydrolyze/degrade OP compounds, such as organophosphate hydrolase (OPH) and organophosphate acid hydrolase (OPAA). This review focuses on discussing various aspects of OPAA as biological recognition unit in terms of its: structure, properties, activity enhancement methods, and utilization for developing OPAA-based biosensing technologies for insecticides, pesticides, and nerve agents.

Enhancement in the production of recombinant human paraoxonase 1 in Escherichia coli: A comprehensive approach of cellular engineering and optimization of protein folding process in vitro.

Human paraoxonase 1(hPON1) belongs to the paraoxonase (PON) family. It is a calcium-dependent enzyme with a size of ∼43 kDa and is composed of 6 bladed beta-barrel structures with two calcium ions in its active site. In humans, it is synthesized in the liver and remains bound with the high-density lipoproteins (HDL) within the blood. It has immense potential to tackle the poisoning associated with the use of organophosphates (OPs) and their derivatives, such as nerve agents, due to role in their degradation. Therefore, hPON1 serves as a potential bio-scavenger that can be used as an antidote or as a surface decontaminating agent in OPs poisoning. However, present systems prove insufficient to produce it in sufficient quantity to make it industrially relevant. Here, our efforts involve producing it recombinantly in an E. coli system with enhanced expression levels by altering cellular and environmental conditions. This has been further improved by the development of in-vitro refolding process for the denatured recombinant hPON1 (rhPON1) protein. This methodology resulted in approximately 200 mg of the enzymatically functional protein from 1 l of E. coli culture. Proper refolding of rhPON1 was confirmed by comparing its enzymatic activity and conformation with serum purified hPON1.

Correlation of HMGB1, PON-1, MCP-1, and Periodontal P. gingivalis with Amniotic Fluid Fecal Dye.

BACKGROUND: This paper aims to investigate the correlation between high mobility group protein-1 (HMG-b1), antioxidant enzyme-1 (paraoxon-1, PON-1), monocyte chemoattractant protein-1 (monocyte chemoattractant protein-1, MCP-1), P. gingivalis, and MSAF. MATERIALS AND METHODS: The total sample size comprised of 73 cases in both groups. These patients were further subdivided into 2 groups: the MSAF group and the control group. 38 women were in the MSAF group and 35 women with term amniotic fluid serum were in the control group. The MSAF group was selected as a full-term singleton amniotic fluid fecal infection group. Clinical data were collected, and specimens were collected. Fecal staining of amniotic fluid and full-term amniotic fluid removes the placenta and umbilical cord blood. The expression of HMGB1 in the placenta was observed by immune-histochemical staining of MSAF and control groups. The content of PON-1 in cord blood was determined by ELISA. RESULTS: Correlation between maternal and neonatal clinical data and MSAF was done; MSAF group mean gestational age was 41.38 ± 1.40 weeks; control group mean gestational age was 39.20 ± 1.24 weeks. This study found no correlation between the birth weight, maternal age, sex, first/transmaternal, hyperthyroidism, hypothyroidism, and anemia between the MSAF and control group with nonsignificant P value (P > 0.05). However, the fatal age, gestational diabetes, gestational hypertension, umbilical cord abnormalities, placental abnormalities, and neonatal asphyxia factors were statistically different with a significant P value of <0.05 between both groups. HMGB1 and Periodontal P. gingivalis are mostly expressed in placental trophoblast, vascular endothelial cells, and amniotic epithelial and interstitial cells. After HE staining of 72 placentas by HE in MSAF and control, 6 had acute chorioamnionitis (5.1 control), 32 had chronic (23.9), 35 had abnormal placentas, and three in MSAF had chorionic columnar metaplasia. In immune-histochemistry experiments, the HMGB1 expression intensity of placental tissue was higher in the MSAF group (P < 0.05); however, the level of PON-1 was lower in the MSAF group as compared to the controls (P < 0.05). CONCLUSIONS: Gestational age and placental abnormalities are clinical high-risk factors for MSAF. HMGB1, PON-1, MCP-1, and Periodontal P. gingivalis may be involved in the development of MSAF, suggesting an oxidative/antioxidant imbalance with inflammation, and may be one of the mechanisms for MSAF development.

Combined Modification of Fiber Materials by Enzymes and Metal Nanoparticles for Chemical and Biological Protection.

To obtain fiber materials with pronounced chemical-biological protection, metal (Zn or Ta) nanoparticles were jointly applied with polyelectrolyte complexes of enzymes and polypeptides being their stabilizers. Computer modeling revealed the preferences between certain polyelectrolyte partners for N-acyl-homoserine lactone acylase and hexahistidine-tagged organophosphorus hydrolase (His6-OPH) possessing the quorum quenching (QQ) behavior with bacterial cells. The combinations of metal nanoparticles and enzymes appeared to function better as compared to the combinations of the same QQ-enzymes with antibiotics (polymyxins), making it possible to decrease the applied quantities by orders of magnitude while giving the same effect. The elimination of Gram-positive and Gram-negative bacterial cells from doubly modified fiber materials notably increased (up to 2.9-fold), whereas His6-OPH retained its hydrolytic activity in reaction with organophosphorus compounds (up to 74% of initially applied activity). Materials with the certain enzyme and Zn nanoparticles were more efficient against Bacillus subtilis cells (up to 2.1-fold), and Ta nanoparticles acted preferentially against Escherichia coli (up to 1.5-fold). Some materials were proved to be more suitable for combined modification by metal nanoparticles and His6-OPH complexes as antimicrobial protectants.

The Removal of Time-Concentration Data Points from Progress Curves Improves the Determination of Km: The Example of Paraoxonase 1.

Several approaches for determining an enzyme's kinetic parameter Km (Michaelis constant) from progress curves have been developed in recent decades. In the present article, we compare different approaches on a set of experimental measurements of lactonase activity of paraoxonase 1 (PON1): (1) a differential-equation-based Michaelis-Menten (MM) reaction model in the program Dynafit; (2) an integrated MM rate equation, based on an approximation of the Lambert W function, in the program GraphPad Prism; (3) various techniques based on initial rates; and (4) the novel program "iFIT", based on a method that removes data points outside the area of maximum curvature from the progress curve, before analysis with the integrated MM rate equation. We concluded that the integrated MM rate equation alone does not determine kinetic parameters precisely enough; however, when coupled with a method that removes data points (e.g., iFIT), it is highly precise. The results of iFIT are comparable to the results of Dynafit and outperform those of the approach with initial rates or with fitting the entire progress curve in GraphPad Prism; however, iFIT is simpler to use and does not require inputting a reaction mechanism. Removing unnecessary points from progress curves and focusing on the area around the maximum curvature is highly advised for all researchers determining Km values from progress curves.

Metal Nanoparticles@Covalent Organic Framework@Enzymes: A Universal Platform for Fabricating a Metal-Enzyme Integrated Nanocatalyst.

Cascade catalysis that combines chemical catalysis and biocatalysis has received extensive attention in recent years, especially the integration of metal nanoparticles (MNPs) with enzymes. However, the compatibility between MNPs and enzymes, and the stability of the integrated nanocatalyst should be improved to promote the application. Therefore, in this study, we proposed a strategy to space-separately co-immobilize MNPs and enzymes to the pores and surface of a highly stable covalent organic framework (COF), respectively. Typically, Pd NPs that were prepared by in situ reduction with triazinyl as the nucleation site were distributed in COF (Tz-Da), and organophosphorus hydrolase (OPH) was immobilized on the surface of Tz-Da by a covalent method to improve its stability. The obtained integrated nanocatalyst Pd@Tz-Da@OPH showed high catalytic efficiency and reusability in the cascade degradation of organophosphate nerve agents. Furthermore, the versatility of the preparation strategy of COF-based integrated nanocatalyst has been preliminarily expanded: (1) Pd NPs and OPH were immobilized in the triazinyl COF (TTB-DHBD) with different pore sizes for cascade degradation of organophosphate nerve agent and the particle size of MNPs can be regulated. (2) Pt NPs and glucose oxidase were immobilized in COF (Tz-Da) to obtain an integrated nanocatalyst for efficient colorimetric detection of phenol.

Ophthalmic drugs: in vitro paraoxonase 1 inhibition and molecular docking studies.

Glaucoma is a neuropathy disorder and is generally treated by drugs. Allergic conjunctivitis is a common ophthalmologic disease. Paraoxonase 1 (PON1) is an organophosphate hydrolyzer and antiatherogenic enzyme. PON1 is known for preventing atherosclerosis through lipid-modifying features, as well as which has decisive actions of antiapoptosis, anti-inflammatory, antithrombosis, and antiadhesion antioxidant activity properties. Thus, reducing the enzyme levels in hyperthyroidism, chronic renal failure, glaucoma, diabetes mellitus, and cardiovascular diseases is a significant risk. This study was tested some ophthalmic drugs used to treat the diseases, such as glaucoma and allergic conjunctivitis, mentioned above, travoprost, latanoprost, ketotifen, emedastine, and olopatadine, for their inhibition activities against PON1. These drugs displayed the potent inhibition effect with IC50 values ranging between 14.95 ± 0.15 and 299.60 ± 4.07 µM and KI constants ranging from 9.71 ± 2.63 to 261.50 ± 59.98 µM. Besides, the molecular docking analyses of the competitive inhibitors, travoprost, emedastine, and olopatadine, were performed to understand the binding interactions on the enzyme's binding site. According to both in vitro and in silico analysis results, travoprost had the most potent effect on PON1 enzyme activity.

Enhancing Paraoxon Binding to Organophosphorus Hydrolase Active Site.

Organophosphorus hydrolase (OPH) is a metalloenzyme that can hydrolyze organophosphorus agents resulting in products that are generally of reduced toxicity. The best OPH substrate found to date is diethyl p-nitrophenyl phosphate (paraoxon). Most structural and kinetic studies assume that the binding orientation of paraoxon is identical to that of diethyl 4-methylbenzylphosphonate, which is the only substrate analog co-crystallized with OPH. In the current work, we used a combined docking and molecular dynamics (MD) approach to predict the likely binding mode of paraoxon. Then, we used the predicted binding mode to run MD simulations on the wild type (WT) OPH complexed with paraoxon, and OPH mutants complexed with paraoxon. Additionally, we identified three hot-spot residues (D253, H254, and I255) involved in the stability of the OPH active site. We then experimentally assayed single and double mutants involving these residues for paraoxon binding affinity. The binding free energy calculations and the experimental kinetics of the reactions between each OPH mutant and paraoxon show that mutated forms D253E, D253E-H254R, and D253E-I255G exhibit enhanced substrate binding affinity over WT OPH. Interestingly, our experimental results show that the substrate binding affinity of the double mutant D253E-H254R increased by 19-fold compared to WT OPH.

Enzymes, Reacting with Organophosphorus Compounds as Detoxifiers: Diversity and Functions.

Organophosphorus compounds (OPCs) are able to interact with various biological targets in living organisms, including enzymes. The binding of OPCs to enzymes does not always lead to negative consequences for the body itself, since there are a lot of natural biocatalysts that can catalyze the chemical transformations of the OPCs via hydrolysis or oxidation/reduction and thereby provide their detoxification. Some of these enzymes, their structural differences and identity, mechanisms, and specificity of catalytic action are discussed in this work, including results of computational modeling. Phylogenetic analysis of these diverse enzymes was specially realized for this review to emphasize a great area for future development(s) and applications.

Endothelial Lipase Modulates Paraoxonase 1 Content and Arylesterase Activity of HDL.

Endothelial lipase (EL) is a strong modulator of the high-density lipoprotein (HDL) structure, composition, and function. Here, we examined the impact of EL on HDL paraoxonase 1 (PON1) content and arylesterase (AE) activity in vitro and in vivo. The incubation of HDL with EL-overexpressing HepG2 cells decreased HDL size, PON1 content, and AE activity. The EL modification of HDL did not diminish the capacity of HDL to associate with PON1 when EL-modified HDL was incubated with PON1-overexpressing cells. The overexpression of EL in mice significantly decreased HDL serum levels but unexpectedly increased HDL PON1 content and HDL AE activity. Enzymatically inactive EL had no effect on the PON1 content of HDL in mice. In healthy subjects, EL serum levels were not significantly correlated with HDL levels. However, HDL PON1 content was positively associated with EL serum levels. The EL-induced changes in the HDL-lipid composition were not linked to the HDL PON1 content. We conclude that primarily, the interaction of enzymatically active EL with HDL, rather than EL-induced alterations in HDL size and composition, causes PON1 displacement from HDL in vitro. In vivo, the EL-mediated reduction of HDL serum levels and the consequently increased PON1-to-HDL ratio in serum increase HDL PON1 content and AE activity in mice. In humans, additional mechanisms appear to underlie the association of EL serum levels and HDL PON1 content.

Engineered Recombinant PON1-OPH Fusion Hybrids: Potentially Effective Catalytic Bioscavengers against Organophosphorus Nerve Agent Analogs.

Organophosphorus nerve agents (OPNAs), including both G- and V-type nerve agents such as sarin, soman, tabun and VX, are extremely neurotoxic organophosphorus compounds. Catalytic bioscavengers capable of hydrolyzing OPNAs are under development because of the low protective effects and adverse side effects of chemical antidotes to OPNA poisoning. However, these bioscavengers have certain limitations for practical application, including low catalytic activity and narrow specificity. In this study, we generated a fusion-hybrid form of engineered recombinant human paraoxonase 1 (rePON1) and bacterial organophosphorus hydrolase (OPH), referred to as GV-hybrids, using a flexible linker to develop more promising catalytic bioscavengers against a broad range of OPNAs. These GV-hybrids were able to synergistically hydrolyze both G-type OPNA analogs (paraoxon: 1.7 ~ 193.7-fold, p-nitrophenyl diphenyl phosphate (PNPDPP): 2.3 ~ 33.0-fold and diisopropyl fluorophosphates (DFP): 1.4 ~ 22.8-fold) and V-type OPNA analogs (demeton-Smethyl (DSM): 1.9 ~ 34.6-fold and malathion: 1.1 ~ 4.2-fold above) better than their individual enzyme forms. Among the GV-hybrid clones, the GV7 clone showed remarkable improvements in the catalytic activity toward both G-type OPNA analogs (kcat/Km (106 M-1 min-1): 59.8 ± 0.06 (paraoxon), 5.2 ± 0.02 (PNPDPP) and 47.0 ± 6.0 (DFP)) and V-type OPNA analogs (kcat/Km (M-1 min-1): 504.3 ± 48.5 (DSM) and 1324.0 ± 47.5 (malathion)). In conclusion, we developed GV-hybrid forms of rePON1 and bacterial OPH mutants as effective and suitable catalytic bioscavengers to hydrolyze a broad range of OPNA analogs.

Hydrolysis of chiral organophosphorus compounds by phosphotriesterases and mammalian paraoxonase-1.

Some organophosphorus compounds (OPs), which are used in the manufacturing of insecticides and nerve agents, are racemic mixtures with at least one chiral center with a phosphorus atom. Acute exposure of humans to these mixtures induces the covalent modification of acetylcholinesterase (AChE) and neuropathy target esterase (NTE) and causes a cholinergic syndrome or organophosphate-induced delayed polyneuropathy syndrome (OPIDP). These irreversible neurological effects are due to the stereoselective interaction of the racemic OPs with these B-esterases (AChE and NTE) and such interactions have been studied in vivo, ex vivo and in vitro, using stereoselective hydrolysis by A-esterases or phosphotriesterases (PTEs) and the PTE from Pseudomonas diminuta, and paraoxonase-1 (PON1) from mammalian serum. PON1 has a limited hydrolytic potential of the racemic OPs, while the bacterial PTE exhibits a significant catalytic activity on the less toxic isomers P(+) of the nerve agents. Avian serum albumin also shows a hydrolyzing capacity of chiral OPs with oxo and thio forms. There are ongoing environmental and bioremediation efforts to design and produce recombinants as bio-scavengers of OPs.

Antioxidant activity of novel quinazolinones bearing sulfonamide: Potential radiomodulatory effects on liver tissues via NF-κB/ PON1 pathway.

In order to discover new antioxidants, fifteen novel quinazolinone derivatives bearing benzenesulfonamide moiety with variable heterocyclic tail, were synthesized and their structures were established on the basis of spectral data. All the synthesized compounds were screened for their antioxidant potential using DPPH assay in comparison to ascorbic acid. The N-(pyrazin-2-yl)-2-[(4-oxo-3-(4-sulfamoylphenyl)-3,4-dihydroquinazolin-2-yl)thio]acetamide 16 was the most active scaffold in this series with greater scavenging activity than that of ascorbic acid. In vivo acute toxicity study of compound 16 indicates its relative safety with a median lethal dose of 200 mg/kg. The possible antioxidant and hepatoprotective activities of compound 16 were evaluated in irradiated mice. Compound 16 caused mitigation of gamma radiation-induced oxidative stress verified by the decline in MDA, ROS and NF-κB levels. Moreover, SOD and PON1 activities, as well as Zn2+ levels, were improved in liver tissues. Furthermore, molecular docking of compound 16 inside the active site of SOD and PON1 demonstrated the same binding interactions as that of the co-crystallized ligands considering the binding possibilities and energy scores. These findings support that compound 16 may represent a structural lead for developing new antioxidants and hepatoprotective agents.

Smart Microbial Cells Couple Catalysis and Sensing to Provide High-Throughput Selection of an Organophosphate Hydrolase.

Enzyme engineering for gain of function requires navigating a large combinatorial sequence space efficiently. Typically, many mutations are needed to get significant improvements, while a single "bad" mutation can inactivate the enzyme. To establish high-throughput screening and achieve enhanced resolution between two variants, genetic libraries of the organophosphate hydrolase enzyme paraoxonase 1 (PON1) were rapidly screened via an engineered positive-feedback circuit: a p-nitrophenol (PNP)-specific transcription factor (TF) regulated expression of PON1, which catalyzed paraoxon breakdown and PNP production. Rare active mutant colonies, picked by simple visual fluorescence of a PON1-green fluorescent protein (GFP) fusion, were characterized. In a single screening round, high (library-scale) throughput enabled the discovery of enhanced paraoxon degradation activity in PON1, including structurally unexpected mutations.