Active Site Of Enzyme Research Articles

Petrosamine Revisited. Experimental and Computational Investigation of Solvatochromism, Tautomerism and Free Energy Landscapes of a Pyridoacridinium Quaternary Salt.

Petrosamine (1)-a colored pyridoacridine alkaloid from the Belizean sponge, Petrosia sp., that is also a potent inhibitor of acetylcholine esterase (AChE)-was investigated by spectroscopic and computational methods. Analysis of the petrosamine-free energy landscapes, pKa and tautomerism, revealed an accurate electronic depiction of the molecular structure of 1 as the di-keto form, with a net charge of q = +1, rather than a dication (q = +2) under ambient conditions of isolation-purification. The pronounced solvatochromism (UV-vis) reported for 1, and related analogs were investigated in detail and is best explained by charge delocalization and stabilization of the ground state (HOMO) of 1 rather than an equilibrium of competing tautomers. Refinement of the molecular structure 1 by QM methods complements published computational docking studies to define the contact points in the enzyme active site that may improve the design of new AChE inhibitors based on the pyridoacridine alkaloid molecular skeleton.

Acetylcholinesterase Inhibition Activity of Hippeastrum papilio (Ravenna) Van Scheepen (Amaryllidaceae) Using Zebrafish Brain Homogenates.

The Amaryllidaceae family constitutes an interesting source of exclusive alkaloids with a broad spectrum of biological activity. Galanthamine, the most relevant one, has been commercialized for the palliative treatment of Alzheimer's disease symptoms since 2001 due to its potential as an acetylcholinesterase (AChE) inhibitor. In vitro screenings against AChE by applying different Amaryllidaceae species and alkaloids have been reported in the literature; however, they are usually carried out using purified market enzymes. The main goal of this work is to evaluate the AChE inhibitory potential of Hippeastrum papilio (Amaryllidaceae) extracts using zebrafish brain homogenates. The biological assays show that the H. papilio bulb extracts present an interesting AChE inhibitory activity in comparison with the positive reference control galanthamine (IC50 values of 1.20 ± 0.10 and 0.79 ± 0.15 μg/mL, respectively). The chemical profile of H. papilio shows that this species has a high amount of galanthamine, which may contribute to the inhibitory effect on AChE activity of zebrafish brains. Computational experiments were used to build the model for zebrafish AChE and to evaluate the interactions between galanthamine and the enzymic active site. This work suggests that zebrafish could represent an important model in the search for bioactive molecules from the Amaryllidaceae family for the treatment of Alzheimer's disease.

Antioxidant and Enzyme Inhibitory Activities of Rhoifolin Flavonoid: In Vitro and in Silico Studies.

Rhoifolin (apigenin-7-O-β-neohesperidoside) belongs to the class of flavonoids and was reported to exhibit anti-inflammatory, cytotoxic, antidiabetic, hepatoprotective, and cardioprotective activities. The current study presents the in-vitro evaluation of the antioxidative effects of rhoifolin by many assays, namely DPPH, CUPRAC, ABTS, phosphomolybdenum, and FRAP. Enzyme inhibitory potential was also evaluated for acetylcholinesterase (AChE), butyrylcholinesterase (BChE), tyrosinase, amylase, and glucosidase enzymes. While results revealed weak antioxidant activities for rhoifolin, the compound demonstrated some promising enzyme inhibitory effects against BChE (4.03 mg GALAE/g) and tyrosinase (7.44 mg KAE/g) but was not active on AChE. Regarding anti-diabetic enzymes, the compound was active on amylase but did not show any inhibition effect on glucosidase. In-silico molecular docking study was performed for rhoifolin on the active site of NADPH oxidase, BChE, and amylase enzymes to verify the observed enzyme inhibitory effect. Good binding affinities were observed for rhoifolin on all the docked enzymes, revealing numerous hydrogen bonds, carbon-hydrogen, van der Waals interactions. This is the first study to evaluate the enzyme inhibition potential of rhoifolin. We concluded that the increase in the degree of glycosylation might decrease the antioxidant abilities of flavonoids and that rhoifolin had moderate enzyme inhibition abilities to be investigated in future studies.

Molecular Bio-inspired Strategies for the Design of Electrocatalytic Systems.

This perspective article delves into the realm of bio-inspired catalysis, highlighting the valuable insights gleaned from enzymatic systems for the design of advanced electrocatalysts. We focus here on three key aspects to mimic: the structure of enzymatic active sites, the essential functions to enable catalytic activity as well as key elementary steps of reaction mechanisms employed by enzymes to ensure maximum efficiency and selectivity. Our research group's contributions to these areas are highlighted, including the synthesis and catalytic activity of cobalt(III) pyridinethiolate complexes, the exploration of bimetallic sites mimicking biological carbon dioxide reductases and of all-ferrous Fe4S4 clusters as mimics of FeP active sites, and the integration of concerted proton-electron transfer (CPET) mediators for the generation of metal hydride species. We emphasize the potential of these bio-inspired approaches in advancing electrocatalyst design and their relevance to molecular catalysis.

Interaction studies of recombinant laccase with co-solutes: Using various spectroscopic, calorimetric, and in silico approaches

The co-solute engineering is a rational approach to elucidate protein-molecule interactions which involves an exhaustive understanding of the structural characteristics of active sites of enzymes and their involvement in enzyme function. Laccase has attracted huge interest among scientists due to its potential uses in diverse fields. In this study, we have exploited a co-solute engineering approach to elucidate the protein interactions with polyethylene glycol (PEG of molecular weight of 400 Da, PEG 400) and amino acid (arginine). This study employed a comprehensive range of spectroscopic techniques (absorbance, fluorescence, FTIR, and circular dichroism), calorimetry, and in silico method (molecular docking) to investigate the protein conformations and determine the thermodynamic binding parameters and affinities in the presence or absence of co-solutes. Furthermore, the presence of co-solutes induced notable conformational changes in the secondary structure of the protein, transitioning it from a β -sheet configuration to an α -helical structure. The objective was to gain insights into the effect of co-solutes on the protein behavior and explore the potential of “Protein-co-solute engineering” for identifying target residues that could be modulated to alter the protein's properties. By employing these multidisciplinary approaches, the study aimed to shed light on the interactions between ligands and proteins and their potential for engineering protein function. A comprehensive understanding of the mechanisms governing protein–ligand interactions is vital for progress in drug discovery. By exploring the physical principles that regulate these interactions, such as binding kinetics, thermodynamics, and driving forces, we can uncover fundamental insights into protein folding and stability, offering a solid foundation for the development of innovative therapeutic strategies.

Copper-instigated modulatory cell mortality mechanisms and progress in oncological treatment investigations.

Copper, a transition metal, serves as an essential co-factor in numerous enzymatic active sites and constitutes a vital trace element in the human body, participating in crucial life-sustaining activities such as energy metabolism, antioxidation, coagulation, neurotransmitter synthesis, iron metabolism, and tetramer deposition. Maintaining the equilibrium of copper ions within biological systems is of paramount importance in the prevention of atherosclerosis and associated cardiovascular diseases. Copper induces cellular demise through diverse mechanisms, encompassing reactive oxygen species responses, apoptosis, necrosis, pyroptosis, and mitochondrial dysfunction. Recent research has identified and dubbed a novel regulatory cell death modality-"cuprotosis"-wherein copper ions bind to acylated proteins in the tricarboxylic acid cycle of mitochondrial respiration, resulting in protein aggregation, subsequent downregulation of iron-sulfur cluster protein expression, induction of proteotoxic stress, and eventual cell death. Scholars have synthesized copper complexes by combining copper ions with various ligands, exploring their significance and applications in cancer therapy. This review comprehensively examines the multiple pathways of copper metabolism, copper-induced regulatory cell death, and the current status of copper complexes in cancer treatment.

Synthesis, molecular docking, and bioactivity study of isoquinoline-sulfonamide hybrid analogues: A promising acetylcholinesterase and butyrylcholinesterase inhibitor candidate

Isoquinoline-sulfonamide hybrid analogues (1–11) were synthesised and tested for their dual cholinesterase inhibitory potentials. All analogues showed a varied degree of inhibitory potential, with IC50 values ranging between 0.30 ± 0.05 to 26.30 ± 0.20 µM (AChE) and 0.80 ± 0.01 to 37.60 ± 0.70 µM (BuChE) as compared to standard drug donepezil (IC50 = 2.16 ± 0.12 and 4.5 ± 0.11 µM respectively). In case of AChE, analogue 2 (IC50 = 0.30 ± 0.05 μM) was most potent, while in case of BuChE, analogues 2, 11, and 5 with IC50 values of 0.80 ± 0.01, 1.20 ± 0.05 and 2.50 ± 0.10 μM respectively, were found most potent. Structure-activity relationship was investigated, which is primarily depending upon nature, number, position, and electron donating/withdrawing effect on phenyl ring. A molecular docking study was carried out to determine the binding interactions of the most potent analogues with the active site of enzymes. The compounds were synthesized by simple modes of synthesis like esterification, hydrazide and finally sulfonamide formation.

Synthesis, in vitro α-glucosidase, α-amylase activities and molecular docking study of hybrid thiazole-sulfonohydrazide analogues

Thiazole-sulfonohydrazide hybrid analogues (1-15), were synthesized and characterized through different techniques such as NMR, HR-EIMS and evaluated against α-glucosidase and α-amylase enzymes. In case of α-glucosidase, analogues 4 (IC50 = 4.60 ± 0.30 µM) while in case of α-amylase, analogue 1 (IC50 = 5.10 ± 0.20 µM) is the most potent among the series confirmed from both the experimental and molecular docking study. Structure activity relationship was carried out for all analogues which mainly depend upon number, nature, position, and electron donating/withdrawing effects of the substituent/s on phenyl ring. Molecular docking study was carried out to show the binding interaction of the most potent analogue with the active site of enzyme.

Synthesis, in vitro α-glucosidase, α-amylase inhibitory potential and molecular docking study of thiadiazole-sulfonamide hybrid analogues

We have synthesized fourteen thiadiazole-sulfonamide hybrid analogues (1–14) , characterized throygh different technequies i.e., NMR, HR-EIMS, and screened against α-glucosidase and α-amylase enzymes. All analogues hava excellent inhibitory potential with IC50 values ranging from 5.50 ± 0.30 to 43.30 ± 0.40 µM (α-glucosidase) and 2.40 ± 0.30 to 21.10 ± 0.50 µM (α-amylase) as compared to standard drug acarbose (IC50 = 38.45 ± 0.80 µM & 11.12 ± 0.15 µM respectivelty). In case of α-glucosidase, analogues 7 (IC50 = 5.50 ± 0.30 µM) while in case of α-amylase, analogues 1 (IC50 = 2.40 ± 0.30 µM) are the most potent among the series. Structure-activity relationships was carried out, which mostly depend on the number, type, location, and electron-donating/withdrawing effects of the substituent(s) on the phenyl ring. To demonstrate the binding interaction of the most potent analogues with the enzyme's active sites, a molecular docking study was conducted. All Compounds were verified for cytotoxicity against 3T3 mouse fibroblast cell line and detected non-toxic. The compounds were synthesized by simple modes of synthesis like thiosemicarbazone, thiadiazole and finally thiadiazole-sulfonamide formation.

Synthesis, in vitro α-glucosidase, urease activities and molecular docking study of benzoxazole bearing Schiff base derivatives

We have synthesis seventeen benzoxazole bearing Schiff base derivatives (1-17), characterized through different spectroscopic techniques i.e., 1H-NMR, 13C-NMR and HREI-MS and were screened against alpha-glucosidase and urease enzymes. All derivatives showed good inhibitory potential with IC50 value ranging from 3.20 ± 0.10 to 38.10 ± 0.20 µM (α-glucosidase) and 2.70 ± 0.40 to 21.20 ± 0.10 µM (urease) in the presence of reference drugs acarbose and thiourea having an IC50 values 38.45 ± 0.80 µM and 21.25 ± 0.15 µM respectively. Methoxy substituted derivative 1 was found most potent against alpha-glucosidase enzyme and derivative 3 was found most potent against urease enzyme. Structure activity relation has been carried out which mainly depend upon the type, number, position and electron donating/withdrawing effects of substituent(s) on phenyl ring. The study was further validated by molecular docking to find the binding interaction of most potent compounds with the enzyme active sites. All Compounds were verified for cytotoxicity against 3T3 mouse fibroblast cell line and detected non-toxic. The compounds were synthesized by simple modes of synthesis like carboxylate, hydrazide and finally Schiff base formation.

Ribavirin inhibits the replication of infectious bursal disease virus predominantly through depletion of cellular guanosine pool.

The antiviral activity of different mutagens against single-stranded RNA viruses is well documented; however, their activity on the replication of double-stranded RNA viruses remains unexplored. This study aims to investigate the effect of different antivirals on the replication of a chicken embryo fibroblast-adapted Infectious Bursal Disease virus, FVSKG2. This study further explores the antiviral mechanism utilized by the most effective anti-IBDV agent. The cytotoxicity and anti-FVSKG2 activity of different antiviral agents (ribavirin, 5-fluorouracil, 5-azacytidine, and amiloride) were evaluated. The virus was serially passaged in chicken embryo fibroblasts 11 times at sub-cytotoxic concentrations of ribavirin, 5-fluorouracil or amiloride. Further, the possible mutagenic and non-mutagenic mechanisms utilized by the most effective anti-FVSKG2 agent were explored. Ribavirin was the least cytotoxic on chicken embryo fibroblasts, followed by 5-fluorouracil, amiloride and 5-azacytidine. Ribavirin inhibited the replication of FVSKG2 in chicken embryo fibroblasts significantly at concentrations as low as 0.05 mM. The extinction of FVSKG2 was achieved during serial passage of the virus in chicken embryo fibroblasts at ≥0.05 mM ribavirin; however, the emergence of a mutagen-resistant virus was not observed until the eleventh passage. Further, no mutation was observed in 1,898 nucleotides of the FVSKG2 following its five passages in chicken embryo fibroblasts in the presence of 0.025 mM ribavirin. Ribavarin inhibited the FVSKG2 replication in chicken embryo fibroblasts primarily through IMPDH-mediated depletion of the Guanosine Triphosphate pool of cells. However, other mechanisms like ribavirin-mediated cytokine induction or possible inhibition of viral RNA-dependent RNA polymerase through its interaction with the enzyme's active sites enhance the anti-IBDV effect. Ribavirin inhibits ds- RNA viruses, likely through IMPDH inhibition and not mutagenesis. The inhibitory effect may, however, be augmented by other non-mutagenic mechanisms, like induction of antiviral cytokines in chicken embryo fibroblasts or interaction of ribavirin with the active sites of RNA-dependent RNA polymerase of the virus.

Structural basis for membrane-proximal proteolysis of substrates by ADAM10

The endopeptidase ADAM10 is a critical catalyst for the regulated proteolysis of key drivers of mammalian development, physiology, and non-amyloidogenic cleavage of APP as the primary α-secretase. ADAM10 function requires the formation of a complex with a C8-tetraspanin protein, but how tetraspanin binding enables positioning of the enzyme active site for membrane-proximal cleavage remains unknown. We present here a cryo-EM structure of a vFab-ADAM10-Tspan15 complex, which shows that Tspan15 binding relieves ADAM10 autoinhibition and acts as a molecular measuring stick to position the enzyme active site about 20Å from the plasma membrane for membrane-proximal substrate cleavage. Cell-based assays of N-cadherin shedding establish that the positioning of the active site by the interface between the ADAM10 catalytic domain and the bound tetraspanin influences selection of the preferred cleavage site. Together, these studies reveal the molecular mechanism underlying ADAM10 proteolysis at membrane-proximal sites and offer a roadmap for its modulation in disease.

Asymmetric Carbohydroxylation of Alkenes Using Photoenzymatic Catalysis.

Alkene difunctionalizations enable the synthesis of structurally elaborated products from simple and ubiquitous starting materials in a single chemical step. Carbohydroxylations of olefins represent a family of reactivity that furnish structurally complex alcohols. While examples of this type of three-component coupling have been reported, catalytic asymmetric examples remain elusive. Here, we report an enzyme-catalyzed asymmetric carbohydroxylation of alkenes catalyzed by flavin-dependent "ene"-reductases to produce enantioenriched tertiary alcohols. Seven rounds of protein engineering reshape the enzyme's active site to increase activity and enantioselectivity. Mechanistic studies suggest that C-O bond formation occurs via a 5-endo-trig cyclization with the pendant ketone to afford an α-oxy radical which is oxidized and hydrolyzed to form the product. This work demonstrates photoenzymatic reactions involving "ene"-reductases can terminate radicals via mechanisms other than hydrogen atom transfer, expanding their utility in chemical synthesis.

Real-time detection of human telomerase DNA synthesis by multiplexed single-molecule FRET

Genomic stability in proliferating cells critically depends on telomere maintenance by telomerase reverse transcriptase. Here we report the development and proof-of-concept results of a single-molecule approach to monitor the catalytic activity of human telomerase in real time and with single-nucleotide resolution. Using zero-mode waveguides and multicolor FRET, we recorded the processive addition of multiple telomeric repeats to individual DNA primers. Unlike existing biophysical and biochemical tools, the novel approach enables the quantification of nucleotide-binding kinetics before nucleotide incorporation. Moreover, it provides a means to dissect the unique translocation dynamics that telomerase must undergo after synthesis of each hexameric DNA repeat. We observed an unexpectedly prolonged binding dwell time of dGTP in the enzyme active site at the start of each repeat synthesis cycle, suggesting that telomerase translocation is composed of multiple rate-contributing sub-steps that evade classical biochemical analysis.

In-vivo anti-diabetic and anti-hyperlipidemic effects of natural metabolites from resin of Commiphora mukul and their in-silico to in-vitro target fishing

Diabetes mellitus is a rapidly spreading global metabolic disorder that has serious social, health, and economic consequences. Herein, we have evaluated in vivo antidiabetic and antihyperlipidemic effects of myrrhanone-B and myrrhanol-B (isolated from Commiphora mukul Hook). We observed that treatment with myrrhanone-B and myrrhanol-B at a dose of 5 and 10 mg/kg body weight for 21 days significantly improved body weight loss, water consumption, and the concentration of blood glucose level (BGL) in alloxan (120 mg/kg) induced diabetic mice, which indicates that the compounds possess strong anti-diabetic activities. In the biochemical analysis, these compounds improved an abnormal level of total cholesterol (TC), triacylglycerol (TG), and low-density lipoprotein cholesterol (LDL-C) to a normal level and increased the high-density lipoprotein cholesterol level (HDLC). Later, drug target of compounds was predicted through in-silico docking which shows that these compounds nicely fit in the active site of α-glucosidase enzyme and mediates excellent interactions with the catalytic residues, Asp214 and Asp349. The in-silico results were confirmed by in-vitro testing of myrrhanone-B and myrrhanol-B against α-glucosidase where both the compounds exhibited excellent inhibitory potency with IC50 values of 19.50 ± 0.71, and 16.11 ± 0.69 µM, respectively. Furthermore, mechanistic study was conducted to observe their binding mechanism, which reflect that myrrhanol-B has mixed type of inhibition (ki = 12.33 ± 0.030 µM), while myrrhanone-B demonstrates competitive type of inhibition (ki =14.53 ± 0.040 µM).

Phylogenetic analysis and docking study of neuraminidase gene of influenza A/H1N1 viruses circulating in Iran from 2010 to 2019

Influenza A viruses (H1N1) have been consistently one of the most evolving viruses that escape from vaccine-induced immunity. Although there has been a rapid rise in human influenza virus knowledge since the 2009 pandemic, the molecular information about Iranian strains is still inadequate. The aim of this study was to analyze the neuraminidase (NA) segment of the Iranian isolates in terms of phylogenetic, antiviral resistance, and vaccine efficiency. Ninety-three NA sequences collected among 1758 nasopharyngeal swab samples during the 2015–2016 influenza season were sequenced and submitted to NCBI. Moreover, all the submitted Iranian influenza H1N1 NA sequences since 2010 till 2019 were included in the study.Software including MEGA-X, MODELLER, UCSF ChimeraX, Auto-Dock 4.2, and other online tools were used to analyze the phylogenetic relationship, vaccine efficiency, and binding affinity to sialic acid of the selected NA proteins. Moreover, the information about antiviral drug resistance mutations of NA were gathered and compared to the Iranian NA segments to check the presence of antiviral drug-resistant strains.The phylogenetic study showed that most Iranian NA sequences (between 2015 and 2016) were located in a single clade and following years were located in its subclade by 3 major mutations (G77R/K, V81A, and J188T). Resistant mutations in drug targets of NA including I117M, D151E, I223V, and S247N were ascertained in 10 isolates during the 2015–2016 flu seasons.Investigation of vaccination effect revealed that Iranian isolates in 2017 and 2018 were best matched to A/Brisbane/02/2018 (H1N1), and in 2019 to A/Guangdong-Maonan/SWL1536/2019 (H1N1).Furthermore, we performed an in-silico analysis of NA enzymatic activity of all Iranian sequences by assessment of enzyme stability, ligand affinity, and active site availability. Overall, the enzyme activity of four Iranian strains (AUG84119, AUG84157, AUG84095, and AUG84100) was assumed as the maximum enzyme activity. This study highlighted the evolutionary trend of influenza A virus/H1N1 circulating in Iran, which provides a preliminary viewpoint for a better comprehension of new emerging strains’ virulence and thus, more appropriate monitoring of influenza virus A/H1N1 during each outbreak season.

Control analysis in the identification of key enzymes driving metabolic adaptations: Towards drug target discovery

Metabolic Control Analysis (MCA) marked a turning point in understanding the design principles of metabolic network control by establishing control coefficients as a means to quantify the degree of control that an enzyme exerts on flux or metabolite concentrations. MCA has demonstrated that control of metabolic pathways is distributed among many enzymes rather than depending on a single rate-limiting step. MCA also proved that this distribution depends not only on the stoichiometric structure of the network but also on other kinetic determinants, such as the degree of saturation of the enzyme active site, the distance to thermodynamic equilibrium, and metabolite feedback regulatory loops. Consequently, predicting the alterations that occur during metabolic adaptation in response to strong changes involving a redistribution in such control distribution can be challenging. Here, using the framework provided by MCA, we illustrate how control distribution in a metabolic pathway/network depends on enzyme kinetic determinants and to what extent the redistribution of control affects our predictions on candidate enzymes suitable as targets for small molecule inhibition in the drug discovery process. Our results uncover that kinetic determinants can lead to unexpected control distribution and outcomes that cannot be predicted solely from stoichiometric determinants. We also unveil that the inference of key enzyme-drivers of an observed metabolic adaptation can be dramatically improved using mean control coefficients and ruling out those enzyme activities that are associated with low control coefficients. As the use of constraint-based stoichiometric genome-scale metabolic models (GSMMs) becomes increasingly prevalent for identifying genes/enzymes that could be potential drug targets, we anticipate that incorporating kinetic determinants and ruling out enzymes with low control coefficients into GSMM workflows will facilitate more accurate predictions and reveal novel therapeutic targets.

Design, synthesis, evaluation of biological activities and molecular docking and dynamic studies of novel acetazolamide analog compounds

Modification of drugs used in the clinic is a frequently used method with regards to medicinal chemistry in the development of new drugs. Acetazolamide is a drug in clinical use as a CA inhibitor. Within the scope of this study, the 'N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl) acetamide’ structure, which is acetazolamide residue, was kept constant; various mercaptan substitutions were made from methylene adjacent to the carbonyl group in the structure. Compounds 4c, 4d, 4e, 4 g, 4h, 4i, and 4j exhibited inhibitor activity against CA enzyme with IC50=0.238 ± 0.010, 0.161 ± 0.007, 0.067 ± 0.002, 0.084 ± 0.003, 0.033 ± 0.001, 0.049 ± 0.002 and 0.187 ± 0.008 µM, respectively. The intermolecular interactions of the promising compounds with aromatase enzyme were investigated through the SP docking approach, which revealed significant binding interaction energies associated with these compounds. To measure the stability of the compounds in the enzyme active site, dynamic studies were performed at 100 ns. In addition to the RMSD, RMSF parameters, the interaction ratios of compound 4h with amino acids in the enzyme active site and the interaction histograms were also investigated. The results obtained are quite promising. Continuous interactions were exhibited with Thr199, Glu106, His96, His94 and His119, which are important for the CA enzyme. Communicated by Ramaswamy H. Sarma

Molecular docking, Synthesis and Characterization of New Indomethacin and Mefenamic Acid Analogues as Potential Anti-inflammatory Agents

In this work the pharmacological study and synthesis of new thiadiazine bearing on triazole which obtained from hippuric acid , indomethacin and mefenamic acid that have carboxylic acid moiety, Drugs with carboxylic groups and thiocarbohydrazide interacted to produce the 4-amino-5-aryl-4H-1,2,4-triazole-3-thiol (1a-c).
 and the starting products 4-amino-5-aryl-4H-1,2,4-triazole-3-thiol) were treated with chloroacetyl chloride to produce final products (2a-c). To confirm the structure of the generated compounds, FT-IR, 1H-NMR, and mass spectroscopy were used to characterize all derivatives (intermediate and final products). The in vivo anti-inflammatory efficacy of some derivatives and thier toxicity to animals (in vivo) were evaluated. And then derivatives were subjected to molecular docking to create safe and efficient molecules. To test each derivative's ability to bind to the enzyme's active site, it was docked into the active sites. To determine the synthetic compound's topological polar surface area, bioavailability, and drug-likeness, An investigation of absorption, distribution, metabolism and elimination was performed. According to the findings, the tested derivatives adhered to the Lipinski rule and were ingested

Reversing the Enantioselectivity of Enzymatic Carbene N-H Insertion Through Mechanism-Guided Protein Engineering.

We report a computationally driven approach to access enantiodivergent enzymatic carbene N-H insertions catalyzed by P411 enzymes. Computational modeling was employed to rationally guide engineering efforts to control the accessible conformations of a key lactone-carbene (LAC) intermediate in the enzyme active site by installing a new H-bond anchoring point. This H-bonding interaction controls the relative orientation of the reactive carbene intermediate, orienting it for an enantioselective N-nucleophilic attack by the amine substrate. By combining MD simulations and site-saturation mutagenesis and screening targeted to only two key residues, we were able to reverse the stereoselectivity of previously engineered S-selective P411 enzymes. The resulting variant, L5_FL-B3, accepts a broad scope of amine substrates for N-H insertion with excellent yields (up to >99 %), high efficiency (up to 12 300 TTN), and good enantiocontrol (up to 7 : 93 er).