Cytochrome P450 enzyme (CYP)-catalyzed functional group transformations are pivotal in the biosynthesis of metabolic intermediates and products, as exemplified by the CYP-catalyzed C7-hydroxylation and the subsequent C7-C8 bond cleavage reaction responsible for the biosynthesis of the well-known antitumor monoterpene indole alkaloid (MIA) camptothecin. To determine the key amino acid residues responsible for the catalytic selectivity of the CYPs involved in MIA biosynthesis, we characterized the enzymes CYP72A728 and CYP72A729 as stereoselective 7-deoxyloganic acid 7-hydroxylases (7DLHs). We then conducted a comparative analysis of the amino acid sequences and the predicted structures of the CYP72A homologs involved in camptothecin biosynthesis, as well as those of the CYP72A homologs implicated in the pharmaceutically significant MIAs biosynthesis in Catharanthus roseus. The crucial amino acid residues for the catalytic selectivity of the CYP72A-catalyzed reactions were identified through fragmental and individual residue replacement, catalytic activity assays, molecular docking, and molecular dynamic simulations analysis. The fragments 1 and 3 of CYP72A565 were crucial for its C7-hydroxylation and C7-C8 bond cleavage activities. Mutating fragments 1 and 2 of CYP72A565 transformed the bifunctional CYP72A565 into a monofunctional 7DLH. Evolutionary analysis of the CYP72A homologs suggested that the bifunctional CYP72A in MIA-producing plants may have evolved into a monofunctional CYP72A. The gene pairs CYP72A728-CYP72A610 and CYP72A729-CYP72A565 may have originated from a whole genome duplication event. This study provides a molecular basis for the CYP72A-catalyzed hydroxylation and C-C bond cleavage activities of CYP72A565, as well as evolutionary insights of CYP72A homologs involved in MIAs biosynthesis.

Tongxie Yaofang (TXYF), a classical traditional Chinese medicine, is commonly used in China to treat ulcerative colitis (UC). The aim of this study was to integrate network pharmacology with molecular docking and molecular dynamics simulations to explore the mechanism of Tongxie Yaofang in the treatment of UC. The traditional Chinese medicine systems pharmacology database was used to retrieve the relevant chemical compositions of the herbs contained in TXYF. The DisGeNET, GeneCards, Online Mendelian Inheritance in Man, and Therapeutic Target Database databases were used to retrieve UC-related targets. To construct protein-protein interaction networks and screen for key targets, gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses of the key targets of TXYF in the treatment of UC were performed using R 4.3.2 software. AutoDock Tools 1.5.7 was used for molecular docking. Molecular dynamics simulations of protein complexes and complexes of proteins with small-molecule ligands and eutectic ligands were carried out with Gromacs 2022 software. Network pharmacology analysis revealed that TXYF could act on UC through multiple targets and pathways. It may exert therapeutic effects mainly through the AGE/RAGE, TOLL, JAK/STAT, and Th17 signaling pathways. The possible targets of TXYF in the treatment of UC could be AKT1, BCL2, EGFR, HMOX1, HSP90AA1, and TGFβ1. Molecular docking analysis revealed that AKT1 had the highest binding energy (-10.55 kcal/mol). Molecular dynamics simulations revealed that the complexes formed by the AKT1 protein and the chemical compounds MOL001910 and MOL00035 had good stability and high binding strength. AKT1 may be the most critical target of TXYF in treating UC, and the key chemical components of TXYF in treating UC may include β-sitosterol (MOL000358) and 11alpha,12alpha-epoxy-3beta-23-dihydroxy-30-norolean-20-en-28,12beta-olide (MOL00 1910). This study revealed that TXYF may exert therapeutic effects on UC through multiple targets, multiple biological functions, and multiple signaling pathways. This study provides a new insight into the pharmacological mechanism of TXYF in treating UC.

1,4-Naphthoquinone scaffold-derived compounds has shown considerable pharmacological properties against cancer, including acute myeloid leukemia (AML) However, its impact and mechanisms in AML are uncertain. In this study, the mechanisms of 1,4-naphthoquinone scaffold-derived compounds against AML were investigated via network pharmacology, molecular docking and molecular dynamics simulation. ASINEX database was used to collect the 1,4-naphthoquinone scaffold-derived compounds, and compounds were extracted from the software to evaluate their drug similarity and toxicity. The potential targets of compounds were retrieved from the SwissTargetPrediction Database and the Similarity Ensemble Approach Database, while the potential targets of AML were obtained from the GeneCards databases and Gene Expression Omnibus. The STRING database was used to construct a protein-protein interaction (PPI) network, topologically and Cyto Hubb plugin of Cytoscape screen the central targets. After selecting the potential key targets, the gene ontology (GO) function annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed for the intersection targets, and a network map of \"compounds-potential targets-pathway-disease\" were constructed. Molecular docking of the compounds with the core target was performed, and core target with the strongest binding force and 1,4-naphthoquinone scaffold-derived compounds was selected for further molecular dynamics simulation and further molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) approach verification. In addition, the Bloodspot database was applied to perform the overall survival of core targets. A total of 19 1,4-naphthoquinone scaffold-derived compounds were chosen out, and then 836 targets of compounds, 96 intersection targets of AML were screened. Core targets include STAT3, TLR4, HSP90AA1, JUN, MMP9, PTPRC, JAK2, PTGS2, KIT and CSF1R. GO functional enrichment analysis revealed that 90 biological processes, 10 cell components and 12 molecular functions were enriched while KEGG pathway enrichment analysis revealed 34 enriched signaling pathways. Analysis of KEGG enrichment hinted that these 10 core genes were located in the pathways in cancer, suggesting that 1,4-naphthoquinone scaffold-derived compounds had potential activity against AML. Molecular docking analysis revealed that the binding energies between 1,4-naphthoquinone scaffold-derived compounds and the core proteins were all higher than - 6 kcal/mol, indicating that the 10 core targets all had strong binding ability with compounds. Moreover, a good binding capacity was inferred from molecular dynamics simulations between compound 7 and MMP9. The total binding free energy calculated using the MM/GBSA approach revealed values of - 6356.865 kcal/mol for the MMP9-7 complex. In addition, Bloodspot database results exhibited that HSP90AA1, MMP9 and PTPRC were associated with overall survival. The findings provide foundations for future studies into the interaction underlying the anti-AML potential of compounds with 1,4-naphthoquinone-based scaffold structures. Compounds with 1,4-naphthoquinone-based scaffold structures exhibits considerable potential in mitigating and treating AML through multiple targets and pathways.

KRAS protein is known to be frequently mutated in various cancers. The most common mutations being at position 12, 13 and 61. The positions 12 and 13 form part of the phosphate binding region (P-loop) of KRAS. Owing to mutation, the protein remains in continuous active state and affects the normal cellular process. Understanding the structural changes owing to mutations in GDP-bound (inactive state) and GTP-bound (active state) may help in the design of better therapeutics. To understand the structural flexibility due to the mutations specifically located at P-loop regions (G12D, G12V and G13D), extensive molecular dynamics simulations (24 μs) have been carried for both inactive (GDP-bound) and active (GTP-bound) structures for the wild type and these mutants. The study revealed that the local structural changes at the site of mutations allosterically guide changes in distant regions of the protein through hydrogen bond and hydrophobic signalling network. The dynamic cross correlation analysis and the comparison of the correlated motions among different systems manifested that changes in SW-I, SW-II, α3 and the loop preceding α3 affects the interactions of GDP/GTP with different regions of the protein thereby affecting its hydrolysis. Further, the Markov state modelling analysis confirmed that the mutations, especially G13D imparts rigidity to structure compared to wild type and thus limiting its conformational state in either intermediate state or active state. The study suggests that along with SW-I and SW-II regions, the loop region preceding the α3 helix and α3 helix are also involved in affecting the hydrolysis of nucleotides and may be considered while designing therapeutics against KRAS.

Human neutrophil elastase (HNE) plays a key role in initiating inflammation in the cardiopulmonary and systemic contexts. Pathological auto-proteolysed two-chain (tc) HNE exhibits reduced binding affinity with inhibitors. Using AutoDock Vina v1.2.0, 66 flavonoid inhibitors, sivelestat and alvelestat were docked with single-chain (sc) HNE and tcHNE. Schrodinger PHASE v13.4.132 was used to generate a 3D-QSAR model. Molecular dynamics (MD) simulations were conducted with AMBER v18. The 3D-QSAR model for flavonoids with scHNE showed r2 = 0.95 and q2 = 0.91. High-activity compounds had hydrophobic A/A2 and C/C2 rings in the S1 subsite, with hydrogen bond donors at C5 and C7 positions of the A/A2 ring, and the C4\' position of the B/B1 ring. All flavonoids except robustaflavone occupied the S1\'-S2\' subsites of tcHNE with decreased AutoDock binding affinities. During MD simulations, robustaflavone remained highly stable with both HNE forms. Principal Component Analysis suggested that robustaflavone binding induced structural stability in both HNE forms. Cluster analysis and free energy landscape plots showed that robustaflavone remained within the sc and tcHNE binding site throughout the 100 ns MD simulation. The robustaflavone scaffold likely inhibits both tcHNE and scHNE. It is potentially superior to sivelestat and alvelestat and can aid in developing therapeutics targeting both forms of HNE.

As a critical sensor protein, NLRP3 detects cellular perturbation caused by diverse exogenous and endogenous stimuli. NLRP3 activation requires domain rotation within the NEK7-bound NLRP3 monomer and assembly. However, a detailed molecular mechanism for NLRP3 assembly and activation remains elusive, particularly in terms of dynamics and energetics. In this work, all-atom molecular dynamics (MD) simulations are executed to describe large-amplitude closed-to-open conformational transitions along the rotational pathway. From the MD trajectories, the computed potential of mean force (PMF) shows that NLRP3 activation through monomeric domain rotation is an uphill process, during which the active conformation of the NLRP3-NEK7 monomer cannot be stabilized. Further binding free-energy calculations for two neighboring NLRP3-NEK7 subunits in a disc assembly with the C10 symmetry reveal that the protein self-assembly starts approximately at the 86.5° position on the rotary pathway, along which the NLRP3 activation becomes a downhill process to the active state at 90.5°. The active NLRP3-NEK7 monomeric conformation in the disc assembly is stabilized because of the interactions between the neighboring subunits, involving mainly FISNA loop 1 in one subunit and a \"crocodile-clip\" structure formed by the NBD helix-loop-strand motif (residues 351-373) and the WHD β-hairpin loop (residues 501-521) in the other. Our simulations also demonstrate that NEK7 plays an important role in the NLRP3 cage dissociation in the centrosome, which is consistent with biological experiments. The computational results provide kinetic, energetic, and structural insights into the molecular mechanisms of the activation of NLRP3 and the NEK7-driven dissociation of inactive NLRP3 cages. The activation mechanism of NLRP3 proposed in this work is significantly different from those of previous structural studies.

Gelation is a critical property of citrus pectin. However, the roles played by neutral sugar side-chains on acid-induced pectin gelation remain poorly understood. Herein, galactan- or/and arabinan-eliminated pectins (P-G, P-A, and P-AG) were used to investigate the effects of side-chains on gelation. The gel hardness values of citrus pectin, P-G, P-A, and P-AG were 42.6, 39.9, 5.3, and 2.1 g, respectively, suggesting that arabinan contributed more to gelation than galactan. We next found that arabinan branches promoted pectin chain entanglement more effectively than arabinan backbones. Destabilizer addition experiments showed that hydrogen bonding, electrostatic interaction, and hydrophobic interaction were the main forces affecting pectin gel networks and strength, which was further validated by molecular dynamic simulations. The total number of hydrogen bonds between the arabinan branches and galactan/HG (65.7) was significantly higher than that between the arabinan backbones and galactan/HG (39.1), indicating that arabinan branches predominated in terms of such interactions. This study thus elucidated the roles played by neutral-sugar side-chains, especially the arabinan branches of acid-induced pectin gels, in term of enhancing high-methoxyl pectin gelation, and offers novel insights into the structure-gelling relationships of citrus pectin.

This study investigated the influence of Konjac Glucomannan (KGM) with varying degrees of polymerization (DKGMx) on the gelatinization and retrogradation characteristics of wheat starch, providing new insights into starch-polysaccharide interactions. This research uniquely focuses on the effects of DKGMx, utilizing multidisciplinary approaches including Rapid Visco Analysis (RVA), Differential Scanning Calorimetry (DSC), rheological testing, Low-Field Nuclear Magnetic Resonance (LF-NMR), and molecular simulations to assess the effects of DKGMx on gelatinization temperature, viscosity, structural changes post-retrogradation, and molecular interactions. Our findings revealed that higher degrees of polymerization (DP) of DKGMx significantly enhanced starch\'s pasting viscosity and stability, whereas lower DP reduced viscosity and interfered with retrogradation. High DP DKGMx promoted retrogradation by modifying moisture distribution. Molecular simulations revealed the interplay between low DP DKGMx and starch molecules. These interactions, characterized by increased hydrogen bonds and tighter binding to more starch chains, inhibited starch molecular rearrangement. Specifically, low DP DKGMx established a dense hydrogen bond network with starch, significantly restricting molecular mobility and rearrangement. This study provides new insights into the role of the DP of DKGMx in modulating wheat starch\'s properties, offering valuable implications for the functional improvement of starch-based foods and advancing starch science.

Tire microplastics (TMPs) and antibiotics are emerging pollutants that widely exist in water environments. The coexistence of these pollutants poses severe threats to aquatic organisms. However, the toxicity characteristics and key molecular factors of the combined exposure to TMPs in aquatic organisms remain unknown. Therefore, the joint toxicity of styrene-butadiene rubber TMPs (SBR-TMPs) and 32 antibiotics (macrolides, fluoroquinolones, β-lactams, sulfonamides, tetracyclines, nitroimidazoles, highly toxic antibiotics, high-content antibiotics, and common antibiotics) in zebrafish was investigated using a full factorial design, molecular docking, and molecular dynamics simulation. Sixty-four combinations of antibiotics were designed to investigate the hepatotoxicity of the coexistence of SBR-TMPs additives and antibiotics in zebrafish. Results indicated that low-order effects of antibiotics (e.g., enoxacin-lomefloxacin and ofloxacin-enoxacin-lomefloxacin) had relatively notable toxicity. The van der Waals interaction between additives and zebrafish cytochrome P450 enzymes primarily affected zebrafish hepatotoxicity. Zebrafish hepatotoxicity was also affected by the ability of SBR-TMPs to adsorb antibiotics, the relation between antibiotics, the affinity of antibiotics docking to zebrafish cytochrome P450 enzymes, electronegativity, atomic mass, and the hydrophobicity of the antibiotic molecules. This study aimed to eliminate the joint toxicity of TMPs and antibiotics and provide more environmentally friendly instructions for using different chemicals.

BACKGROUND: Previous clinical and basic studies have revealed that ginseng might have cardioprotective properties against anthracycline-induced cardiotoxicity (AIC). However, the underlying mechanism of ginseng action against AIC remains insufficiently understood. The aim of this study was to explore the related targets and pathways of ginseng against AIC using network pharmacology, molecular docking, cellular thermal shift assay (CETSA) and molecular dynamics (MD) simulations.
RESULTS: Fourteen drug-disease common targets were identified. Enrichment analysis showed that the AGE-RAGE in diabetic complications, fluid shear stress and atherosclerosis, and TNF signaling pathway were potentially involved in the action of ginseng against AIC. Molecular docking demonstrated that the core components including Kaempferol, beta-Sitosterol, and Fumarine had notable binding activity with the three core targets CCNA2, STAT1, and ICAM1. Furthermore, the stable complex of STAT1 and Kaempferol with favorable affinity was further confirmed by CETSA and MD simulation.
CONCLUSIONS: This study suggested that ginseng might exert their protective effects against AIC through the derived effector compounds beta-Sitosterol, Kaempferol and Fumarine by targeting CCNA2, STAT1, and ICAM1, and modulating AGE-RAGE in diabetic complications, fluid shear stress and atherosclerosis, and TNF signaling pathways.