Binding Conformation Research Articles

Developing Stapled Aptamers with a Constrained Conformation for Osteogenesis Imperfect Therapeutics.

Despite the extensive development of aptamers in basic research, only a limited number have successfully progressed to clinical trials. This limitation is primarily attributed to the inherent instability of aptamers' conformation, resulting in low affinity, poor serum stability, and inconsistent potency, posing a significant challenge to their stabilization. Herein, we established a feasible strategy to develop staple aptamers using the predicted binding conformations and titration cross-linking (TTC) method. Through this strategy, we successfully synthesized various stapled sclerostin aptamers with over 70% yield. Importantly, we demonstrated that stapled aptamers significantly enhanced their affinity (approximately 20-fold) and serum stability (negligible degradation within 32 h). Moreover, in an osteogenesis imperfecta mouse model (Col1a2+/G610C mice), the stapled aptamer (named c-0127OA) exhibited a potent antagonistic effect on sclerostin, leading to enhanced anabolic bone anabolic potential. Collectively, our established stapling strategy is effective in stabilizing aptamers' conformation, with c-0127OA emerging as a promising therapeutic candidate for osteogenesis imperfecta.

Role of Tyrosine Phosphorylation in PTP-PEST.

We study the influence of tyrosine phosphorylation on PTP-PEST, a cytosolic protein tyrosine phosphatase. Utilizing a combination of experimental data and computational modeling, specific tyrosine sites, notably, Y64 and Y88, are identified for potential phosphorylation. Phosphorylation at these sites affects loop dynamics near the catalytic site, altering interactions among key residues and modifying the size of the binding pocket. This, in turn, impacts substrate binding, as indicated by changes in the binding energy. Our findings provide insights into the structural and functional consequences of tyrosine phosphorylation on PTP-PEST, enhancing our understanding of its effects on substrate binding and catalytic conformation.

Hydrazide-hydrazones as novel antioxidants - in vitro, molecular docking and DFT studies

The pathogenesis of many diseases, such as obesity, depression, cancer, cataract, and neurodegenerative diseases, is related to the generation of reactive oxygen species (ROS). Structures possessing radical-scavenging properties act as antioxidants and they could prevent the progression of the aforementioned diseases. Therefore, the current work was focused on the resynthesis and the antioxidant evaluation of 13 hydrazide-hydrazones. Two in vitro tests - DPPH and ABTS, were applied for the determination of the antioxidant capacities. The free-radical scavenging assays displayed that the hydrazide-hydrazone synthesized after condensation with a salicylaldehyde (5b) is the most potent antioxidant. The in vitro evaluation through the ABTS test showed that the former structure has greater antioxidant properties compared with the used standard - Trolox (6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid). In concentrations of 31 µM, the pyrrole-based structure showed 35.77% radical-scavenging effects. A possible binding conformation of the most active hydrazide-hydrazone in the active site of NADPH oxidase was visualised through docking simulations. The active amino acids involved in the stabilisation of the complexes were discussed. DFT studies demonstrated that 5b is a stable structure with good hydrogen and electron donating properties. Overall, the pyrrole-based hydrazide-hydrazone possesses promising antioxidant properties, however further in vitro/in vivo biological evaluations are required.

Recognition and Cleavage of Human tRNA Methyltransferase TRMT1 by the SARS-CoV-2 Main Protease.

The SARS-CoV-2 main protease (Mpro, or Nsp5) is critical for the production of functional viral proteins during infection and, like many viral proteases, can also target host proteins to subvert their cellular functions. Here, we show that the human tRNA methyltransferase TRMT1 can be recognized and cleaved by SARS-CoV-2 Mpro. TRMT1 installs the N2,N2-dimethylguanosine (m2,2G) modification on mammalian tRNAs, which promotes global protein synthesis and cellular redox homeostasis. We find that Mpro can cleave endogenous TRMT1 in human cell lysate, resulting in removal of the TRMT1 zinc finger domain. TRMT1 proteolysis results in elimination of TRMT1 tRNA methyltransferase activity and reduced tRNA binding affinity. Evolutionary analysis shows that the TRMT1 cleavage site is highly conserved in mammals, except in Muroidea, where TRMT1 is likely resistant to cleavage. In primates, regions outside the cleavage site with rapid evolution could indicate adaptation to ancient viral pathogens. Furthermore, we determined the structure of a TRMT1 peptide in complex with Mpro, revealing a substrate binding conformation distinct from the majority of available Mpro-peptide complexes. Kinetic parameters for peptide cleavage show that the TRMT1(526-536) sequence is cleaved with comparable efficiency to the Mpro-targeted nsp8/9 viral cleavage site. Mutagenesis studies and molecular dynamics simulations together indicate that kinetic discrimination occurs during a later step of Mpro-mediated proteolysis that follows substrate binding. Our results provide new information about the structural basis for Mpro substrate recognition and cleavage, the functional roles of the TRMT1 zinc finger domain in tRNA binding and modification, and the regulation of TRMT1 activity by SARS-CoV-2 Mpro. These studies could inform future therapeutic design targeting Mpro and raise the possibility that proteolysis of human TRMT1 during SARS-CoV-2 infection suppresses protein translation and oxidative stress response to impact viral pathogenesis.

Multi-Target Mechanisms of Si-Ni-San on Anxious Insomnia: An Example of Network-pharmacology and Molecular Docking Analysis.

Based on comprehensive network-pharmacology and molecular docking analysis, this study was intended to unveil the multiple mechanisms of Si-Ni-San (SNS) in treating anxious insomnia. The compounds of SNS were meticulously analyzed, selected and standardized with references to their pharmacological attributes. The components included chaihu (Bupleurum chinense DC.), baishao (Paeonia lactiflora Pall.), zhishi (Citrus aurantium L.) and gancao (Glycyrrhiza uralensis Fisch. ex DC.). We used the Traditional Chinese Medicine System Pharmacology (TCMSP) Database, Traditional Chinese Medicines Integrated Database (TCMID), GeneCards database, therapeutic target database (TTD) and comparative toxicogenomic database (CTD) to construct the components-compounds-targets networks and used Cytoscape 3.9.1 software to visualize the outcome. Afterwards, the STRING database and Cytoscape 3.9.1 software were utilized to construct and visualize the protein-protein interaction (PPI) network analysis. In addition, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were also conducted through the Database for Annotation, Visualization, and Integrated Discovery (DAVID). The molecular docking program was carried out using AutoDock 4.2 software to understand interactions between target receptors and compound ligands selected for study. We thoroughly sorted and filtered 31 pharmacologically active compounds from SNS. Subsequently, several potential target genes were predicted, of which there were 59 target genes distinctly associated with anxious insomnia. The PPI analysis indicated that the core target proteins included AKT1, IL6, TNF, SLC6A4, MAOA and GABRA2. The results of our study indicated that SNS potentially remediates anxious insomnia by reducing inflammation, neurodegeneration, and cell apoptosis of neurons. In addition, GO and KEGG enrichment analysis results indicated that SNS could modulate multiple aspects of anxious insomnia through mechanisms related to pathways of neuroactive ligand-receptor interaction. These pathways include various kinds of synaptic transmission pathways, and anti-inflammatory activity associated with response pathways. When we compared the components-compounds-targets networks and the compounds-targets-synaptic pathways networks, the five active compounds, including beta-Sitosterol, Kaempferol, Tetramethoxyluteolin, Isorhamnetin and Shinpterocarpin, were selected to conduct molecular docking experiments. Eleven target proteins, (AKT1, SLC6A4, ADRB2, MAOA, ACHE, ESR1, CYP3A4, CHRNA7, GABRA2, HTR2A and NOS3), which also play significant roles in regulating serotonergic, cholinergic, dopaminergic and GABAergic systems in the PPI network, were selected to act as receptors in molecular docking trials. The results showed that docking pairs isorhamnetin-AKT1, isorhamnetin-SLC6A4, β-sitosterol-MAOA, β- sitosterol-ACHE, isorhamnetin-CHRNA7 and shinpterocarpin-GABRA2 provided the most stable conformations of ligand-receptor binding between key compounds and core target proteins in the SNS. In the study, we offer a computational result, revealing that SNS may alleviate sleep disorders associated with anxiety through a "multi-compounds, multi-targets, and multi-pathways" mechanism. The network-pharmacology and molecular docking outcomes could theoretically confirm the anti-anxiety and anti-insomnia effects of SNS. Although this research is purely statistical and systematic without empirical validation, it serves as a stepping stone and cornerstone for subsequent experimental investigations.

Identification of novel and potent inhibitors of SARS-CoV-2 main protease from DNA-encoded chemical libraries.

In vitro screening of large compound libraries with automated high-throughput screening is expensive and time-consuming and requires dedicated infrastructures. Conversely, the selection of DNA-encoded chemical libraries (DECLs) can be rapidly performed with routine equipment available in most laboratories. In this study, we identified novel inhibitors of SARS-CoV-2 main protease (Mpro) through the affinity-based selection of the DELopen library (open access for academics), containing 4.2 billion compounds. The identified inhibitors were peptide-like compounds containing an N-terminal electrophilic group able to form a covalent bond with the nucleophilic Cys145 of Mpro, as confirmed by x-ray crystallography. This DECL selection campaign enabled the discovery of the unoptimized compound SLL11 (IC50 = 30 nM), proving that the rapid exploration of large chemical spaces enabled by DECL technology allows for the direct identification of potent inhibitors avoiding several rounds of iterative medicinal chemistry. As demonstrated further by x-ray crystallography, SLL11 was found to adopt a highly unique U-shaped binding conformation, which allows the N-terminal electrophilic group to loop back to the S1' subsite while the C-terminal amino acid sits in the S1 subsite. MP1, a close analog of SLL11, showed antiviral activity against SARS-CoV-2 in the low micromolar range when tested in Caco-2 and Calu-3 (EC50 = 2.3 µM) cell lines. As peptide-like compounds can suffer from low cell permeability and metabolic stability, the cyclization of the compounds will be explored in the future to improve their antiviral activity.

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.

Integrating strategy to investigate the formation and stabilization mechanisms of Curcumin-Cyclodextrin inclusion complexes: experimental characterizations and multi-scale computational simulations.

The Cyclodextrin (CD) encapsulation technology can significantly enhance the water solubility of Curcumin (CUR) and effectively protect it against chemical degradations. In the current study, a combination of experimental and multi-scale computational approaches was used to investigate the binding mechanism between CUR and β-CDs (β-CD, 2-HP-β-CD, Me-β-CD, and DM-β-CD). Phase solubility studies showed that β-CDs exhibited a strong binding affinity with CUR and significantly enhanced its solubility. SEM, PXRD, DSC, and TG analysis confirmed the formation of inclusion complexes, resulting in the transition of CUR from crystalline to an amorphous state. FTIR, 1H, and 13CNMR spectra deduced that CUR may have multiple binding conformations with β-CDs. The rationality of molecular dynamics simulation systems was confirmed by comparing the simulated IR spectrum from quantum mechanics with the experimental IR spectrum. MM-PBSA and umbrella sampling simulation calculated the binding free energy of the CUR/CD inclusion complexes, ranking Me-β-CD > DM-β-CD > 2-HP-β-CD > β-CD, and clarified that van der Waals interaction played a major role in stabilizing the inclusion complexes. RDF analysis confirmed that the release of high-energy water molecules drove the formation of the CUR/CD inclusion complex, and the β-CD substituents affected their exterior hydration layer. Additionally, principal component analysis (PCA) and non-covalent interaction analysis revealed three CUR/CD binding modes: bead-on-string, terminal insertion, and V-shaped-folding. The research strategy adopted here can serve as a paradigm for investigating the encapsulation mechanism of poorly soluble drugs with CDs.

Molecular mechanism of Dang-Shen-Yu-Xing decoction against Mycoplasma bovis pneumonia based on network pharmacology, molecular docking, molecular dynamics simulations and experimental verification.

Mycoplasma bovis pneumonia is a highly contagious respiratory infection caused by Mycoplasma bovis. It is particularly prevalent in calves, posing a significant threat to animal health and leading to substantial economic losses. Dang-Shen-Yu-Xing decoction is often used to treat this condition in veterinary clinics. It exhibits robust anti-inflammatory effects and can alleviate pulmonary fibrosis. However, its mechanism of action remains unclear. Therefore, this study aimed to preliminarily explore the molecular mechanism of Dang-Shen-Yu-Xing decoction for treating mycoplasma pneumonia in calves through a combination of network pharmacology, molecular docking, molecular dynamics simulation methods, and experimental validation. The active components and related targets of Dang-Shen-Yu-Xing decoction were extracted from several public databases. Additionally, complex interactions between drugs and targets were explored through network topology, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes enrichment analyses. Subsequently, the binding affinity of drug to disease-related targets was verified through molecular docking and molecular dynamics simulation. Finally, the pharmacodynamics were verified via animal experiments. The primary network topology analysis revealed two core targets and 10 key active components of Dang-Shen-Yu-Xing decoction against Mycoplasma bovis pneumonia. Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that the mechanism of Dang-Shen-Yu-Xing decoction for treating mycoplasma bovis pneumonia involved multiple signaling pathways, with the main pathways including PI3K-Akt and IL17 signaling pathways. Moreover, molecular docking predicted the binding affinity and conformation of the core targets of Dang-Shen-Yu-Xing decoction, IL6, and IL10, with the associated main active ingredients. The results showed a strong binding of the active ingredients to the hub target. Further, molecular docking dynamics simulation revealed three key active components of IL10 induced by Dang-Shen-Yu-Xing decoction against Mycoplasma bovis pneumonia. Finally, animal experiments confirmed Dang-Shen-Yu-Xing decoction pharmacodynamics, suggesting that it holds potential as an alternative therapy for treating mycoplasma bovis pneumonia.

Virtual screening, molecular docking, and molecular dynamics simulation studies on the hypoglycemic function of oat peptides

Oat contains a large amount of polysaccharides and peptides, among which oat peptides have the function of reducing blood sugar levels in the body. This paper reviewed the peptides obtained from oat extraction and identification from literature and constructed the corresponding oat peptide database. Based on the DPP4 protein, a virtual screening of the peptide database was performed. One hundred nanosecond molecular dynamics simulations were performed for the six peptides obtained from the screening. The interaction information between different peptide molecules and DPP4 was analyzed from the stable binding conformations after the simulation, and the binding free energy between different peptide molecules and DPP4 was calculated. The results show that the peptide molecules obtained from the virtual screening can all stably bind to the DPP4 protein, among which two peptide molecules have relatively strong affinity with DPP4 and can be used as lead molecules for the subsequent design and modification of DPP4 inhibitors. The simulation results are informative for a deeper understanding of the structural characteristics of DPP4 and the molecular recognition mechanism between DPP4 and oat peptides.

Structure-Based Design and Development of Phosphine Oxides as a Novel Chemotype for Antibiotics that Dysregulate Bacterial ClpP Proteases.

A series of arylsulfones and heteroarylsulfones have previously been demonstrated to dysregulate the conserved bacterial ClpP protease, causing the unspecific degradation of essential cellular housekeeping proteins and ultimately resulting in cell death. A cocrystal structure of a 2-β-sulfonylamide analog, ACP1-06, with Escherichia coli ClpP showed that its 2-pyridyl sulfonyl substituent adopts two orientations in the binding site related through a sulfone bond rotation. From this, a new bis-aryl phosphine oxide scaffold, designated as ACP6, was designed based on a "conformation merging" approach of the dual orientation of the ACP1-06 sulfone. One analog, ACP6-12, exhibited over a 10-fold increase in activity over the parent ACP1-06 compound, and a cocrystal X-ray structure with ClpP confirmed its predicted binding conformation. This allowed for a comparative analysis of how different ligand classes bind to the hydrophobic binding site. The study highlights the successful application of structure-based rational design of novel phosphine oxide-based antibiotics.

Identification of Potential Tryptase Inhibitors from FDA-Approved Drugs Using Machine Learning, Molecular Docking, and Experimental Validation.

This study explores the innovative use of machine learning (ML) to identify novel tryptase inhibitors from a library of FDA-approved drugs, with subsequent confirmation via molecular docking and experimental validation. Tryptase, a significant mediator in inflammatory and allergic responses, presents a therapeutic target for various inflammatory diseases. However, the development of effective tryptase inhibitors has been challenging due to the enzyme's complex activation and regulation mechanisms. Utilizing a machine learning model, we screened an extensive FDA-approved drug library to identify potential tryptase inhibitors. The predicted compounds were then subjected to molecular docking to assess their binding affinity and conformation within the tryptase active site. Experimental validation was performed using RBL-2H3 cells, a rat basophilic leukemia cell line, where the efficacy of these compounds was evaluated based on their ability to inhibit tryptase activity and suppress β-hexosaminidase activity and histamine release. Our results demonstrated that several FDA-approved drugs, including landiolol, laninamivir, and cidofovir, significantly inhibited tryptase activity. Their efficacy was comparable to that of the FDA-approved mast cell stabilizer nedocromil and the investigational agent APC-366. These findings not only underscore the potential of ML in accelerating drug repurposing but also highlight the feasibility of this approach in identifying effective tryptase inhibitors. This research contributes to the field of drug discovery, offering a novel pathway to expedite the development of therapeutics for tryptase-related pathologies.

Conformationally Constrained Isoquinolinones as Orally Efficacious Hepatitis B Capsid Assembly Modulators.

Isoquinolinone-based HBV capsid assembly modulators that bind at the dimer:dimer interface of HBV core protein have been shown to suppress viral replication in chronic hepatitis B patients. Analysis of their binding mode by protein X-ray crystallography has identified a region of the small molecule where the application of a constraint can lock the preferred binding conformation and has allowed for further optimization of this class of compounds. Key analogues demonstrated single digit nM EC50 values in reducing HBV DNA in a HepDE19 cellular assay in addition to favorable ADME and pharmacokinetic properties, leading to a high degree of oral efficacy in a relevant in vivo hydrodynamic injection mouse model of HBV infection, with 12e effecting a 3 log10 decline in serum HBV DNA levels at a once daily dose of 1 mg/kg. Additionally, maintenance of activity was observed in clinically relevant HBV core protein variants T33N and I105T.

Distinct binding conformations of epinephrine with α- and β-adrenergic receptors.

Agonists targeting α2-adrenergic receptors (ARs) are used to treat diverse conditions, including hypertension, attention-deficit/hyperactivity disorder, pain, panic disorders, opioid and alcohol withdrawal symptoms, and cigarette cravings. These receptors transduce signals through heterotrimeric Gi proteins. Here, we elucidated cryo-EM structures that depict α2A-AR in complex with Gi proteins, along with the endogenous agonist epinephrine or the synthetic agonist dexmedetomidine. Molecular dynamics simulations and functional studies reinforce the results of the structural revelations. Our investigation revealed that epinephrine exhibits different conformations when engaging with α-ARs and β-ARs. Furthermore, α2A-AR and β1-AR (primarily coupled to Gs, with secondary associations to Gi) were compared and found to exhibit different interactions with Gi proteins. Notably, the stability of the epinephrine-α2A-AR-Gi complex is greater than that of the dexmedetomidine-α2A-AR-Gi complex. These findings substantiate and improve our knowledge on the intricate signaling mechanisms orchestrated by ARs and concurrently shed light on the regulation of α-ARs and β-ARs by epinephrine.

Gardenin B, A Natural Inhibitor for USP7: In vitro Evaluation and In silico Identification

Background: Ubiquitin-specific protease 7 (USP7) is one of the most widely studied deubiquitin enzymes (DUBs). The protein level of USP7 is highly expressed in a variety of malignant cancers, which suggests that it is a prognostic marker of cancers and a potential drug target for oncotherapy. Objective: The aim of this study was to identify natural and effective USP7 inhibitors, in order to understand the activation of the USP7/p53 pathway by natural inhibitors. Methods: In this study, USP7 enzyme activity screening assay system and p53 luciferase reporter assay system have been applied for the discovery of natural USP7 inhibitors targeting the catalytic active site. Molecular docking and molecular dynamics (MD) simulation revealed the combined mechanism between USP7 with gardenin B. Results: The gardenin B was screened from our home-lab natural products (160 flavonoids) and had cytotoxicity in HCT116 cells (IC50 = 46.28 ± 2.16 μM). Preliminary in vitro studies disclosed its antiproliferative activity and activated p53 signaling pathway in HCT116 cells. We found that the complex formed by gardenin B and 5vsk (Ledock score = -6.86, MM/GBSA score = -53.35) had the optimal binding conformation. Moreover, the MD simulation showed that the π-π interactions between gardenin B with His461 and Phe409, and the hydrogen bonds interaction between gardenin B with Leu406 played an important role in maintaining the close binding of the complexes. Conclusion: In conclusion, gardenin B could be used as a natural product inhibitor of USP7 for further optimized design and development potential.

In Silico and In Vitro Investigation of Phytoconstituents from Flacourtia Jangomas as a Potential Candidate for the Treatment of Type 2 Diabetes Mellitus

AbstractFlacourtia jangomas is a plant used for centuries in Brazil, China, India, and the Malaya Peninsula to treat conditions such as diabetes, asthma, anaemia, and diarrhoea. Despite its therapeutic and economic benefits, the plant has not been popular among farmers due to its limited yield and lack of knowledge about its potential. Targeting PPARγ agonists is a promising approach for discovering and developing effective drugs to treat type 2 diabetes. Due to the activation of the PPARγ through PPARγ agonists, target genes implicated in inflammation, adipogenesis, lipid metabolism, and glucose metabolism are transcriptionally regulated. This study analysed the curated datasets of F. jangomas to identify the binding conformations of the phytoconstituents with different T2DM targets through molecular docking, molecular dynamics, and ADMET prediction approach. To determine the most favourable binding conformations of phytoconstituents that bind with the PPARγ receptors, density functional theory and principal component analysis were performed. The study reveals that the phytoconstituents of F. jangomas can act as PPARγ agonists and could be used to design and optimise the potential antidiabetic agents inspired by natural products. These findings provide a promising approach towards developing effective antidiabetic drugs.

Potential mechanism prediction of indole-3-propionic acid against diminished ovarian reserve via network pharmacology, molecular docking and experimental verification

BackgroundOxidative stress (OS) is one of the major causes of ovarian aging and dysfunction. Indole-3-propionic acid (IPA) is an indole compound derived from tryptophan with free radical scavenging and antioxidant properties, and thus may have potential applications in protecting ovarian function, although the exact mechanisms are unknown. This study aims to preliminarily elucidate the potential mechanisms of IPA that benefit ovarian reserve function through network pharmacology, molecular docking, and experimental verification.MethodsThe related protein targets of IPA were searched on SwissTargetPrediction, TargetNet, BATMAN-TCM, and PharmMapper databases. The potential targets of diminished ovarian reserve (DOR) were identified from OMIM, GeneCards, DrugBank, and DisGeNET databases. The common targets were uploaded directly to the STRING database to construct PPI networks. We then performed GO and KEGG enrichment analysis on the targets. Subsequently, molecular docking and molecular dynamics simulation were used to validate the binding conformation of IPA to candidate targets. Furthermore, we carried out in vitro experiments to validate the prediction results of network pharmacology.ResultsWe identified a total of 61 potential targets for the interaction of IPA with DOR. The PPI network topological parameter analysis yielded 13 hub genes for DOR treatment. The GO biological process enrichment analysis identified 293 entries, mainly enriched in aging, signal transduction, response to hypoxia, negative regulation of apoptotic process, and positive regulation of cell proliferation. The KEGG enrichment analysis mainly included lipid and atherosclerosis, progesterone-mediated oocyte maturation, AGE-RAGE, relaxin, estrogen, and other signaling pathways. The molecular docking further revealed the direct binding of IPA with six hub proteins including NOS3, AKT1, EGFR, PPARA, SRC, and TNF. In vitro experiments showed that IPA pretreatment attenuated H2O2-induced cellular oxidative stress damage, while IPA exerted cytoprotective and antioxidant damage effects by regulating the six hub genes and antioxidant proteins.ConclusionWe systematically illustrated the potential protective effects of IPA against DOR through multiple targets and pathways using network pharmacology, and further verified the cytoprotective effect and antioxidant properties of IPA through in vitro experiments. These findings provide new insights into the targets and molecular mechanisms whereby IPA improves DOR.

Activity-based metaproteomics driven discovery and enzymological characterization of potential α-galactosidases in the mouse gut microbiome

The gut microbiota offers an extensive resource of enzymes, but many remain uncharacterized. To distinguish the activities of similar annotated proteins and mine the potentially applicable ones in the microbiome, we applied an effective Activity-Based Metaproteomics (ABMP) strategy using a specific activity-based probe (ABP) to screen the entire gut microbiome for directly discovering active enzymes and their potential applications, not for exploring host-microbiome interactions. By using an activity-based cyclophellitol aziridine probe specific to α-galactosidases (AGAL), we successfully identified and characterized several gut microbiota enzymes possessing AGAL activities. Cryo-electron microscopy analysis of a newly characterized enzyme (AGLA5) revealed the covalent binding conformations between the AGAL5 active site and the cyclophellitol aziridine ABP, which could provide insights into the enzyme’s catalytic mechanism. The four newly characterized AGALs have diverse potential activities, including raffinose family oligosaccharides (RFOs) hydrolysis and enzymatic blood group transformation. Collectively, we present a ABMP platform that facilitates gut microbiota AGALs discovery, biochemical activity annotations and potential industrial or biopharmaceutical applications.

Virtual Screening and Molecular Docking: Discovering Novel METTL3 Inhibitors.

Methyltransferase-like 3 (METTL3) is an RNA methyltransferase that catalyzes the N6 -methyladenosine (m6A) modification of mRNA in eukaryotic cells. Past studies have shown that METTL3 is highly expressed in various cancers and is closely related to tumor development. Therefore, METTL3 inhibitors have received widespread attention as effective treatments for different types of tumors. This study proposes a hybrid high-throughput virtual screening (HTVS) protocol that combines structure-based methods with geometric deep learning-based DeepDock algorithms. We identified unique skeleton inhibitors of METTL3 from our self-built internal database. Among them, compound C3 showed significant inhibitory activity on METTL3, and further molecular dynamics simulations were performed to provide more details about the binding conformation. Overall, our research demonstrates the effectiveness of hybrid virtual algorithms, which is of great significance for understanding the biological functions of METTL3 and developing treatment methods for related diseases.

Molecular insights reveal how the glycolipids in cell membrane mitigates nanomaterial's invasion

Nanomaterial-cellular membrane interaction is crucial for the cytotoxicity of such materials in theoretical investigations. However, previous research often used cellular membrane models with one or few lipid types, which deviates significantly from realistic membrane compositions. Here, employing molecular dynamics (MD) simulations, we investigate the impact of a typical nanomaterial, boron nitride (BN), on a cellular membrane model based on the realistic small intestinal epithelial cell (SIEC) membrane. This membrane contains a complex composition, including abundant glycolipids. Our MD simulations reveal that BN nanosheet can partially insert into the SIEC membrane, maintaining a stable binding conformation without causing obvious structural changes. Dynamic analyses suggest that van der Waals (vdW) interactions drive the binding process between BN and the SIEC membrane. Further simulation of the interaction between BN nanosheet and deglycosylated SIEC membrane confirms that BN nanosheet cause significant structural damage to deglycosylated SIEC membranes, completely inserting into the membrane, extracting lipids, and burying some lipid hydrophilic heads within the membrane interior. Quantitative analyses of mean squared displacements (MSD) of membranes, membrane thicknesses, area per lipid, and order parameters indicate that BN nanosheet causes more substantial damage to deglycosylated SIEC membrane than to intact SIEC membrane. This comparison suggests the molecular mechanism involved in mitigating BN invasion by SIEC membrane that the polysaccharide heads of glycolipids in the SIEC membrane form a significant steric hindrance on membrane surface, not only hindering the insertion of BN, but also resisting the lipid extraction by BN. Free energy calculations further support this conclusion. Overall, our MD simulations not only shed new light into the reduced impact of BN nanosheet on the realistic SIEC membrane but also highlight the importance of glycolipids in protecting cell membranes from nanomaterial invasion, contributing to a deeper understanding of nanomaterial-realistic cell membrane interactions.