Active Site Residues Research Articles

Multifunctional in vitro, in silico and DFT analyses on antimicrobial BagremycinA biosynthesized by Micromonospora chokoriensis CR3 from Hieracium canadense

Among the actinomycetes in the rare genera, Micromonospora is of great interest since it has been shown to produce novel therapeutic compounds. Particular emphasis is now on its isolation from plants since its population from soil has been extensively explored. The strain CR3 was isolated as an endophyte from the roots of Hieracium canadense, and it was identified as Micromonospora chokoriensis through 16S gene sequencing and phylogenetic analysis. The in-vitro analysis of its extract revealed it to be active against the clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and Candida tropicalis (15 mm). No bioactivity was observed against Gram-negative bacteria, Escherichia coli ATCC 25922, and Klebsiella pneumoniae ATCC 706003. The Micromonospora chokoriensis CR3 extract was also analyzed through the HPLC-DAD-UV–VIS resident database, and it gave a maximum match factor of 997.334 with the specialized metabolite BagremycinA (BagA). The in-silico analysis indicated that BagA strongly interacted with the active site residues of the sterol 14-α demethylase and thymidylate kinase enzymes, with the lowest binding energies of − 9.7 and − 8.3 kcal/mol, respectively. Furthermore, the normal mode analysis indicated that the interaction between these proteins and BagA was stable. The DFT quantum chemical properties depicted BagA to be reasonably reactive with a HOMO-LUMO gap of (ΔE) of 4.390 eV. BagA also passed the drug-likeness test with a synthetic accessibility score of 2.06, whereas Protox-II classified it as a class V toxicity compound with high LD50 of 2644 mg/kg. The current study reports an endophytic actinomycete, M. chokoriensis, associated with H. canadense producing the bioactive metabolite BagA with promising antimicrobial activity, which can be further modified and developed into a safe antimicrobial drug.

Effects of the Y432S Cancer-Associated Variant on the Reaction Mechanism of Human DNA Polymerase κ.

Human DNA polymerases are vital for genetic information management. Their function involves catalyzing the synthesis of DNA strands with unparalleled accuracy, which ensures the fidelity and stability of the human genomic blueprint. Several disease-associated mutations and their functional impact on DNA polymerases have been reported. One particular polymerase, human DNA polymerase kappa (Pol κ), has been reported to be susceptible to several cancer-associated mutations. The Y432S mutation in Pol κ, associated with various cancers, is of interest due to its impact on polymerization activity and markedly reduced thermal stability. Here, we have used computational simulations to investigate the functional consequences of the Y432S using classical molecular dynamics (MD) and coupled quantum mechanics/molecular mechanics (QM/MM) methods. Our findings suggest that Y432S induces structural alterations in domains responsible for nucleotide addition and ternary complex stabilization while retaining structural features consistent with possible catalysis in the active site. Calculations of the minimum energy path associated with the reaction mechanism of the wild type (WT) and Y432S Pol κ indicate that, while both enzymes are catalytically competent (in terms of energetics and the active site's geometries), the cancer mutation results in an endoergic reaction and an increase in the catalytic barrier. Interactions with a third magnesium ion and environmental effects on nonbonded interactions, particularly involving key residues, contribute to the kinetic and thermodynamic distinctions between the WT and mutant during the catalytic reaction. The energetics and electronic findings suggest that active site residues favor the catalytic reaction with dCTP3- over dCTP4-.

Engineered Phenylalanine Ammonia‐Lyases for the Enantioselective Synthesis of Aspartic Acid Derivatives

Biocatalytic hydroamination of alkenes is an efficient and selective method to synthesize natural and unnatural amino acids. Phenylalanine ammonia‐lyases (PALs) have been previously engineered to access a range of substituted phenylalanines and heteroarylalanines, but their substrate scope remains limited, typically including only arylacrylic acids. Moreover, the enantioselectivity in the hydroamination of electron‐deficient substrates is often poor. Here, we report the structure‐based engineering of PAL from Planctomycesbrasiliensis (PbPAL), enabling preparative‐scale enantioselective hydroaminations of previously inaccessible yet synthetically useful substrates, such as amide‐ and ester‐containing fumaric acid derivatives. Through the elucidation of cryo‐electron microscopy (cryo‐EM) PbPAL structure and screening of the structure‐based mutagenesis library, we identified the key active site residue L205 as pivotal for dramatically enhancing the enantioselectivity of hydroamination reactions involving electron‐deficient substrates. Our engineered PALs demonstrated exclusive a‐regioselectivity, high enantioselectivity, and broad substrate scope. The potential utility of the developed biocatalysts was further demonstrated by a preparative‐scale hydroamination yielding tert‐butyl protected l‐aspartic acid, widely used as intermediate in peptide solid‐phase synthesis.

Engineered Phenylalanine Ammonia-Lyases for the Enantioselective Synthesis of Aspartic Acid Derivatives.

Biocatalytic hydroamination of alkenes is an efficient and selective method to synthesize natural and unnatural amino acids. Phenylalanine ammonia-lyases (PALs) have been previously engineered to access a range of substituted phenylalanines and heteroarylalanines, but their substrate scope remains limited, typically including only arylacrylic acids. Moreover, the enantioselectivity in the hydroamination of electron-deficient substrates is often poor. Here, we report the structure-based engineering of PAL from Planctomycesbrasiliensis (PbPAL), enabling preparative-scale enantioselective hydroaminations of previously inaccessible yet synthetically useful substrates, such as amide- and ester-containing fumaric acid derivatives. Through the elucidation of cryo-electron microscopy (cryo-EM) PbPAL structure and screening of the structure-based mutagenesis library, we identified the key active site residue L205 as pivotal for dramatically enhancing the enantioselectivity of hydroamination reactions involving electron-deficient substrates. Our engineered PALs demonstrated exclusive a-regioselectivity, high enantioselectivity, and broad substrate scope. The potential utility of the developed biocatalysts was further demonstrated by a preparative-scale hydroamination yielding tert-butyl protected l-aspartic acid, widely used as intermediate in peptide solid-phase synthesis.

Screening and rational identification of a novel angiotensin-converting enzyme C-domain inhibitory peptide from Fabaceae food peptide library

Angiotensin-converting enzyme (ACE), consisting of N-domain and C-domain, is a key regulator of blood pressure. The use of cACE-specific inhibitors helps minimize side effects in clinical applications. Legumes are a good source of proteins containing ACE inhibitory peptides; however, no studies have reported the identification of cACE-specific inhibitory peptides from Fabaceae. In this study, thermal hydrolysates from seeds, sprouts, pods, seedlings, and flowers of legumes were analyzed. Flowers of legumes exhibited a C-domain-preference ACE inhibition and anti-hypertensive effect in rats. Screening the legume peptide library identified a novel cACE inhibitory peptide, SJ-1. This study reported the first identification of cACE inhibitory peptide from Fabaceae foods. SJ-1, identified from the legume flowers, interacted with active site residues of cACE, leading to the inhibition of ACE activity, downregulation of bradykinin levels, and reduction of blood pressure. These findings also suggested the potential of legume proteins as a source of cACE inhibitory peptides.

Targeting an Old Foe for Cancer: A Molecular Dynamics Perspective to Unravel the Specific Binding Nature of 2-Methoxy Estradiol to Human β-Tubulin Isotypes.

Microtubules, composed of α- and β-tubulin subunits are crucial for cell division with their dynamic tissue-specificity which is dictated by expression of isotypes. These isotypes differ in carboxy-terminal tails (CTTs), rich in negatively charged acidic residues in addition to the differences in the composition of active site residues. 2-Methoxy estradiol (2-ME) is the first antimicrotubule agent that showed less affinity toward hemopoietic-specific β1 isotype consequently preventing myelosuppression toxicity. The present study focuses on the MD-directed conformational analysis of 2-ME and estimation of its binding affinity in the colchicine binding pocket of various β-tubulin isotypes combined with the α-tubulin isotype, α1B. AlphaFold 2.0 was used to predict the 3D structure of phylogenetically divergent human β-tubulin isotypes in dimer form with α1B. The dimeric complexes were subjected to induced-fit docking with 2-ME. The statistical analysis of docking showed differences in the binding characteristics of 2-ME with different isotypes. The replicas of atom-based molecular dynamic simulations of the best conformation of 2-ME provided insights into the molecular-level details of its binding pattern across the isotypes. Furthermore, the MM/GBSA analyses revealed the specific binding energy profile of 2-ME in β-tubulin isotypes. It also highlighed, 2-ME exhibits the lowest binding affinity toward the β1 isotype as supported by experimental study. The present study may offer useful information for designing next-generation antimicrotubule agents that are more specific and less toxic.

Conformational Dynamics in Corynebacterium glutamicum Diaminopimelate Epimerase: Insights from Ligand Parameterization, Atomistic Simulation, and Markov State Modeling.

The microbial enzyme diaminopimelate epimerase (DapF), a vital enzyme in the lysine biosynthetic pathway, catalyzes the conversion of L, L-diaminopimelate (L, L-DAP) to D, L-diaminopimelate (D, L-DAP) using a catalytic cysteine dyad with one cysteine in thiol state and another in thiolate. Under oxidizing conditions, the catalytic cysteines of apo DapF form a disulfide bond that alters the structure and function of DapF. Given its potential as a target for antimicrobial resistance treatments, understanding DapF's functional dynamics is imperative. In the present work, we employ microsecond-scale all-atom molecular dynamics simulations of product-bound DapF and apo-DapF under oxidized and reduced conditions. We employ a polarized charge model for the ligand and the active site residues, which was necessary to preserve the electrostatic environment in the active site and retain the ligand in the active site. The product-bound DapF and apo-DapF in oxidized and reduced conditions exhibit a closed, semi-open, and open conformation, respectively, as identified using the internal coordinates of the dimeric enzyme and the principal component analysis. The conformational switch is guided by the dynamic catalytic (DC) loop, loop II, and loop III movements in the active site. The time scale of the close-to-open conformational transition is estimated to be 0.8 μs through Markov state modeling (MSM) and transition path theory (TPT). The present study explains the role of various active site residues and loops in ligand binding and protein dynamics in the DapF enzyme under different redox conditions. Such information will be helpful in future inhibitor design studies targeting the DapF enzyme.

An active site mutation induces oxygen reactivity in D-arginine dehydrogenase: A case of superoxide diverting protons †

Enzymes are potent catalysts that increase biochemical reaction rates by several orders of magnitude. Flavoproteins are a class of enzymes whose classification relies on their ability to react with molecular oxygen (O2) during catalysis using ionizable active site residues. Pseudomonas aeruginosa D-arginine dehydrogenase (PaDADH) is a flavoprotein that oxidizes D-arginine for P. aeruginosa survival and biofilm formation. The crystal structure of PaDADH reveals the interaction of the glutamate 246 (E246) side chain with the substrate and at least three other active site residues, establishing a hydrogen bond network in the active site. Additionally, E246 likely ionizes to facilitate substrate binding during PaDADH catalysis. This study aimed to investigate how replacing the E246 residue with leucine affects PaDADH catalysis and its ability to react with O2 using steady-state kinetics coupled with pH profile studies. The data reveal a gain of O2 reactivity in the E246L variant, resulting in a reduced flavin semiquinone species and superoxide (O2•-) during substrate oxidation. The O2•- reacts with active site protons, resulting in an observed non-stoichiometric slope of 1.5 in the enzyme's log (kcat/Km) pH profile with D-arginine. Adding superoxide dismutase results in an observed correction of the slope to 1.0. This study demonstrates how O2•- can alter the slopes of limbs in the pH profiles of flavin-dependent enzymes and serves as a model for correcting non-stoichiometric slopes in elucidating reaction mechanisms of flavoproteins.

Identifying Potential Alzheimer's Disease Therapeutics through GSK-3β Inhibition: A Molecular Docking and Dynamics Approach

Emerging as a promising drug target for Alzheimer's disease (AD) therapy, glycogen synthase kinase 3β (GSK-3β) has garnered attention. This study sought to rigorously scrutinize a compendium of natural compounds retrieved from the ZINC database through pharmacodynamic experiments, employing a 1H-indazole-3-carboxamide (INDZ) scaffold, to identify compounds capable of inhibiting the GSK-3β protein. Utilizing a multi-step approach, the study involved pharmacophore analysis, followed by molecular docking to select five promising ligands for further investigation. Subsequently, ESMACS simulations were employed to assess the stability of the ligand-protein interactions. Evaluation of the binding modes and free energy of the ligands revealed that five compounds (2a-6a) exhibited crucial interactions with the active site residues. Furthermore, various methodologies, including hydrogen bond and clustering analyses, were utilized to ascertain their inhibitory potential and elucidate the factors contributing to ligand binding in the protein's active site. The findings from MMPBSA/GBSA analysis indicated that these five selected small molecules closely approached the IC50 value of the reference ligand (OH8), yielding energy values of −34.85, −32.58, −31.71, and −30.39kcal/mol, respectively. Additionally, an assessment of the interactions using hydrogen bond and dynamic analyses delineated the effective binding of the ligands with the binding pockets in the protein. Through computational analysis, we obtained valuable insights into the molecular mechanisms of GSK-3β, aiding in the development of more potent inhibitors.

Molecular screening of phytocompounds targeting the interface between influenza A NS1 and TRIM25 to enhance host immune responses

BackgroundInfluenza A virus causes severe respiratory illnesses, especially in developing nations where most child deaths under 5 occur due to lower respiratory tract infections. The RIG-I protein acts as a sensor for viral dsRNA, triggering interferon production through K63-linked poly-ubiquitin chains synthesized by TRIM25. However, the influenza A virus's NS1 protein hinders this process by binding to TRIM25, disrupting its association with RIG-I and preventing downstream interferon signalling, contributing to the virus's evasion of the immune response. MethodsIn our study we used structural-based drug designing, molecular simulation, and binding free energy approaches to identify the potent phytocompounds from various natural product databases (>100,000 compounds) able to inhibit the binding of NS1 with the TRIM25. ResultsThe molecular screening identified EA-8411902 and EA-19951545 from East African Natural Products Database, NA-390261 and NA-71 from North African Natural Products Database, SA-65230 and SA- 4477104 from South African Natural Compounds Database, NEA- 361 and NEA- 4524784 from North-East African Natural Products Database, TCM-4444713 and TCM-6056 from Traditional Chinese Medicines Database as top hits. The molecular docking and binding free energies results revealed that these compounds have high affinity with the specific active site residues (Leu95, Ser99, and Tyr89) involved in the interaction with TRIM25. Additionally, analysis of structural dynamics, binding free energy, and dissociation constants demonstrates a notably stronger binding affinity of these compounds with the NS1 protein. Moreover, all selected compounds exhibit exceptional ADMET properties, including high water solubility, gastrointestinal absorption, and an absence of hepatotoxicity, while adhering to Lipinski’s rule. ConclusionOur molecular simulation findings highlight that the identified compounds demonstrate high affinity for specific active site residues involved in the NS1-TRIM25 interaction, exhibit exceptional ADMET properties, and adhere to drug-likeness criteria, thus presenting promising candidates for further development as antiviral agents against influenza A virus infections.

Repurposing DrugBank compounds as potential Plasmodium falciparum class 1a aminoacyl tRNA synthetase multi-stage pan-inhibitors with a specific focus on mitomycin

Plasmodium falciparum aminoacyl tRNA synthetases (PfaaRSs) are potent antimalarial targets essential for proteome fidelity and overall parasite survival in every stage of the parasite’s life cycle. So far, some of these proteins have been singly targeted yielding inhibitor compounds that have been limited by incidences of resistance which can be overcome via pan-inhibition strategies. Hence, herein, for the first time, we report the identification and in vitro antiplasmodial validation of Mitomycin (MMC) as a probable pan-inhibitor of class 1a (arginyl(A)-, cysteinyl(C), isoleucyl(I)-, leucyl(L), methionyl(M), and valyl(V)-) PfaaRSs which hypothetically may underlie its previously reported activity on the ribosomal RNA to inhibit protein translation and biosynthesis. We combined multiple in silico structure-based discovery strategies that first helped identify functional and druggable sites that were preferentially targeted by the compound in each of the plasmodial proteins: Ins1-Ins2 domain in Pf-ARS; anticodon binding domain in Pf-CRS; CP1-editing domain in Pf-IRS and Pf-MRS; C-terminal domain in Pf-LRS; and CP-core region in Pf-VRS. Molecular dynamics studies further revealed that MMC allosterically induced changes in the global structures of each protein. Likewise, prominent structural perturbations were caused by the compound across the functional domains of the proteins. More so, MMC induced systematic alterations in the binding of the catalytic nucleotide and amino acid substrates which culminated in the loss of key interactions with key active site residues and ultimate reduction in the nucleotide-binding affinities across all proteins, as deduced from the binding energy calculations. These altogether confirmed that MMC uniformly disrupted the structure of the target proteins and essential substrates. Further, MMC demonstrated IC50 < 5μM against the Dd2 and 3D7 strains of parasite making it a good starting point for malarial drug development. We believe that findings from our study will be important in the current search for highly effective multi-stage antimalarial drugs.

Structural and biochemical characterization of a nucleotide hydrolase from Streptococcus pneumonia.

In this report, we structurally and biochemically characterized the unknown gene product SP1746 from Streptococcus pneumoniae serotype 4. Various crystal structures of SP1746 in the apo form and in complex with different nucleotides were determined. SP1746 is a globular protein, which belongs to the histidine-aspartate (HD) domain superfamily with two Fe3+ ions in the active site that are coordinated by key active site residues and water molecules. All nucleotides bind in a similar orientation in the active site with their phosphate groups anchored to the diiron cluster. Biochemically, SP1746 hydrolyzes different nucleotide substrates. SP1746 most effectively hydrolyzes diadenosine tetraphosphate (Ap4A) to two ADPs. Based on the aforementioned data, we annotated SP1746 as an Ap4A hydrolase, belonging to the YqeK family. Our invitro data indicate a potential role for SP1746 in regulating Ap4A homeostasis, which requires validation with invivo experiments in bacteria in the future.

Synthesis, Molecular Docking Studies and Biological Evaluation of Thiazolyl Hydrazone Derivatives of Chromone-3-carbaldehyde as Potent Anti-Oxidant and Anti-Inflammatory Agents.

A series of 15 thiazolyl hydrazone derivatives of chromone-3- carbaldehyde have been designed and synthesized by the cyclization of thiosemicarbazone derivatives of chromone-3-carbaldehydes with 4'-substituted-2-bromo acetophenones. All these derivatives were evaluated for antioxidant activity by their direct scavenging activity objects to reactive oxygen species such as DPPH, and nitric oxide, as well as in vitro antiinflammatory activity by a protein denaturation method. Most of these synthesized compounds have shown significant antioxidant activity, among which the compounds 5b, 5c, 5e, 5g, and 5j showed very good antioxidant activities in comparison with the standard ascorbic acid. The in vitro anti-inflammatory activity revealed that the compounds 5b, 5g, and 5h possessed significant activity compared to standard diclofenac sodium. Additionally, molecular docking studies of these molecules using ovalbumin as the protein showed remarkable interactions with its active site residues, and the results indicated that the binding mode of these compounds closely resembled that of the reference compound, diclofenac sodium. Thus, these compounds represent an attractive template for the evaluation of new antiinflammatory and antioxidant agents and might be useful for exploring new therapeutic tools.

Interplay Between Substrate Polarity and Protein Dynamics in Evolved Kemp Eliminases

Extensive molecular dynamics (MD) simulations on designed and evolved enzymes for the Kemp Elimination were performed. Over thirty different systems including combinations of three protein scaffolds used in computational design (KE70, KE59 and HG3), several directed evolution variants and ligands (5‐nitro‐benzoisoxazole, and 5‐nitro‐benzotriazole), were screened computationally. The study was focused mostly on computational and evolved variants for which X‐ray structures are available. MD simulations were used to monitor the organization of the active sites over time periods of 100 ns. A general trend was observed in the simulations: the optimal organization of active site residues, corresponding to the most active catalysts, is only maintained with polarized, TS‐like substrates, reinforcing the crucial role of protein dynamics and electrostatic preorganization for efficient biocatalysis. Implications for enzyme design protocols are discussed.

Appraisal of the Neuroprotective Potentials of Isoeugenol Using In-vitro, In-vivo and In-silico Approaches

Appraisal of the Neuroprotective Potentials of Isoeugenol Using In-vitro, In-vivo and In-silico Approaches

Isolation and Functional Characterization of Erythrofibrase: An Alfa-Fibrinogenase Enzyme from Trimeresurus erythrurus Venom of North-East India.

Green pit viper bites induce mild toxicity with painful local swelling, blistering, cellulitis, necrosis, ecchymosis and consumptive coagulopathy. Several bite cases of green pit vipers have been reported in several south-east Asian countries including the north-eastern region of India. The present study describes isolation and characterization of a haemostatically active protein from Trimeresurus erythrurus venom responsible for coagulopathy. Using a two-step chromatographic method, a snake venom serine protease erythrofibrase was purified to homogeneity. SDS-PAGE of erythrofibrase showed a single band of ~30 kDa in both reducing and non-reducing conditions. The primary structure of erythrofibrase was determined by ESI LC-MS/MS, and the partial sequence obtained showed 77% sequence similarity with other snake venom thrombin-like enzymes (SVTLEs). The partial sequence obtained had the typical 12 conserved cysteine residues, as well as the active site residues (His57, Asp102 and Ser195). Functionally, erythrofibrase showed direct fibrinogenolytic activity by degrading the Aα chain of bovine fibrinogen at a slow rate, which might be responsible for causing hypofibrinogenemia and incoagulable blood for several days in envenomated patients. Moreover, the inability of Indian polyvalent antivenom (manufactured by Premium Serum Pvt. Ltd., Maharashtra, India) to neutralize the thrombin-like and plasmin-like activity of erythrofibrase can be correlated with the clinical inefficacy of antivenom therapy. This is the first study reporting an α-fibrinogenase enzyme erythrofibrase from T. erythrurus venom, which is crucial for the pathophysiological manifestations observed in envenomated victims.

Structural insights into the potential binding sites of Cathepsin D using molecular modelling techniques

Alzheimer’s disease (AD) is the most prevalent type of dementia caused by the accumulation of amyloid beta (Aβ) peptides. The extracellular deposition of Aβ peptides in human AD brain causes neuronal death. Therefore, it has been found that Aβ peptide degradation is a possible therapeutic target for AD. CathD has been known to breakdown amyloid beta peptides. However, the structural role of CathD is not yet clear. Hence, for the purpose of gaining a deeper comprehension of the structure of CathD, the present computational investigation was performed using virtual screening technique to predict CathD's active site residues and substrate binding mode. Ligand-based virtual screening was implemented on small molecules from ZINC database against crystal structure of CathD. Further, molecular docking was utilised to investigate the binding mechanism of CathD with substrates and virtually screened inhibitors. Localised compounds obtained through screening performed by PyRx and AutoDock 4.2 with CathD receptor and the compounds having highest binding affinities were picked as; ZINC00601317, ZINC04214975 and ZINCC12500925 as our top choices. The hydrophobic residues Viz. Gly35, Val31, Thr34, Gly128, Ile124 and Ala13 help stabilising the CathD-ligand complexes, which in turn emphasises substrate and inhibitor selectivity. Further, MM-GBSA approach has been used to calculate binding free energy between CathD and selected compounds. Therefore, it would be beneficial to understand the active site pocket of CathD with the assistance of these discoveries. Thus, the present study would be helpful to identify active site pocket of CathD, which could be beneficial to develop novel therapeutic strategies for the AD.

The Role of PNPLA3_rs738409 Gene Variant, Lifestyle Factors, and Bioactive Compounds in Nonalcoholic Fatty Liver Disease: A Population-Based and Molecular Approach towards Healthy Nutrition.

This study aimed to investigate the impact of a common non-synonymous gene variant (C>G, rs738409) in patatin-like phospholipase domain-containing 3 (PNPLA3), leading to the substitution of isoleucine with methionine at position 148 (PNPLA3-I148M), on susceptibility to nonalcoholic fatty liver disease (NAFLD) and explore potential therapeutic nutritional strategies targeting PNPLA3. It contributed to understanding sustainable dietary practices for managing NAFLD, recently referred to as metabolic-dysfunction-associated fatty liver. NAFLD had been diagnosed by ultrasound in a metropolitan hospital-based cohort comprising 58,701 middle-aged and older Korean individuals, identifying 2089 NAFLD patients. The interaction between PNPLA3 and lifestyle factors was investigated. In silico analyses, including virtual screening, molecular docking, and molecular dynamics simulations, were conducted to identify bioactive compounds from foods targeting PNPLA3(I148M). Subsequent cellular experiments involved treating oleic acid (OA)-exposed HepG2 cells with selected bioactive compounds, both in the absence and presence of compound C (AMPK inhibitor), targeting PNPLA3 expression. Carriers of the risk allele PNPLA3_rs738409G showed an increased association with NAFLD risk, particularly with adherence to a plant-based diet, avoidance of a Western-style diet, and smoking. Delphinidin 3-caffeoyl-glucoside, pyranocyanin A, delta-viniferin, kaempferol-7-glucoside, and petunidin 3-rutinoside emerged as potential binders to the active site residues of PNPLA3, exhibiting a reduction in binding energy. These compounds demonstrated a dose-dependent reduction in intracellular triglyceride and lipid peroxide levels in HepG2 cells, while pretreatment with compound C showed the opposite trend. Kaempferol-7-glucoside and petunidin-3-rutinoside showed potential as inhibitors of PNPLA3 expression by enhancing AMPK activity, ultimately reducing intrahepatic lipogenesis. In conclusion, there is potential for plant-based diets and specific bioactive compounds to promote sustainable dietary practices to mitigate NAFLD risk, especially in individuals with genetic predispositions.

Synthesis, structural characterization, and quantum chemical study of the 7-acetyl-5-nitrobenzofurans as anticancer agents with antioxidant properties

Synthesis of 7-acetyl-2-aryl-5-nitrobenzofurans 2a–j involved sequential Sonogashira cross-coupling of 2-hydroxy-3-iodo-5-nitroacetophenone with terminal acetylenes and in situ Cacchi-type cycloisomerization of 5-alkynylated intermediates. Hirshfeld's fingerprint diagrams and surface analysis showed varied intermolecular interactions. Additionally, intramolecular interactions were examined using DFT and QTAIM calculations. Benzofuran derivatives were tested for antiproliferative efficacy in vitro against MDA-MB-231 and Caski cell lines. The 1-(5-nitro-2-(p-tolyl)benzofuran-7-yl)ethan-1-one 2h showed promising antiproliferative activity against Caski and MDA-MB-231 cell lines, with IC50 values of 26.96 ± 4.97 µM and 5.13 ± 0.86 µM, respectively, compared to Camptothecin (IC50 = 8.96 ± 0.23 and 24.69 ± 0.26 µM). Derivatives with 2-(4-fluorophenyl)-2c, 2-(4-methoxyphenyl)- 2g, and 2-(3,5-bis(trifluoromethyl)phenyl)-2j groups showed significant antioxidant activity, with Fe-reducing power values of 520.14 ± 1.34 µM, 433.50 ± 1.30 µM, and 466.08 ± 1.91 µM, respectively, compared to ascorbic acid (IC50 = 538.05 µM). The most active molecules that docked with protease (2c, 2g, and 2j) and FKBP12-mTOR (2d, 2h and 2j) active site residues showed significant interactions which were confirmed by Molecular dynamic simulation. ADME investigation indicated drug-like characteristics, safe in silico toxicity, and no carcinogenic or mutagenicity.

Two novel angiotensin-converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV) inhibiting peptides from tilapia (Oreochromis mossambicus) skin and their molecular docking mechanism.

In the study, papain was used to hydrolyze tilapia (Oreochromis mossambicus) skin to obtain a tilapia skin hydrolysate (TSH) with dual angiotensin-converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV) inhibitory activities. The resulting TSH was sequentially fractionated by ultrafiltration, size exclusion separation chromatography, and reverse-phase high-performance liquid chromatography. Its inhibitory effects on ACE and DPP-IV were determined by commercial reagent kits. Two peptides purified from TSH were identified as Gly-Pro-Leu-Gly-Ala-Leu (GPLGAL) and Lys-Pro-Ala-Gly-Asn (KPAGN) by the ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Inhibitory concentration (IC50) of GPLGAL on ACE and DPP-IV were 117.20±1.69 and 187.10±2.75µM, respectively. IC50 of KPAGN on ACE and DPP-IV were 137.40±2.33 and 259.20±2.85µM, respectively. The molecular simulation demonstrated that the binding affinities of GPLGAL to ACE and DPP-IV proteins were -8.5 and -7.4kcal/mol, respectively, whereas those of KPAGN to ACE and DPP-IV proteins were -7.9 and -6.7kcal/mol, respectively. GPLGAL interacted with 21 amino acid residues of the ACE active site, whereas KPAGN engaged with 19 amino acid residues. Additionally, GPLGAL interacted with 10 amino acid residues of the DPP-IV active site, whereas KPAGN engaged with 13 amino acid residues. The two peptides predominantly occupied the active sites of ACE (His513, Tyr523, and Ala354) and DPP-IV (Tyr662 and Arg125) through hydrogen bonding. This leads to the deactivation of ACE and DPP-IV. PRACTICAL APPLICATION: Accelerate tilapia skin development and high-value utilization; provide foundation for preparing the peptides with dual ACE and DPP-IV inhibiting activity.