In the original publication [...].

Cholinesterases (ChEs) are irreversibly inhibited by organophosphorous compounds (OP). Then, OP-inhibited ChEs undergo a reaction that progressively decreases reactivatability. This process called “aging” results from dealkylation of the adduct. Aged ChEs are resistant to antidotal oximes. Structural and conformational changes in the aged phosphylated ChE active site pocket impair enzyme reactivatability. Thus, reactivation of aged ChEs was a challenge for more than 70 years. However, it was postulated that realkylation of aged adducts could lead to reactivation of enzymes. This hypothesis was confirmed in 2018 when a new generation of reactivators, electrophilic quinone methide precursors (QMPs), capable of resuscitating or resurrecting aged ChEs were synthesized. The QMP-mediated resurrection process of ChEs by these first “resurrectors” is still very slow. Thus, substantial optimization in the chemical design of new drugs and drug-targeted delivery are needed before the resurrection approach can be translated into clinically viable therapies. However, despite limitations, the first achievements resolving the non-reactivatability issue of OP-aged cholinesterases and successful administration of these new reactivators can be regarded as a major step forward in improving the therapy of OP poisoning.

Predicting the substrate reactivity strength for a given biocatalyst remains a central challenge in computational biocatalysis. Here, we present Subdate, a modular workflow that combines descriptor-guided organization of substrate analogs with ab initio metadynamics simulations to prioritize reactive candidates. The workflow integrates i) substrate library construction, ii) conformer generation and descriptors set definition, iii) library clustering, iv) representative-substrate selection, and v) reaction-barrier quantitative prediction. Applied to selected biocatalysts (human butyrylcholinesterase and the catalytic antibody A17) sharing an SN2 reaction mechanism, Subdate quantitatively identifies reactivity trends that match experimental kinetic measurements. The developed workflow provides a mechanism-aware strategy for reactive substrate prioritization for efficient sampling through the chemical library in biocatalysis.

The existence of multiple molecular forms of enzymes, genetic polymorphism and functional promiscuity raise the question of the identity of active center(s) responsible for several activities. In the present review, we recapitulate the general strategy, implementing the simple and rigorous inhibition kinetic method for probing the existence of single or multiple active sites on enzyme molecules. The model enzyme we chose to illustrate this approach is human butyrylcholinesterase, an enzyme that shows a complex functional (promiscuity), structural (multiple oligomeric forms) and genetic polymorphisms (numerous allelozymes and isoenzymes).This classical active site discrimination method is based on the analysis of enzyme irreversible inhibition profiles of enzymes under first-order conditions by monitoring the progressive enzyme activity decay with two reporter substrates of different specificity. The use of chiral irreversible inhibitors and/or chiral reporter substrates provides additional kinetic information about preferential enantioselectivity or binding complementarity of the target enzyme, allowing selection of the best inhibitors or substrates. Then, additional investigations, using structural methods (X-ray structure analysis, mass spectrometry), in silico simulations and classical biochemical methods (electrophoresis, PCR) provide definitive answers.

The high catalytic power of cholinesterases results from an efficient interplay between optimised structure, molecular dynamics and fine adjustment of substrates in the active site. Kinetic and molecular modelling investigations of the catalysis of 'inverse' ester substrates compared to 'normal' ester substrates by butyrylcholinesterase (BChE) were performed to shed light on the effect of isomeric inversion of the ester bond on binding and catalytic steps. The substrates used were phenyl-acetate (PhA), -propionate (PhPr), -butyrate (PhBu). The 'inverse' substrates used were benzoic acid methyl ester (BAME), phenyl acetic acid methyl ester (PAAME) and phenyl propionic acid methyl ester (PPAME). Inversion of the ester bond dramatically affected catalytic efficiency. Comparative analysis of the kinetics of both types of substrates showed that hydrolysis of 'inverse' substrates was only possible at high enzyme concentrations, and the rates were first-order, indicating (S)max << Km. The ratio of specific activity (kcat/Km) between normal and 'inverse' substrates increased 12-fold from PhA/BAME to PhPr/PAAME. PhBu was the best substrate, while PPAME was not hydrolysed by BChE. Thus, introduction of methylene group(s) in the acyl moiety of substrates progressively increased kcat/Km, while introduction of methylene group(s) in phenol/alcohol moiety of 'inverse' substrate considerably decreased kcat/Km, indicating dramatic loss of substrate complementarity to the active site. QM/MM and molecular dynamics simulations revealed the molecular basis for altered kinetics of 'inverse' substrate hydrolysis. The observed effects stemmed from reduced stability of reaction intermediates and transition states, along with inversion of the productive complex conformation. BChE exhibited no selectivity for this conformation or alternative binding modes.

Affinity gel electrophoresis was introduced about 50 years ago. Proteins interact with a ligand immobilized in the support. Specific interactions cause a decrease in electrophoretic mobility. The presence of a free ligand, competing with an immobilized ligand, restores electrophoretic mobility. In affinity capillary electrophoresis, the ligand is mobile, and its interaction with a specific protein changes the mobility of the protein–ligand complex. This review mostly focuses on gel affinity electrophoresis. The theoretical basis of this technique, ligand immobilization strategies, and principles for determination of ligand affinity are addressed. Factors affecting specificity and strength of interactions are discussed, in particular, the structure of the affinity matrix, pH, temperature, hydrostatic pressure, solvent, co-solvents, electric field, and other physico-chemical conditions. Capillary affinity electrophoresis principles and uses are also briefly introduced. Affinity gel electrophoresis can be used for qualitative and quantitative purposes. This includes detection of specific proteins in complex media, investigation of specific interactions, protein heterogeneity, molecular and genetic polymorphism, estimation of dissociation constants of protein–ligand complexes, and conformational stability of binding sites. Future prospects, in particular for screening of engineered mutants and potential new drugs, coupling to other analytical methods, and ultra-microtechnological developments, are addressed in light of trends and renewal of this old technique.

Malathion has the reputation of being a safe pesticide. There are no reported cases of cholinergic toxicity in people exposed to low environmental doses of malathion. Our goal was to explain the safety of malathion in terms of the mechanism of malathion detoxication. The structure of malathion includes a built-in safety feature, specifically two ethyl esters. The ethyl esters are decarboxylated by human esterases to negatively charged malathion which does not react with acetylcholinesterase. Acetylcholinesterase is the toxicologically relevant target for organophosphates such as malathion. A toxic form of malathion is produced by Cytochrome P450 enzymes which convert malathion to malaoxon. Malaoxon is toxic because it inhibits acetylcholinesterase. We used high pressure liquid chromatography on a Prodigy 5 µm ODS column to monitor the production of enzyme-catalyzed decarboxylation of the malathion ethyl esters. The products of malathion decarboxylation were identified by mass spectrometry using a Thermo RSLC Ultimate 3000 ultra-high pressure chromatography system with a Thermo Easy-Spray PepMap RSLC C18 separation column attached to an Orbitrap Fusion Lumos Tribrid mass spectrometer. Decarboxylation and enzyme inhibition were assayed with recombinant human acetylcholinesterase (rHuAChE), human butyrylcholinesterase (HuBChE), and recombinant human liver carboxylesterase (rHuCE1). A trace contaminant in 98.5% pure malathion was identified by mass spectrometry. Consistent with the fact that negatively charged compounds are not inhibitors of HuAChE, HuBChE, or HuCE1, we found that negatively charged, decarboxylated malathion did not inhibit the activity of rHuAChE, HuBChE, or rHuCE1. Carboxylesterase detoxified malathion 100,000-fold faster compared to rHuAChE and HuBChE. Low dose exposures to malathion are not directly toxic The toxic metabolite, malaoxon, is produced very slowly. By comparison, detoxified malathion acids are formed rapidly. In conclusion, our data suggest that the safety of low dose environmental exposures to malathion is explained by the fact that malathion is detoxified faster than it is activated to the toxic malaoxon. Our review of the literature finds no convincing evidence that low dose malathion exposure causes cancer.

The nano-technological approach and supramolecular chemistry principles relation to the encapsulation of enzymes pave the way for creating next-generation nano-system-functionalized nano-compartments. The most promising approach for prophylaxis and the treatment of organophosphate (OP) poisoning is the use of stable, bioavailable nano-compartments containing OP-scavenging enzymes. Such enzymes, like butyrylcholinesterase (BChE), wild type and mutants, could also be used for the detoxification of other poisonous esters. There are two types of IRD-labeled human BChE-containing nano-scavengers: PEGylated liposomes and polyethyleneglycol-polypropylenesulfide polymersomes, which were developed with diameter close to 100 nm. BChE-polymersomes have higher encapsulation efficiency (95%) and slower release rate of enzymes (more than 7 days) compared to BChE-liposomes. The catalytic properties of encapsulated enzymes were analyzed for nano-compartment formulations, lipophilicity, the structure of block copolymers, and for different ester substrate polarity: positively charged butyrylthiocholine iodide, neutral phenyl acetate, and negatively charged aspirin. The highest kcat (more than three times) compared to non-encapsulated BChE was for polymersomes based on diblock PEG-PPS polymersomes towards the neutral phenyl acetate substrate.

Butyrylcholinesterase (BChE) is widely distributed in human tissues, although its physiological roles remain incompletely defined. It contributes modestly to cholinergic transmission and participates in lipid and ghrelin metabolism. BChE is pharmacologically and toxicologically significant due to its ability to hydrolyze various esters and neutralize toxic compounds such as carbamates and organophosphate (OP) pesticides and nerve agents. This review explores current insights into BChE functions and regulatory physiological mechanisms, with particular emphasis on its interaction with microRNAs (miRNAs) and defense against toxicants. BChE serves as a bioscavenger of OPs and reversible inhibitors, including several drugs and environmental chemicals. Moreover, its plasma activity is recognized as a valuable biomarker for disease prognosis, treatment monitoring, and the assessment of OP poisoning severity. Recent findings revealed a strong connection between specific miRNAs and BChE regulation, positioning these small non-coding RNAs as potential indicators of metabolic dysfunction and diverse pathological conditions. Additionally, miRNAs appear to modulate BChE expression in response to stress, inflammation, and immune responses. These discoveries highlight the potential of miRNA-based therapeutic approaches targeting BChE pathways in various clinical settings.

A new strategy for effective antidotes against organophosphorus compounds (OPs) is enzyme-loaded nanoscavenger technology. Cell membrane-coated nanoforms of bioscavengers reduce adverse effects and ensure stability, immunotolerance, and prolonged protective action. For the first time, synthesis of double layers polymersomes coated by red blood cells (erythrocytes) ghosts, based on amphiphilic di- and triblock polyethylene glycol-polysulfide copolymers (PEG-b-PPS) was carried out to make enzyme microreactors. The enzyme was an evolved multiple mutant of the archaea Saccharolobus solfataricus phosphotriesterase (PTE). Two approaches were used for making this formulation: lipid fusion and hypotonic swelling. Respectively two types of PTE-loaded nanoscavengers (i) hybrid camouflaging RBC-PTE-loaded polymersomes and (ii) PTE-polymersomes captured by erythrocytes ghosts (PTE-RBC-microreactors) were prepared with high encapsulation efficiency, loading capacity and enzyme activity. CD-1 mice were challenged with paraoxon as a model OP. Pre-treatment of animals with i.v. injected PTE-RBC-microreactors led to LD50 shifts up to 15.5 times compared to control mice. In post-exposure treatments, LD50 shifts were up to 9.8 times. Pharmacokinetics of PTE-RBC-microreactors showed half times of 20 min and 1 hour for distribution and elimination phases, respectively. These first results with erythrocyte ghost microreactors are promising and open a new way to personalized medical countermeasures for detoxification of OPs and other toxicants.

The review analyzes recent advances, challenges, and practical applications in the field of enzymes within the framework of chemical enzymology and enzyme engineering. The achievements in the fundamental understanding of molecular mechanisms of the catalytic cycle of enzymatic reactions made using quantum mechanics/molecular mechanics methods with supercomputer technologies and bioinformatic approaches are considered. The design of protein biocatalysts with new properties is a fundamentally significant methodology of the bioengineering approach to solving practical problems, which is demonstrated by a number of examples. The increasing role of biocatalysis in medicine and biomedical research is illustrated by addressing the problems of antibiotic synthesis and overcoming antibiotic resistance of bacteria, mechanisms of neurodegenerative diseases and development of drugs to treat Alzheimer's disease, biocatalytic processes of DNA repair and the role of mechanisms of functioning of heme peroxidases in the human body. The use of enzymes to degrade endogenous and exogenous toxicants has been greatly developed in recent decades. The advances and problems of using enzymes in therapy and drug delivery are analyzed. The fundamental role of enzymes in modern analysis and diagnosis is noted. The review considers a new trend in the development of bioanalytical methods using aptamers, multi-analysis systems on biochips, surface-enhanced Raman scattering systems, and bioelectroanalysis. The bibliography includes 460 references.

Mining of organophosphorous (OPs)-degrading bacterial enzymes in collections of known bacterial strains and in natural biotopes are important research fields that lead to the isolation of novel OP-degrading enzymes. Then, implementation of strategies and methods of protein engineering and nanobiotechnology allow large-scale production of enzymes, displaying improved catalytic properties for medical uses and protection of the environment. For medical applications, the enzyme formulations must be stable in the bloodstream and upon storage and not susceptible to induce iatrogenic effects. This, in particular, includes the nanoencapsulation of bioscavengers of bacterial origin. In the application field of bioremediation, these enzymes play a crucial role in environmental cleanup by initiating the degradation of OPs, such as pesticides, in contaminated environments. In microbial cell configuration, these enzymes can break down chemical bonds of OPs and usually convert them into less toxic metabolites through a biotransformation process or contribute to their complete mineralization. In their purified state, they exhibit higher pollutant degradation efficiencies and the ability to operate under different environmental conditions. Thus, this review provides a clear overview of the current knowledge about applications of OP-reacting enzymes. It presents research works focusing on the use of these enzymes in various bioremediation strategies to mitigate environmental pollution and in medicine as alternative therapeutic means against OP poisoning.

The article is devoted to the creation of enzymatic nanoreactors based on polystyrene-block-poly(acrylic acid) (PS-b-PAA) copolymers containing bioscavengers capable of neutralizing toxic esters both in the body and in the environment. Block copolymers of different amphiphilicity, hydrophilicity and molecular weights were synthesized and characterized using gel permeation chromatography, NMR and UV spectroscopy. Polymeric nanocontainers in the absence and presence of human butyrylcholinesterase were made by film hydration and characterized by dynamic light scattering and microscopy methods. Enzyme activity was determined using the Ellman method. For the first time, factors that need to be taken into account for the creation of effective enzymatic nanoreactors based on PS-b-PAA are presented. The data obtained open up the possibility of PS-b-PAA nanoreactor use for future in vivo bioscavenger studies.

The study examines the impact of microstructure and polymethyl methacrylate (PMMA) grafting on the degradability of Zn-Mg alloys. The mechanical properties of a Zn alloy containing 0.68 wt% Mg and extruded at 200 °C are enhanced for degradable load-bearing applications, addressing a crucial need in the field. The material exhibits a bimodal grain size distribution that is random texture, consisting of secondary phases, grains, and sub-grains. With an elongation to failure of 16 %, the yield and ultimate tensile strengths are 325.9 and 414.5 MPa, respectively, and the compressive yield strength is 450.5 MPa. The "grafting-from" method was used to coat a few micrometers thick of PMMA on both bulk and scaffold Zn alloys to mitigate the corrosion rate. The last one is a porous structure, with a porosity of 65.8 %, considered as in the first approach of an orthopedic implant. After being immersed for 720 h, the PMMA-grafted bulk alloy's corrosion rate decreased from 0.43 to 0.25 mm/y. Similarly, the scaffold alloy's corrosion rate reduced from 1.24 to 0.49 mm/y. These results indicate that the method employed could be used for future orthopedic applications.

ViralZone (http://viralzone.expasy.org) is a knowledge repository for viruses that links biological knowledge and databases. It contains data on virion structure, genome, proteome, replication cycle and host-virus interactions. The new update provides better access to the data through contextual popups and higher resolution images in Scalable Vector Graphics (SVG) format. These images are designed to be dynamic and interactive with human viruses to give users better access to the data. In addition, a new coronavirus-specific resource provides regularly updated data on variants and molecular biology of SARS-CoV-2. Other virus-specific resources have been added to the database, particularly for HIV, herpesviruses and poxviruses.

The β-adrenergic drug Mirabegron, a drug initially used for the treatment of an overactive bladder, has new potential indications and is hydrolyzed by butyrylcholinesterase (BChE). This compound is one of the only arylacylamide substrates to be catabolized by BChE. A steady-state kinetic analysis at 25 °C and pH 7.0 showed that the enzyme behavior is Michaelian with this substrate and displays a long pre-steady-state phase characterized by a burst. The induction time, τ, increased with substrate concentration (τ ≈ 18 min at maximum velocity). The kinetic behavior was interpreted in terms of hysteretic behavior, resulting from a slow equilibrium between two enzyme active forms, E and E′. The pre-steady-state phase with the highest activity corresponds to action of the E form, and the steady state corresponds to action of the E′ form. The catalytic parameters were determined as kcat = 7.3 min−1 and Km = 23.5 μM for the initial (burst) form E, and kcat = 1.6 min−1 and Km = 3.9 μM for the final form E′. Thus, the higher affinity of E′ for Mirabegron triggers the slow enzyme state equilibrium toward a slow steady state. Despite the complexity of the reaction mechanism of Mirabegron with BChE, slow BChE-catalyzed degradation of Mirabegron in blood should have no impact on the pharmacological activities of this drug.

Human butyrylcholinesterase (BChE) is an efficient bioscavenger of toxicants. Highly purified BChE was labelled with the near infrared fluorescent IRDye800CW. The goal was to determine the pharmacokinetics and fate of enzyme in mice. BChE-IRDye800CW was encapsulated in polyethylene glycol-polypropylene sulfide-based spherical polymersome nanoreactors with the following characteristics: 140 nm diameter, xi = -6 mV, PDI <= 0.2, 1 year stability. Encapsulation did not alter the functional properties of BChE. Free and encapsulated enzyme were injected intravenously to CD-1 mice (single dose of enzyme 1.5 mg/kg and PEG-PPS polymersomes 25 mg/ kg) and were analyzed for 8 days using an in vivo imaging system. Results showed that the pharmacokinetic distribution alpha-phase of encapsulated BChE (t1/2 = 17.6 h) was longer than for free enzyme (t1/2 = 6.6 h). The mean half-time for elimination beta-phase was 2-time longer for encapsulated enzyme than for free enzyme (150 vs 72 h). Transient changes in infrared fluorescence in organs showed that BChE is eliminated from liver. However, free and encapsulated enzymes were cleared via different pathways. This first study of pharmacokinetics and fate of BChE encapsulated in polymersomes initiates research of new formulations of bioscavengers aimed at increasing the residence time of enzymes in the blood stream.

Currently, there are problems to standardize methods for determining the concentration of nanoparticles and creation of etalon materials for calibrating measured concentrations. Accurate determination of nanoparticle concentration is necessary to assess the maximum dose of administered nanotherapeutics for diagnostics and therapy in vivo, to determine the order of reaction in enzymatic nanoreactors. In addition, this parameter determines biological effects, such as the formation of a protein corona on the outer surface of nanoparticles that precedes nanoparticles’ absorption and internalization in cells. This review discusses the most common methods for determining the concentration of nanoparticles based on direct visualization, using microscopy, light absorption or light scattering, direct counting of nanoparticles, and gravimetry. Results may differ from one method to the other. Thus, the use of a combination of several methods provides more reliable results. The advantages, disadvantages and ways to improve accuracy of results are also presented.

The thorough characterization of polymer chains grafted through a “grafting-from” process onto substrates based on the determination of number (Mn) and weight (Mw) average molar masses, as well as dispersity (Ɖ), is quite challenging. It requires the cleavage of grafted chains selectively at the polymer–substrate bond without polymer degradation to allow their analysis in solution with steric exclusion chromatography, in particular. The study herein describes a technique for the selective cleavage of PMMA grafted onto titanium substrate (Ti-PMMA) using an anchoring molecule that combines an atom transfer radical polymerization (ATRP) initiator and a UV-cleavable moiety. This technique allows the demonstration of the efficiency of the ATRP of PMMA on titanium substrates and verification that the chains were grown homogeneously.

Butyrylcholinesterase (BChE) is present in plasma and numerous cells and organs. Its physiological function(s) is(are) still unclear. However, this enzyme is of pharmacological and toxicological importance. It displays a broad specificity and is capable of hydrolyzing a wide range of substrates with turnovers differing by several orders of magnitude. Nowaday, these substrates include more than two dozen carboxyl-ester drugs, numerous acetylated prodrugs, and transition state analogues of acetylcholine. In addition, BChE displays a promiscuous hydrolytic activity toward amide bonds of arylacylamides, and slowly hydrolyzes carbamyl- and phosphoryl-esters. Certain pseudo-substrates like carbamates and organophosphates are major drugs of potential medical interest. The existence of a large genetic poly-allelism, affecting the catalytic properties of BChE is at the origin of clinical complications in the use of certain drugs catabolized by BChE. The number of drugs and prodrugs hydrolyzed by BChE is expected to increase in the future. However, very few quantitative data (Km, kcat) are available for most marketed drugs, and except for myorelaxants like succinylcholine and mivacurium, the impact of BChE genetic mutations on catalytic parameters has not been evaluated for most of these drugs.