Active Pocket Research Articles

A Comprehensive Evaluation of a Coumarin Derivative and Its Corresponding Palladium Complex as Potential Therapeutic Agents in the Treatment of Gynecological Cancers: Synthesis, Characterization, and Cytotoxicity

Background: The aim of this research is the synthesis and characterization of coumarin-palladium complex and the investigation of the cytotoxicity of both the ligand and the complex. Methods: The palladium( II) complex (CC) was obtained in the reaction between (E)-3-(1-((4-hydroxy-3-methoxyphenyl)amino)ethylidene)-2,4-dioxochroman-7-yl-acetate (CL) and potassium-tetrachloropalladate(II) and characterized using IR and NMR spectra, experimentally and theoretically. Cytotoxicity of CL and CC were determined for human cervical carcinoma HeLa, ovarian cancer A2780, hormone dependent breast cancer MCF7, and colorectal cancer HCT116 lines. The interaction of investigated compounds with HSA was followed by spectrofluorimetric method. The binding mechanism in the active pocket was assessed via molecular docking simulations. Results: A low mean absolute error between experimental and theoretical data proved that the optimized structure corresponded to the experimental one. Both compounds showed a satisfactory selectivity index towards neoplastic cells. The binding affinity of tested compounds to the HSA were confirmed. The molecular docking showed a much lower change in the Gibbs free energy of binding for CC compared to CL. Conclusions: The obtained results revealed that CL and CC exhibit significant effects on several cancer cell lines and good binding properties to HSA, while molecular docking discovered that CC has the most pronounced activity against alpha-fetoprotein.

Identification of novel inhibitors targeting PI3Kα via ensemble-based virtual screening method, biological evaluation and molecular dynamics simulation.

PIK3CA gene encoding PI3K p110α is one of the most frequently mutated and overexpressed in majority of human cancers. Development of potent and selective novel inhibitors targeting PI3Kα was considered as the most promising approaches for cancer treatment. In this investigation, a virtual screening platform for PI3Kα inhibitors was established by employing machine learning methods, pharmacophore modeling, and molecular docking approaches. 28 potential PI3Kα inhibitors with different scaffolds were selected from the databases with 295,024 compounds. Among the 28 hits, hit15 exhibited the best inhibitory effect against PI3Kα with IC50 value less than 1.0µM. The molecular dynamics simulation indicated that hit15 could stably bind to the active site of PI3Kα, interact with some residues by hydrophobic, electrostatic and hydrogen bonding interactions, and finally induced PI3Kα active pocket substantial conformation changes. Stable H-bond interactions were formed between hit15 and residues of Lys776, Asp810 and Asp933. The binding free energy of PI3Kα-hit15 was -65.3kJ/mol. The free energy decomposition indicated that key residues of Asp805, Ile848 and Ile932 contributed stronger energies to the binding free energy. The above results indicated that hit15 with novel scaffold was a potent PI3Kα inhibitor and considered as a promisingcandidateforfurther drugdevelopment to treat various cancers with PI3Kα over activated.

Characterization of Four Carboxylesterases Involved in Detoxification of β-Cypermethrin, λ-Cyhalothrin, and Malathion in Helicoverpa armigera.

Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) is a globally devastating pest and has evolved with varying levels of resistance to a broader spectrum of insecticides. CarEs are major enzymes involved in insecticide detoxification and metabolic resistance. Four CarE genes were identified and cloned from H. armigera, and the expression pattern analyses indicated that they were predominantly expressed in the detoxifying tissues and larval feeding periods. The insecticide inductive assays further suggested that these CarE genes with expressions were significantly upregulated after beta-cypermethrin, lambda-cyhalothrin, malathion, and chlorpyrifos exposures. The purified recombinant proteins of both CarE016B and CarE016C by bacterial expression exhibited significantly higher catalytic efficiency toward α-naphthyl acetate and also showed relatively stronger binding with both β-cypermethrin and λ-cyhalothrin compared to the CarE006C and CarE015A. In vitro metabolism assay with HPLC suggests that CarE016B and CarE016C have the ability to metabolize β-cypermethrin, λ-cyhalothrin, and malathion with varying hydrolase activities. GC-MS/MS analysis identified 3-phenoxybenzaldehyde (3-PBAld) as the metabolite of both β-cypermethrin and λ-cyhalothrin. Homology modeling and molecular docking analyses further indicate that these three types of insecticides could be anchored into the active pocket of both CarEs. Collectively, these results demonstrate that both CarE016B and CarE016C play a critical role in the detoxification of pyrethroid and organophosphate insecticides in H. armigera. This study provides the foundations for a comprehensive understanding of the role of the CarEs family in insecticide detoxification and resistance in H. armigera.

Target-centric analysis of hepatitis B: identifying key molecules and pathways for treatment

Hepatitis B virus (HBV) poses a significant global health challenge, potentially leading to severe liver conditions, with currently limited effective treatment options available. Xiao-Chai-Hu-Tang (XCHT), a well-known Traditional Chinese Medicine (TCM) prescription, shows promise in clinical trials for treating HBV. Therefore, screening the complex components of XCHT, identifying the active compounds, and closely exploring the targets associated with hepatitis B may constitute an effective strategy for the development of new therapeutic drugs for the treatment of this disease. A systematic pharmacology and GEO chip analysis identified key targets and pathways for hepatitis B treatment and effective ingredients. Molecular docking and molecular dynamics simulation techniques were used to explore the affinity and stability of active compounds with core targets, while assessing the druggability and safety of the active compounds. The therapeutic effect of the active compound protoporphyrin in XCHT on hepatitis B were mediated through key targets such as AKT1, MAPK1, and LCK, as well as key signaling pathways like PI3K-Akt signaling pathway and Ras signaling pathway. Protoporphyrin effectively bond to active pockets of core targets and demonstrated favorable druggability and a high safety threshold. The study provided valuable insights into the development of effective treatments for hepatitis B.

L858R/L718Q and L858R/L792H Mutations of EGFR Inducing Resistance Against Osimertinib by Forming Additional Hydrogen Bonds.

Acquired resistance to first-line treatments in various cancers both promotes cancer recurrence as well as limits effective treatment. This is true for epidermal growth factor receptor (EGFR) mutations, for which secondary EGFR mutations are one of the principal mechanisms conferring resistance to the covalent inhibitor osimertinib. Thus, it is very important to develop a deeper understanding of the secondary mutational resistance mechanisms associated with EGFR mutations arising in tumors treated with osimertinib to expedite the development of innovative therapeutic drugs to overcome acquired resistance. This work uses all-atom molecular dynamics (MD) simulations to investigate the conformational variation of two reported EGFR mutants (L858R/L718Q and L858R/L792H) that resist osimertinib. The wild-type EGFR kinase domain and the L858R mutant are used as the reference. Our MD simulation results revealed that both the L718Q and L792H secondary mutations induce additional hydrogen bonds between the residues in the active pocket and the residues with the water molecules. These additional hydrogen bonds reduce the exposure area of C797, the covalent binding target of osimertinib. The additional hydrogen bonds also influence the binding affinity of the EGFR kinase domain by altering the secondary structure and flexibility of the amino acid residues in the domain. Our work highlights how the two reported mutations may alter both residue-residue and residue-solvent hydrogen bonds, affecting protein binding properties, which could be helpful for future drug discovery.

Design, Synthesis, and Docking Study of Potentially Active Antimicrobial Pyrazolo[1,5‐a]pyrimidine Derivatives

AbstractCompound 4,4′‐(1,4‐phenylenebis(thiazole‐4,2‐diyl))bis(1H‐pyrazol‐5‐amine) (5) was used in the preparation of novel pyrazolo[1,5‐a]pyrimidines incorporating a thiazole moiety (6, 7b, 8, 9, and 13–18). the reactions were described via the following reactions: acetylacetone, acetanilide, diethyl malonate, ethyl cyanoacetate, 3‐(dimethylamino)‐1‐phenylprop‐2‐en‐1‐one, 2‐((dimethylamino)methylene)‐3‐oxopentane‐nitrile, 3‐(dimethylamino)‐1‐(thiophen‐3‐yl)prop‐2‐en‐1‐one, 2‐((dimethylamino)methylene)‐5,5‐dimethylcyclo‐hexane‐1,3‐dione, dimethylformamide‐dimethylacetal, and with a mixture of p‐nitro benzaldehyde/cyclohexanone, respectively. Gram‐positive bacteria B. subtilis and B. thuringiensis, as well as Gram‐negative bacteria E. coli and P. aeruginosa, were tested for the newly synthesized compounds' in vitro antibacterial activity. Their in vitro antifungal activity against fungus strains B. Fabae and F. oxysporum was also assessed. The compounds 16–18 had the strongest activity against Gram‐positive bacteria, with MIC values = 3.125 µg/mL against B. subtilis, which is equipotent to the drug reference Chloramphenicol, and MIC values = 6.25 µg/mL against B. thuringiensis, which is equipotent to the drug reference Cephalothin. Compounds (13–18) were shown to be superimposable to CNB based on their docking results, with the exception of derivative 18. Furthermore, they demonstrated a strong binding mode with DNA gyrase B's active pocket (PDB: 1KZN) by forming several contacts with the enzyme's essential amino acids in a way that was comparable to CNB.

Structure-guided design and photochemical synthesis of new carbamo(dithioperoxo)thioates with improved potencies to SARS-CoV-2 3CLpro

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has triggered a protracted global pandemic from 2019 to 2022, and posed a significant threat to human health. One of the non-structural proteins 3CLpro of SARS-CoV-2 is considered as a validated target for the development of inhibitors against the virus. Disulfiram has been reported as a covalent inhibitor of 3CLpro; however, its structure lacks bonding site with active pockets of 3CLpro and its highly symmetric structure doesn’t match well with the irregular cavity of the active center, limiting its therapeutic applications. To enhance their affinity for the 3CLpro target, in this study, two kinds of disulfiram derivatives, designed based on the reevaluation and optimization of disulfiram, have been synthesized through photoredox chemistry, and the novel carbamo(dithioperoxo)thioates 4g-m were found to display 5–17 folds potency against SARS-CoV-2 3CLpro compared to the parent disulfiram, with resulting half-maximal inhibitory concentration (IC50) values ranging from 0.14–0.47 μM. Carbamo(dithioperoxo)thioates 4i containing a 4-hydroxy piperidine and a 4-trifluoromethyl phenyl ring, was identified as the most potent inhibitor (IC50 = 0.14 μM). Furthermore, molecular dynamics simulations, binding free energy analysis and mass analysis were performed and provided insights on the stability, conformational behavior, and interactions of 4g with 3CLpro. The green synthetic methodology, the privileged carbamo(dithioperoxo)thioate scaffold, and the molecular mechanisms presented might serve as a useful system for the further discovery of highly potent inhibitors targeting SARS-CoV-2 3CLpro.

Discovery of potential FDA-approved SARS-CoV-2 Papain-like protease inhibitors: A multi-phase in silico approach

Papain-like protease (PLpro) is a crucial enzyme for SARS-CoV-2 replication and immune evasion. Inhibiting PLpro could be a promising strategy to fight against COVID-19. This study aimed to identify potent inhibitors of PLpro among FDA-approved drugs using an in silico approach. The study also aimed to examine and confirm the binding of the selected compounds to the active pocket of PLpro using a multi-phased in silico approach, involving the screening of 3009 FDA-approved drugs to pinpoint the most similar compounds to, TTT, the co-crystallized ligand TTT of PLpro. The selected compounds were subjected to further analysis, including molecular docking, molecular dynamics simulations, MM-GPSA (molecular mechanics generalized Born surface area), and PLIP (Protein-Ligand Interaction Profiler) studies, to examine and confirm their binding to the active pocket of PLpro. Seven candidates (Vismodegib, Celecoxib, Ketoprofen, Indomethacin, Naphazoline, Valdecoxib, and Eslicarbazepine) showed promising in silico activities against the PLpro. The computational analysis confirmed the binding of Celecoxib to the active pocket of PLpro, suggesting its potential in the fight against COVID-19. This study identified seven FDA-approved drugs as potential inhibitors of PLpro, providing a feasible approach for drug repurposing against COVID-19. The results obtained from the in silico approach hold promise, but further in vitro and in vivo studies are warranted to validate the potential of these compounds.

Anti colorectal cancer activity and in silico studies of novel pyridine nortopsentin analog as cyclin dependent kinase 6 inhibitor

Nortopsentins are a vital class of deep-sea sponge metabolites which can be used as leads for antitumor agents. Although their action has been studied in several diseases’ contexts, their cytotoxic activity against colorectal carcinoma has not yet been fully investigated. Therefore, a series of 2,6-bis(1H-indol-3-yl)-4-(substituted-phenyl)pyridin-5-carbonitriles 4a–j (nortopsentin analogs) was investigated for their cytotoxic activity against colorectal carcinoma. The analog 4i showed the highest antitumor activity via inducing cell cycle arrest at G1 phase. Cell cycle arrest was induced due to expression downregulation of CDK2, CDK4, and CDK6. In addition, 4i suppressed the enzymatic activity of CDK6. The theoretical study of some basic quantum factors and the geometric shape of compound 4i proved that the compound is stable and a soft molecule, in which the EHOMO and ELUMO energies were negative and had a small ∆E gap. 4i also demonstrated a high potential for oral bioavailability due to its adherence to Lipinski’s rule of five. The molecular docking studies of 4i analog showed good binding mode with CDK6 active pocket through the formation of multiple interactions with its key amino acids.

Identifying Novel Inhibitors for Dengue NS2B-NS3 Protease by Combining Topological similarity, Molecular Dynamics, MMGBSA and SiteMap Analysis.

DENV NS2B-NS3 protease inhibitors were designed based upon the reference molecule, 4-(1,3-dioxoisoindolin-2-yl)-N-(4-ethylphenyl) benzenesulfonamide, reported by our team with the aim to optimize lead compound via rational approach. Top five best scoring molecules with zinc ids ZINC23504872, ZINC48412318, ZINC00413269, ZINC13998032 and ZINC75249613 bearing 'pyrimidin-4(3H)-one' basic scaffold have been identified as a promising candidate against DENV protease enzyme. The shape and electrostatic complementary between identified HITs and reference molecules were found to be Tanimotoshape 0.453, 0.690, 0.680, 0.685 & 0.672 respectively and Tanimotoelectrostatic 0.211, 0.211, 0.441, 0.442, 0.442 and 0.442 respectively. The molecular docking studies suggested that the identified HITs displayed the good interactions with active site residues and lower binding energies. The stability of docked complexes was assessed by MD simulations studies. The RMSD values of protein backbone (1.6779, 3.1563, 3.3634, 3.3893 & 3.0960 Å) and protein backbone RMSF values (1.0126, 1.0834, 1.0890, 0.9974 & 1.0080 Å respectively) for all top five HITs were stable and molecules did not fluctuate from the active pocket during entire 100ns MD run. The druggability Dscore below 1 indicate the tightly binding of ligand at the active site. Dscore for ZINC23504872 was found to be 1.084 while for the second class of compounds ZINC48412318, ZINC00413269, ZINC13998032 and ZINC75249613, 0.503, 0.484, 0.487 and 0.501 Dscores were observed. In-silico ADMET calculations suggested that all five HITs were possessed the drug likeliness properties and did not violate the Lipinski's rule of five. Summing up, these in-silico generated data suggested that the identified molecules bearing pyrimidin-4(3H)-one would be promising scaffold for DENV protease inhibitors. However, experimental results are needed to prove the obtained results.

A Lipase Gene of Thermomyces lanuginosus: Sequence Analysis and High-Efficiency Expression in Pichia pastoris.

Lipase, a type of enzyme that decomposes and synthesizes triglycerides, plays an important role in lipid processing. In this study, a heat-resisting lipase gene (lip4) from Thermomyces lanuginosus was subcloned into the pPICZαA vector and then transformed into Pichia pastoris X33. The recombinant yeast cell concentration reached the maximum (119.5 g/L) at 144 h, and the lipase (Lip4) activity reached the maximum (3900 U/mL) at 168 h in 10 L bioreactor. Through bioinformatics analysis, S168, as the key site of Lip4, participated in the formation of the catalytic triads S168-D223-H280 and G166-H167-S168-L169-G170. Furthermore, S168 and seven conserved amino acids of G104/288, S105, A195, P196, V225 and I287 constitute the active center of Lip4. Specifically, the structure modeling showed two α-helices of the lid domain, outside the active pocket domain, controlling the entry of the substrate on Lip4. The potential glycosylation of Asn-33 may be involved in exhibiting the high stable temperature for lipase activity. Therefore, the eukaryotic system was constructed to express Lip4 efficiently, and the amino acid sites related to the catalytic efficiency of Lip4 were clarified, providing a new way for its subsequent property research and industrial application.

Deep quantification of substrate turnover defines protease subsite cooperativity.

Substrate specificity determines protease functions in physiology and in clinical and biotechnological applications, yet quantitative cleavage information is often unavailable, biased, or limited to a small number of events. Here, we develop qPISA (quantitative Protease specificity Inference from Substrate Analysis) to study Dipeptidyl Peptidase Four (DPP4), a key regulator of blood glucose levels. We use mass spectrometry to quantify >40,000 peptides from a complex, commercially available peptide mixture. By analyzing changes in substrate levels quantitatively instead of focusing on qualitative product identification through a binary classifier, we can reveal cooperative interactions within DPP4's active pocket and derive a sequence motif that predicts activity quantitatively. qPISA distinguishes DPP4 from the related C. elegans DPF-3 (a DPP8/9-orthologue), and we relate the differences to the structural features of the two enzymes. We demonstrate that qPISA can direct protein engineering efforts like the stabilization of GLP-1, a key DPP4 substrate used in the treatment of diabetes and obesity. Thus, qPISA offers a versatile approach for profiling protease and especially exopeptidase specificity, facilitating insight into enzyme mechanisms and biotechnological and clinical applications.

Molecular Engineering L-Aspartate-Alpha-Decarboxylase to Enhance Catalytic Stability and Performance.

L-aspartate-alpha-decarboxylase (ADC) catalyzes the decarboxylation of L-aspartate to produce β-alanine, which is the decisive step in the biosynthesis of β-alanine. However, the low catalytic stability and efficiency of ADC limit its industrial applications. In this study, a variant of ADC from Bacillus subtilis were used as a starting point for engineering. After constructing a random mutagenesis library by error-prone PCR, followed by high-throughput screening,four substitutions (S7 N, K63 N, A99T, and K113R) were identified. By screening saturation mutagenesis libraries on these positions and computational analysis, two recombined variants N3(S7 N/K63 N/I88 M/A99E/K113R/I126*) and Y1(S7Y/K63 N/I88 M/A99E/K113R/I126*) with improved performance were obtained. Compared to the wild type, the catalytic efficiency and catalytic stability of the best two variants were enhanced up to 95 %(variant N3) and up to 89 %(variant Y1), respectively. In addition, Y1 exhibited 3.37 times improved half-life and 2-fold improved total turnover number. Hydrophilicity analysis and molecular dynamics (MD) simulation revealed that the increased hydrophilicity and steric hindrance of key amino acid residues would affect the catalytic activity and stability. The improved catalytic performance of the variants could be attributed to their enhanced binding capacity to the substrate within the active pocket and the alleviation of mechanism-based inactivation.

Mitochondrial resilience and antioxidant defence against HIV-1: unveiling the power of Asparagus racemosus extracts and Shatavarin IV.

Asparagus racemosus (AR), an Ayurvedic botanical, possesses various biological characteristics, yet its impact on HIV-1 replication remains to be elucidated. This study aimed to investigate the inhibitory effects of AR root extracts and its principal bioactive molecule, Shatavarin IV (Shatavarin), on HIV-1 replication and their role in mitigating mitochondrial dysfunction during HIV-1 infection, utilizing both in vitro and in silico methodologies. The cytotoxicity of the extracts was evaluated using MTT and ATPlite assays. In vitro anti-HIV-1 activity was assessed in TZM-bl cells against X4 and R5 subtypes, and confirmed in peripheral blood mononuclear cells using HIV-1 p24 antigen capture ELISA and viral copy number assessment. Mechanistic insights were obtained through enzymatic assays targeting HIV-1 Integrase, Protease and Reverse Transcriptase. Shatavarin's activity was also validated via viral copy number and p24 antigen capture assays, along with molecular interaction studies against key HIV-1 replication enzymes. HIV-1 induced mitochondrial dysfunction was evaluated by detecting mitochondrial reactive oxygen species (ROS), calcium accumulation, mitochondrial potential, and caspase activity within the infected cells. Non-cytotoxic concentrations of both aqueous and hydroalcoholic extracts derived from Asparagus racemosus roots displayed dose-dependent inhibition of HIV-1 replication. Notably, the hydroalcoholic extract exhibited superior Reverse Transcriptase activity, complemented by moderate activity observed in the Protease assay. Molecular interaction studies revealed that Shatavarin IV, the key bioactive constituent of AR, formed hydrogen bonds within the active binding pocket site residues crucial for HIV replication enzyme catalysis, suggesting its potential in attenuating HIV-1 infection. Mitochondrial dysfunction induced by HIV-1 infection, marked by increased oxidative stress, mitochondrial calcium overload, loss of mitochondrial membrane potential, and elevated caspase activity, was effectively mitigated by treatment with AR extracts and Shatavarin IV. These findings underscore the potential of AR extracts and Shatavarin IV as antiviral agents, while enhancing mitochondrial function during HIV-1 infection. In conclusion, Asparagus racemosus extracts, particularly Shatavarin IV, demonstrate promising inhibitory effects against HIV-1 replication while concurrently ameliorating mitochondrial dysfunction induced by the virus. These findings suggest the therapeutic potential of AR extracts and Shatavarin in combating HIV-1 infection and improving mitochondrial health.

In vitro and In Silico Molecular Modeling Studies of Newly Synthesized Pyrrole Derivatives for their Antimicrobial and Anticancer Properties

Background: Pyrroles are biologically active scaffolds that can have a number of different effects. They also have unique pharmacophores inside their ring system that make it easier to make molecules that are more active. Significantly, in the past ten years, research has been conducted on the anti-bacterial properties, with a particular emphasis on drug-resistant Gram-positive and Gram-negative pathogens such as mycobacteria. Additionally, scaffolds based on pyrroles were utilized in the production of anti-tumor medicines that function by modulating or suppressing genes. Objective: This research aimed to create new pyrrole derivatives by chemical means and evaluate their antimicrobial and anticancer properties. Methods: By reacting diverse 1H-pyrrole-2-carbohydrazide derivatives with benzaldehyde under normal conditions, a number of pyrrole variations were created. IR, mass spectroscopy, NMR, and elemental analysis were used to characterize the synthesized compounds. The serial dilution method was used to assess antimicrobial activity, along with a molecular docking study against the enzyme α-topoisomerase II (α-Topo II), which effectively controls the topology of DNA. This enzyme is strongly expressed in rapidly dividing cells and is crucial for transcription, replication, and chromosome structure. Pyrrole and its synthetic derivatives are known to exhibit strong anticancer activity by specifically targeting α-Topo II, which has led multiple researchers to investigate α-Topo II inhibitors as potential anticancer medicines. Consequently, designing pyrroline derivatives is interesting since the succinimide portion of the joined heteroaromatic molecule can selectively engage with the ATP binding pocket via the hydrogen bond network. Newer synthesized compounds were also docked via Schrodinger 2022-4 into the active pocket of the three-dimensional crystallographic structure of the ATP site of human topoisomerase IIα (htopo IIα) (PDB ID: 1zxm). Results: Among the newly synthesized pyrrole derivatives, compounds demonstrated significant anti- microbial activity. In addition, the AutoDock Vina application was used to perform in-silico docking computations. Compounds 1a and 1h were found to have a stronger antiproliferative effect than compounds against MCF-07 breast cancer cell linen our study, ligands 1a and 1b exhibited better binding energies of -5.049 and -5.035 kcal/mol, respectively. Most of the synthesized pyrrole derivatives showed promising antiproliferative effects; however, further in vivo investigations are needed to confirm or refute these results. Conclusion: The results of this investigation show that pyrrole derivatives have the potential to be effective antimicrobial and anticancer agents. While resolving safety issues, the structural alterations carried out in this study enhanced the compounds' medicinal capabilities. To clarify the underlying mechanisms of action and enhance the pharmacological characteristics of these new pyrrole derivatives, further research is necessary.

Synthesis, C-N/N-N bond conformational analysis and evaluation of naphtho[2,3-d][1,2,3]triazole-4,9-dione tethered N-acyl hydrazones as α-amylase inhibitors: Insights from molecular modeling and ADMET analysis

In an effort to expand the repertoire of potent α-amylase inhibitors, we sought to develop novel inhibitors by combining 1,4-naphthoquinone, 1,2,3-triazole, and N-acyl hydrazone scaffolds in a single matrix. To achieve this, twelve novel naphtho[2,3-d][1,2,3]triazole-4,9‑dione tethered N-acyl hydrazones were synthesized through condensation reaction of 2-(4,9-dioxo-4,9-dihydro-1H-naphtho[2,3-d][1,2,3]triazol-1-yl)acetohydrazide with various substituted aryl aldehydes. Structural elucidation for all the compounds was performed using 1D, 2D-NMR, FTIR, and mass spectral analyses. The synthesized molecules were evaluated for their ability to inhibit α-amylase activity using acarbose as the standard drug. All the derivatives exhibited potent inhibition of α-amylase, with IC50 values ranging between 17.26 ± 0.07 to 25.62 ± 0.03 μg/mL. Notably, compound 9c possessing –meta substituted –NO2 group displayed the highest activity (IC50 = 17.26 ± 0.07 μg/mL) among the series. Structure-activity relationship (SAR) revealed the pivotal role of aryl ring substitutions in determining inhibitory efficacy. To validate these findings and to assess the binding stability of 9c within the catalytic site α-amylase dervied for A. oryzae (PDB ID:7TAA), in silico studies were performed. The compound 9c effectively occupies the enzyme's active pocket, with minimal RMSD fluctuations observed over 100 ns simulation, indicating stable protein-ligand complex. ADMET predictions suggested favorable drug-like properties, underscoring the potential of these compounds as novel α-amylase inhibitors for managing type 2 diabetes mellitus

Intricate Evolution of Multifunctional Lipoxygenase in Red Algae.

Lipoxygenases (LOXs) from lower organisms have substrate flexibility and function versatility in fatty acid oxidation, but it is not clear how these LOXs acquired the ability to execute multiple functions within only one catalytic domain. This work studied a multifunctional LOX from red alga Pyropia haitanensis (PhLOX) which combined hydroperoxidelyase (HPL) and allene oxide synthase (AOS) activity in its active pocket. Molecular docking and site-directed mutagenesis revealed that Phe642 and Phe826 jointly regulated the double peroxidation of fatty acid, Gln777 and Asn575 were essential to the AOS function, and the HPL activity was improved when Asn575, Gln777, or Phe826 was replaced by leucine. Phylogenetic analysis indicated that Asn575 and Phe826 were unique amino acid sites in the separated clades clustered with PhLOX, whereas Phe642 and Gln777 were conserved in plant or animal LOXs. The N-terminal START/RHO_alpha_C/PITP/Bet_v1/CoxG/CalC (SRPBCC) domain of PhLOX was another key variable, as the absence of this domain disrupted the versatility of PhLOX. Moreover, the functions of two homologous LOXs from marine bacterium Shewanella violacea and red alga Chondrus crispus were examined. The HPL activity of PhLOX appeared to be inherited from a common ancestor, and the AOS function was likely acquired through mutations in some key residues in the active pocket. Taken together, our results suggested that some LOXs from red algae attained their versatility by amalgamating functional domains of ancestral origin and unique amino acid mutations.

Mining unique cysteine synthetases and computational study on thoroughly eliminating feedback inhibition through tunnel engineering.

L-cysteine is an essential component in pharmaceutical and agricultural industries, and synthetic biology has made strides in developing new metabolic pathways for its production, particularly in archaea with unique O-phosphoserine sulfhydrylases (OPSS) as key enzymes. In this study, we employed database mining to identify a highly catalytic activity OPSS from Acetobacterium sp. (AsOPSS). However, it was observed that the enzymatic activity of AsOPSS suffered significant feedback inhibition from the product L-cysteine, exhibiting an IC50 value of merely 1.2 mM. A semi-rational design combined with tunnel analysis strategy was conducted to engineer AsOPSS. The best variant, AsOPSSA218R was achieved, totally eliminating product inhibition without sacrificing catalytic efficiency. Molecular docking and molecular dynamic simulations indicated that the binding conformation of AsOPSSA218R with L-cys was altered, leading to a reduced affinity between L-cysteine and the active pocket. Tunnel analysis revealed that the AsOPSSA218R variant reshaped the landscape of the tunnel, resulting in the construction of a new tunnel. Furthermore, random acceleration molecular dynamics simulation and umbrella sampling simulation demonstrated that the novel tunnel improved the suitability for product release and effectively separated the interference between the product release and substrate binding processes. Finally, more than 45 mM of L-cysteine was produced in vitro within 2 h using the AsOPSSA218R variant. Our findings emphasize the potential for relieving feedback inhibition by artificially generating new product release channels, while also laying an enzymatic foundation for efficient L-cysteine production.

ACE inhibitory effect and saltiness-enhancing properties of chicken-derived umami peptides: Digestive stability, inhibition kinetics, multiple ligand docking and central composite design

Angiotensin-I-converting enzyme (ACE) inhibitory activity and saltiness-enhancing properties of chicken-derived umami peptides were investigated. DGGRYY and NEFGYSNR were screened and the IC50 values were 28.71 μM and 283.24 μM, indicating their potential as novel ACE inhibitors. DGGRYY and NEFGYSNR have good pH and thermal stability. After gastrointestinal digestion, the ACE-inhibitory activity of DGGRYY retained about 53 %, whereas NEFGYSNR retained about 57 %. The inhibition pattern of both peptides was determined to be uncompetitive, consisting with the result of multiple ligand docking that the binding sites were outside the ACE active pocket. Trp59, Tyr62, Asp121, Arg124, and Ser516 were the key binding sites that contributed to the total binding energy. In addition, saltiness and palatability models were established according to sensory analysis and central composite design. In 0.1 % ~ 0.3 % NaCl solutions, the addition of DGGRYY and NEFGYSNR could enhance the salty intensity and compensate for the palatability loss caused by salt reduction.

Comprehensive Analysis of NADH:Ubiquinone Oxidoreductase Subunit B3 in Gynecological Tumors and Identification of Its Natural Inhibitor Wedelolactone.

The aim of this study was to explore the role of NADH:ubiquinone oxidoreductase subunit B3 (NDUFB3) in human gynecological malignancies and to screen potential natural compounds targeting it. GEPIA and HPA databases were used to study the expression characteristics of NDUFB3. GO and KEGG enrichment analyses were performed using the R software clusterProfiler package. GSEA for NDUFB3 was performed using the LinkedOmics database. Natural compounds targeting NDUFB3 were screened by virtual screening and molecular docking. After NDUFB3 was depleted or wedelolactone treatment, cell proliferation was detected by CCK-8 assay. The production of reactive oxide species (ROS) in tumor cells was detected by dihydroethidium fluorescent probe. The cell cycle and apoptosis were evaluated by flow cytometry. It was revealed that NDUFB3 was highly expressed in ovarian cancer (OV), uterine corpus endometrial carcinoma (UCEC), and cervical squamous cell carcinoma (CESC). NDUFB3 expression was associated with multiple immunomodulators in CESC, OV, and UCEC. NDUFB3 was predicted to modulate MAPK signaling pathways in CESC, OV, and UCEC. Knocking down NDUFB3 inhibited the proliferation of CESC, OV, and UCEC cells, increased intracellular ROS production, and induced cell cycle arrest and apoptosis. Wedelolactone was a potential small molecule with a strong ability to bind with the active pocket of NDUFB3, and wedelolactone could kill CESC, OV, and UCEC cells partly via NDUFB3. In conclusion, NDUFB3 may be a potential biomarker and a new target for gynecological tumors, and wedelolactone may exert antitumor activity via targeting NDUFB3.