Exploring the activation mechanism of metabotropic glutamate receptor 2.

Homomeric dimerization of metabotropic glutamate receptors (mGlus) is essential for the modulation of their functions and represents a promising avenue for the development of novel therapeutic approaches to address central nervous system diseases. Yet, the scarcity of detailed molecular and energetic data on mGlu2 impedes our in-depth comprehension of their activation process. Here, we employ computational simulation methods to elucidate the activation process and key events associated with the mGlu2, including a detailed analysis of its conformational transitions, the binding of agonists, Gi protein coupling, and the guanosine diphosphate (GDP) release. Our results demonstrate that the activation of mGlu2 is a stepwise process and several energy barriers need to be overcome. Moreover, we also identify the rate-determining step of the mGlu2's transition from the agonist-bound state to its active state. From the perspective of free-energy analysis, we find that the conformational dynamics of mGlu2's subunit follow coupled rather than discrete, independent actions. Asymmetric dimerization is critical for receptor activation. Our calculation results are consistent with the observation of cross-linking and fluorescent-labeled blot experiments, thus illustrating the reliability of our calculations. Besides, we also identify potential key residues in the Gi protein binding position on mGlu2, mGlu2 dimer's TM6-TM6 interface, and Gi α5 helix by the change of energy barriers after mutation. The implications of our findings could lead to a more comprehensive grasp of class C G protein-coupled receptor activation.

Post-abortion needs-based education via the WeChat platform to lessen fear and encourage effective contraception: a post-abortion care service intervention-controlled trial.

OBJECTIVE: Our study aims to investigate post-abortion needs-based education via the WeChat platform for women who had intended abortion in the first trimester, whether they are using effective contraception or becoming pregnant again. DESIGN: This single hospital intervention-controlled trial used a nearly 1:1 allocation ratio. Women who had intended abortions were randomly assigned to a Wechat group (needs-based education) and a control group (Traditional education). The women's ability to use effective contraception was the main result. Whether they unknowingly became pregnant again was the second result. Another result was patient anxiousness. Before and after education, women filled out questionnaires to assess their contraception methods and anxiety. METHODS: Based on the theoretical framework of contraceptions of IBL (inquiry-based learning), post-abortion women were included in WeChat groups. We use WeChat Group Announcement, regularly sending health education information, one-on-one answers to questions, and consultation methods to explore the possibilities and advantages of WeChat health education for women after abortion. A knowledge paradigm for post-abortion health education was established: From November 2021 until December 2021, 180 women who had an unintended pregnancy and undergone an induced or medical abortion were recruited, their progress was tracked for four months, and the PAC service team monitored the women's speech, discussed and classified the speech entries and summarized the common post-abortion needs in 8 aspects. At least 2 research group members routinely extracted records and categorized the outcomes. RESULTS: Before education, there were no appreciable variations between the two groups regarding sociodemographic characteristics, obstetrical conditions, abortion rates, or methods of contraception (P > 0.05). Following education, the WeChat group had a greater rate of effective contraception (63.0%) than the control group (28.6%), and their SAS score dropped statistically more than that of the control group (P < 0.05). Following the education, there were no unwanted pregnancies in the WeChat group, whereas there were 2 in the traditional PAC group. Only 5 participants in the WeChat group and 32 in the conventional PAC group reported mild anxiety after the education.

Application of Coarse-Grained (CG) Models to Explore Conformational Pathway of Large-Scale Protein Machines.

Protein machines are clusters of protein assemblies that function in order to control the transfer of matter and energy in cells. For a specific protein machine, its working mechanisms are not only determined by the static crystal structures, but also related to the conformational transition dynamics and the corresponding energy profiles. With the rapid development of crystallographic techniques, the spatial scale of resolved structures is reaching up to thousands of residues, and the concomitant conformational changes become more and more complicated, posing a great challenge for computational biology research. Previously, a coarse-grained (CG) model aiming at conformational free energy evaluation was developed and showed excellent ability to reproduce the energy profiles by accurate electrostatic interaction calculations. In this study, we extended the application of the CG model to a series of large-scale protein machine systems. The spike protein trimer of SARS-CoV-2, ATP citrate lyase (ACLY) tetramer, and P4-ATPases systems were carefully studied and discussed as examples. It is indicated that the CG model is effective to depict the energy profiles of the conformational pathway between two endpoint structures, especially for large-scale systems. Both the energy change and energy barrier between endpoint structures provide reasonable mechanism explanations for the associated biological processes, including the opening of receptor binding domain (RBD) of spike protein, the phospholipid transportation of P4-ATPase, and the loop translocation of ACLY. Taken together, the CG model provides a suitable alternative in mechanistic studies related to conformational change in large-scale protein machines.

Discovery of novel IDO1 inhibitors via structure-based virtual screening and biological assays.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a heme-containing enzyme that catalyzes the first and rate-limiting step in catabolism of tryptophan via the kynurenine pathway, which plays a pivotal role in the proliferation and differentiation of T cells. IDO1 has been proven to be an attractive target for many diseases, such as breast cancer, lung cancer, colon cancer, prostate cancer, etc. In this study, docking-based virtual screening and bioassays were conducted to identify novel inhibitors of IDO1. The cellular assay demonstrated that 24 compounds exhibited potent inhibitory activity against IDO1 at micromolar level, including 8 compounds with IC50 values below 10 µM and the most potent one (compound 1) with IC50 of 1.18 ± 0.04 µM. Further lead optimization based on similarity searching strategy led to the discovery of compound 28 as an excellent inhibitor with IC50 of 0.27 ± 0.02 µM. Then, the structure-activity relationship of compounds 1, 2, 8 and 14 analogues is discussed. The interaction modes of two compounds against IDO1 were further explored through a Python Based Metal Center Parameter Builder (MCPB.py) molecular dynamics simulation, binding free energy calculation and electrostatic potential analysis. The novel IDO1 inhibitors of compound 1 and its analogues could be considered as promising scaffold for further development of IDO1 inhibitors.

Systematically identifying the anti-inflammatory constituents of Cimicifuga dahurica by UPLC-Q/TOF-MS combined with network pharmacology analysis.

Cimicifuga dahurica (Turcz.) Maxim, which is also regarded as the main origin of "Shengma" in the Chinese Pharmacopoeia, has been used as a cooling and detoxification agent for thousands of years. Our previous phytochemical investigations of C. dahurica extracts (CDEs) led to the isolation of a series of 9,19-cycloalkane triterpenoids and phenolic acids showing a potential anti-inflammatory activity. However, the chemical profiling of CDEs and the material basis of its anti-inflammatory effect in vivo has not been clarified. In the present study, the CDE chemical profile and prototype components in rat plasma were identified via ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. As a result, a total of 106 components were identified or tentatively characterized in CDEs, including 54 triterpenoids, 35 phenolic acids, eight amides and nine other type constituents (39 compounds were confirmed with the reference standards). In addition, 20 prototype components (15 triterpenoids and five phenolic acids) were identified in rat plasma, which potentially related to the anti-inflammatory effects of CDEs. Moreover, the anti-inflammatory activities of the main prototype components were further evaluated by their inhibitory effects on the production of NO, as well as the expressions of iNOS and COX-2 in lipopolysaccharide-stimulated RAW264.7 cells, which indicated that 9,19-cycloalkane triterpenoids may play an anti-inflammatory role by down-regulating the expression of iNOS.

Metabolic profiles of Jin-hong tablets in rats by ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry.

Jin-hong tablets (JHTs), a well-known traditional Chinese patent medicine (TCPM), have been effectively used for the treatment of chronic superficial gastritis (CSG). The metabolic profile of TCPMs is performed to determine their bioactive components. In this study, a five-step strategy based on ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry and metabolynx™ software combined with mass defect filter technique was developed to delineate the metabolic profile of JHT in vivo. As a result, a total of 163 JHT-related xenobiotics (38 prototypes and 125 metabolites) were identified or tentatively characterized in rat biological samples, and the phase I and II metabolism processes mainly included demethylation, hydroxylation, sulfation, and glucuronidation. In addition, after oral administration of JHT, a large amount of alkaloid-related ingredients was detected in rat plasma samples, indicating that alkaloids may play an important role in the treatment of CSG with JHT. This study is beneficial for understanding the JHT's in vivo metabolic profiles and characteristics, which helps to reveal its in vivo effective components and provides a solid basis for further studies on its functional mechanism.

4-Hydroxy Pyridones from Heterologous Expression and Cultivation of the Native Host.

4-Hydroxy pyridones are a class of fungi-derived polyketide-nonribosomal peptide products featuring a core of 4-hydroxy-2-pyridone which have a wide range of biological activities. Genome mining of in-house strains using polyketide synthase-nonribosomal peptide synthase as a query identified an endophyte Tolypocladium sp. 49Y, which possesses a potential 4-hydroxy pyridone biosynthetic gene cluster. Heterologous expression in Aspergillus oryzae NSAR1 revealed that this gene cluster is functional and able to produce a rare type of 4-hydroxy pyridones called tolypyridones (compounds 3 and 4). Tolypocladium sp. 49Y was grown in a variety of media which led to the isolation of six 4-hydroxy pyridones (5-10) and one pyrrolidone (11) from a rice culture, and compounds 3 and 9 showed antifungal activity. These latter compounds are different from those obtained by heterologous expression. This study shows that both heterologous expression and cultivation of the native host are complementary approaches to discover new natural products.

Computational study on the selective inhibition mechanism of MS402 to the first and second bromodomains of BRD4.

As a member of the bromodomain and extraterminal domain (BET) family, BRD4 is considered as a potential target for cancer treatment. However, because of the highly conservation of its two homologous bromodomains (BD1/BD2), selective inhibition of each bromodomain remains a challenge. MS402 is a domain-selective inhibitor of BRD4-BD1 over BRD4-BD2 reported recently. Understanding the selectivity mechanism would be very useful for the further design of more potent BD1-selectivity inhibitors. Molecular dynamics simulation, adaptive biasing force and multiple-walker adaptive biasing force were performed to study the inhibition and domain-selective mechanism of MS402 toward BRD4-BD1 over BRD4-BD2 here. Results demonstrate BRD4-BD1 binds to MS402 with lower binding free energy than BRD4-BD2. Residues Gln85, Pro86, Asn140, and Ile146 are crucial for MS402's selectively binding to BRD4-BD1. MS402 needs to overcome more energy barrier to dissociate from BD1 than from BD2 pocket. These findings will be helpful for rational structural modification of existing inhibitors to increase their BD1-selectivity.

Fluorophore-Dependent Cleavage of Disulfide Bond Leading to a Highly Selective Fluorescent Probe of Thioredoxin.

Finding specific small molecule probes of a biological target is extremely desired but remains a big challenge. We reported herein a highly selective fluorescent probe derivatized from the nile blue fluorophore, NBL-SS, for thioredoxin (Trx), a ubiquitous redox-regulating protein essentially involved in cell growth, differentiation, and death. NBL-SS displayed multiple favorable properties, such as red emission, fast response, and high fluorescence signal, which enabled the probe to readily image Trx functions in live cells and in vivo. The fluorophore-dependent selectivity indicates that manipulation of weak interactions between probes and their target biomacromolecules could further improve the probes' specificity. In addition, our discovery, i.e., the preference reduction of simple disulfide bonds by Trx over glutathione, also advances the development of disulfide cleavage-based probes, prodrugs, and theranostic agents.

Targeting Thioredoxin Reductase by Ibrutinib Promotes Apoptosis of SMMC-7721 Cells.

Ibrutinib (IBT), the first-in-class inhibitor of Bruton's tyrosine kinase (BTK), has demonstrated clinical activity against various B-cell malignancies. Aside from its therapeutic mechanism through BTK inhibition, IBT has other target sites reported for cancer therapy, leading us to investigate whether IBT has unreported targets. Our study revealed that IBT can inhibit SMMC-7721 cells through irreversible inhibition of mammalian thioredoxin reductase enzymes. Further study demonstrated that IBT can cause cellular reactive oxygen species elevation and induce cancer cell apoptosis. The discovery of a new target of IBT sheds light on better understanding its anticancer mechanisms and provides a theoretical foundation for its further use in clinical therapy.

Virtual screening-guided discovery of thioredoxin reductase inhibitors.

The selenoprotein thioredoxin reductase (TXNRD) is a promising therapeutic target for cancer. To discover novel TXNRD inhibitors, a library of α, ß-unsaturated carbonyl compounds were applied in structure-based virtual screening for the selection of hit compounds. Fifteen top-ranked compounds were further validated experimentally, exhibiting potent inhibition of TXNRD and remarkable cytotoxicity to cancer cells. The further binding mode analysis indicated that multiple noncovalent interactions between the inhibitors and the active pocket of TXNRD facilitated the formation of covalent bonds between the Sec498 on TXNRD and the α, ß-unsaturated carbonyl groups on inhibitors. Results from both simulations and experiments demonstrated that Sec498 is the prime interaction site for the inhibition of TXNRD. Taking compound 7 as an example, the inhibition of TXNRD by compounds promoted oxidative stress-mediated apoptosis of cancer cells. Given these findings, novel TXNRD inhibitors may be discovered and introduced to the growing fields of small molecule drugs against TXNRD.

Molecular dynamics simulation, binding free energy calculation and unbinding pathway analysis on selectivity difference between FKBP51 and FKBP52: Insight into the molecular mechanism of isoform selectivity.

As co-chaperones of the 90-kDa heat shock protein(HSP90), FK506 binding protein 51 (FKBP51) and FK506 binding protein 52 (FKBP52) modulate the maturation of steroid hormone receptor through their specific FK1 domains (FKBP12-like domain 1). The inhibitors targeting FK1 domains are potential therapies for endocrine-related physiological disorders. However, the structural conservation of the FK1 domains between FKBP51 and FKBP52 make it difficult to obtain satisfactory selectivity in FK506-based drug design. Fortunately, a series of iFit ligands synthesized by Hausch et al exhibited excellent selectivity for FKBP51, providing new opportunity for design selective inhibitors. We performed molecular dynamics simulation, binding free energy calculation and unbinding pathway analysis to reveal selective mechanism for the inhibitor iFit4 binding with FKBP51 and FKBP52. The conformational stability evaluation of the "Phe67-in" and "Phe67-out" states implies that FKBP51 and FKBP52 have different preferences for "Phe67-in" and "Phe67-out" states, which we suggest as the determinant factor for the selectivity for FKBP51. The binding free energy calculations demonstrate that nonpolar interaction is favorable for the inhibitors binding, while the polar interaction and entropy contribution are adverse for the inhibitors binding. According to the results from binding free energy decomposition, the electrostatic difference of residue 85 causes the most significant thermodynamics effects on the binding of iFit4 to FKBP51 and FKBP52. Furthermore, the importance of substructure units on iFit4 were further evaluated by unbinding pathway analysis and residue-residue contact analysis between iFit4 and the proteins. The results will provide new clues for the design of selective inhibitors for FKBP51.

Xanthatin Promotes Apoptosis via Inhibiting Thioredoxin Reductase and Eliciting Oxidative Stress.

Xanthatin (XT), a naturally occurring sesquiterpene lactone presented in cocklebur ( Xanthium strumarium L.), is under development as a potential anticancer agent. Despite the promising anticancer effect of XT, the molecular mechanism underlying its cellular action has not been well elucidated. The mammalian thioredoxin reductase (TrxR) enzymes, the essential seleno-flavoproteins containing a penultimate selenocysteine (Sec) residue at the C-terminus, represent a promising target for cancer chemotherapeutic agents. In this study, XT inhibits both the purified TrxR and the enzyme in cells. The possible binding mode of XT with the TrxR protein is predicted by the covalent docking method. Mechanism studies reveal that XT targets the Sec residue of TrxR and inhibits the enzyme activity irreversibly. Simultaneously, the inhibition of TrxR by XT promotes the oxidative stress-mediated apoptosis of HeLa cells. Importantly, the knockdown of the enzyme sensitizes the cells to XT treatment. Targeting TrxR thus discloses a novel molecular mechanism in accounting for the cellular action of XT and provides insights into the development of XT as an anticancer agent.

Understanding the structural and energetic basis of PD-1 and monoclonal antibodies bound to PD-L1: A molecular modeling perspective.

BACKGROUND: The inhibitors blocking the interaction between programmed cell death protein 1(PD-1) and programmed death-ligand 1(PD-L1) can activate the immune response of T cell and eliminate cancer cells. The crystallographic studies have provided structural insights of the interactive interfaces between PD-L1 and its protein ligands. However, the hotspot residues on PD-L1 as well as structural and energetic basis for different protein ligands still need to be further investigated. METHODS: Molecular modeling methods including molecular dynamics simulation, per-residue free energy decomposition, virtual alanine scanning mutagenesis and residue-residue contact analysis were used to qualitatively and quantitatively analyze the interactions between PD-L1 and different protein ligands. RESULTS: The results of virtual alanine scanning mutagenesis suggest that Y56, Q66, M115, D122, Y123, R125 are the hotspot residues on PD-L1. The residue-residue contact analysis further shows that PD-1 interacts with PD-L1 mainly by F and G strands while monoclonal antibodies like avelumab and BMS-936559 mainly interact with PD-L1 by CDR2 and CDR3 loops of the heavy chain. CONCLUSIONS: A structurally similar ß-hairpin peptide with 13 or 14 residues was extracted from each protein ligand and these ß-hairpin peptides were found tightly binding to the putative hotspot residues on PD-L1. GENERAL SIGNIFICANCE: This study recognizes the hotspot residues on PD-L1 and uncovers the common structural and energetic basis of different protein ligands binding to PD-L1. These results will be valuable for the design of small molecule or peptide inhibitors targeting on PD-L1.

Investigate the mechanisms of Chinese medicine Fuzhengkangai towards EGFR mutation-positive lung adenocarcinomas by network pharmacology.

BACKGROUND: Chinese traditional herbal medicine Fuzhengkangai (FZKA) formulation combination with gefitinib can overcome drug resistance and improve the prognosis of lung adenocarcinoma patients. However, the pharmacological and molecular mechanisms underlying the active ingredients, potential targets, and overcome drug resistance of the drug are still unclear. Therefore, it is necessary to explore the molecular mechanism of FZKA. METHODS: A systems pharmacology and bioinformatics-based approach was employed to investigate the molecular pathogenesis of EGFR-TKI resistance with clinically effective herb formula. The differential gene expressions between EGFR-TKI sensitive and resistance cell lines were calculated and used to find overlap from targets as core targets. The prognosis of core targets was validated from the cancer genome atlas (TCGA) database by Cox regression. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment is applied to analysis core targets for revealing mechanism in biology. RESULTS: The results showed that 35 active compounds of FZKA can interact with eight core targets proteins (ADRB2, BCL2, CDKN1A, HTR2C, KCNMA1, PLA2G4A, PRKCA and LYZ). The risk score of them were associated with overall survival and relapse free time (HR = 6.604, 95% CI: 2.314-18.850; HR = 5.132, 95% CI: 1.531-17.220). The pathway enrichment suggested that they involved in EGFR-TKI resistance and non-small cell lung cancer pathways, which directly affect EGFR-TKI resistance. The molecular docking showed that licochalcone a and beta-sitosterol can closely bind two targets (BCL2 and PRKCA) that involved in EGFR-TKI resistance pathway. CONCLUSIONS: This study provided a workflow for understanding mechanism of CHM for against drug resistance.

Ligand induced change of ß2 adrenergic receptor from active to inactive conformation and its implication for the closed/open state of the water channel: insight from molecular dynamics simulation, free energy calculation and Markov state model analysis.

The reported crystal structures of ß2 adrenergic receptor (ß2AR) reveal that the open and closed states of the water channel are correlated with the inactive and active conformations of ß2AR. However, more details about the process by which the water channel states are affected by the active to inactive conformational change of ß2AR remain illusive. In this work, molecular dynamics simulations are performed to study the dynamical inactive and active conformational change of ß2AR induced by inverse agonist ICI 118,551. Markov state model analysis and free energy calculation are employed to explore the open and close states of the water channel. The simulation results show that inverse agonist ICI 118,551 can induce water channel opening during the conformational transition of ß2AR. Markov state model (MSM) analysis proves that the energy contour can be divided into seven states. States S1, S2 and S5, which represent the active conformation of ß2AR, show that the water channel is in the closed state, while states S4 and S6, which correspond to the intermediate state conformation of ß2AR, indicate the water channel opens gradually. State S7, which represents the inactive structure of ß2AR, corresponds to the full open state of the water channel. The opening mechanism of the water channel is involved in the ligand-induced conformational change of ß2AR. These results can provide useful information for understanding the opening mechanism of the water channel and will be useful for the rational design of potent inverse agonists of ß2AR.

Revealing the conformational dynamics of UDP-GlcNAc recognition by O-GlcNAc transferase via Markov state model.

The O-linked N-acetylglucosamine (O-GlcNAc) glycosylation is a critical post-translational modification and closely linked to various physiological and pathological conditions. The O-GlcNAc transferase (OGT) functions as the only glycosyltransferase of O-GlcNAc glycosylation by transferring GlcNAc from UDP-GlcNAc to serine or threonine residues on protein substrates. The interaction mode of UDP-GlcNAc against OGT has been preliminarily revealed by the crystal structures, yet an atomic-level comprehension for the conformational dynamics of the recognition process remains elusive. Here, we construct the Markov state model based on extensive all-atom molecular dynamics (MD) simulations with an aggregated simulation time of ∼9 µs, and reveal that the UDP-GlcNAc recognition process by OGT encompasses four key metastable states, occurring within an estimated timescale of ∼10 µs. During UDP-GlcNAc recognition process, we find the pyrophosphate moiety (P2O52-) initially anchors to the active pocket via salt bridge and hydrogen bonds, facilitating subsequent binding of the uridine and GlcNAc moieties. Furthermore, the functional roles of K842 involved in the salt bridge with P2O52- were evaluated through extra mutant MD simulations. Overall, our study provides valuable insights into the UDP-GlcNAc recognition mechanism by OGT, which could further aid in mechanistic studies of O-GlcNAc glycosylation and drug development targeting on OGT.

Studies of the Mechanism of Nucleosome Dynamics: A Review on Multifactorial Regulation from Computational and Experimental Cases.

The nucleosome, which organizes the long coil of genomic DNA in a highly condensed, polymeric way, is thought to be the basic unit of chromosomal structure. As the most important protein-DNA complex, its structural and dynamic features have been successively revealed in recent years. However, its regulatory mechanism, which is modulated by multiple factors, still requires systemic discussion. This study summarizes the regulatory factors of the nucleosome's dynamic features from the perspective of histone modification, DNA methylation, and the nucleosome-interacting factors (transcription factors and nucleosome-remodeling proteins and cations) and focuses on the research exploring the molecular mechanism through both computational and experimental approaches. The regulatory factors that affect the dynamic features of nucleosomes are also discussed in detail, such as unwrapping, wrapping, sliding, and stacking. Due to the complexity of the high-order topological structures of nucleosomes and the comprehensive effects of regulatory factors, the research on the functional modulation mechanism of nucleosomes has encountered great challenges. The integration of computational and experimental approaches, the construction of physical modes for nucleosomes, and the application of deep learning techniques will provide promising opportunities for further exploration.

A predicted structure of NADPH Oxidase 1 identifies key components of ROS generation and strategies for inhibition.

NADPH oxidase 1 (NOX1) is primarily expressed in epithelial cells and responsible for local generation of reactive oxygen species (ROS). By specifically manipulating the local redox microenvironment, NOX1 actively engages in epithelial immunity, especially in colorectal and pulmonary epithelia. To unravel the structural basis of NOX1 engaged epithelial immune processes, a predicted structure model was established using RaptorX deep learning models. The predicted structure model illustrates a 6-transmembrane domain structure, a FAD binding domain, and an NADPH binding/NOXO1 interacting region. The substrate/cofactor binding scheme with respect to this proposed model highly correlates with published reports and is verified in our site-directed mutagenesis assays. An electron transport chain, from NADPH to FAD and the two heme groups, was well supported by the predicted model. Through molecular docking analysis of various small molecule NOX1 inhibitors and subsequent experimental validation, we identified pronounced active sites for potent NOX1 inhibition. Specifically, LEU60, VAL71, MET181, LEU185, HIS208, PHE211, TYR214, and TYR280 in the transmembrane domain form an active pocket for insertion of the small molecule inhibitors to inhibit electron transfer between the heme groups, thus affecting extracellular ROS generation. Altogether, our study provides structural information to help elucidate the role of NOX1 in epithelial generation of ROS and sheds light on the development of therapeutics for NOX1 related illnesses.