Plasma-Droplet Fusion-Mass Spectrometry Reveals Sub-Millisecond Protein Unfolding Dynamics Induced by Reactive Oxygen Species.

Investigating the connection between reactive oxygen species (ROS) and oxidative protein unfolding is critical to reveal the mechanisms underlying disease involving elevated ROS and protein misfolding. This could inform the development of therapeutics targeting cells based on their redox status. In this study, we developed a plasma-droplet fusion-mass spectrometry platform to rapidly assess protein resilience to ROS. This home-built system fuses ROS generated from the microplasma source with protein microdroplets from a tunable nanospray source. At the droplet-plasma intersection, ROS interact with proteins before entering the mass spectrometer for mass identification and structural characterization. Benefiting from the small-sized microdroplet with adjustable traveling velocity, the platform enables the first sub-millisecond kinetic study of ROS-induced protein unfolding, with a rate constant of approximately 1.81 ms-1. Capturing ROS-induced protein unfolding intermediates and the resultant ligand release dynamics can be extended to many more protein systems. We foresee broad applications for establishing previously undetected protein unfolding events when biologically impactful ROS are enriched in time and space with functional proteins and complexes.

Assembly of Glycopeptides in Living Cells Resembling Viral Infection for Cargo Delivery.

Self-assembly in living cells represents one versatile strategy for drug delivery; however, it suffers from the limited precision and efficiency. Inspired by viral traits, we here report a cascade targeting-hydrolysis-transformation (THT) assembly of glycosylated peptides in living cells holistically resembling viral infection for efficient cargo delivery and combined tumor therapy. We design a glycosylated peptide via incorporating a ß-galactose-serine residue into bola-amphiphilic sequences. Co-assembling of the glycosylated peptide with two counterparts containing irinotecan (IRI) or ligand TSFAEYWNLLSP (PMI) results in formation of the glycosylated co-assemblies SgVEIP, which target cancer cells via ß-galactose-galectin-1 association and undergo galactosidase-induced morphological transformation. While GSH-reduction causes release of IRI from the co-assemblies, the PMI moieties release p53 and facilitate cell death via binding with protein MDM2. Cellular experiments show membrane targeting, endo-/lysosome-mediated internalization and in situ formation of nanofibers in cytoplasm by SgVEIP. This cascade THT process enables efficient delivery of IRI and PMI into cancer cells secreting Gal-1 and overexpressing ß-galactosidase. In vivo studies illustrate enhanced tumor accumulation and retention of the glycosylated co-assemblies, thereby suppressing tumor growth. Our findings demonstrate an in situ assembly strategy mimicking viral infection, thus providing a new route for drug delivery and cancer therapy in the future.

Composite Multidimensional Ion Mobility-Mass Spectrometry for Improved Differentiation of Stereochemical Modifications.

Stereochemical modifications (SCMs), mostly present in the form of d-amino acid substitution, have been increasingly identified from a wide range of neuropeptides and disease-associated biomarker proteins. Traditional mass spectrometry-based SCM identification has been effectively enhanced with technological and strategic advancements in ion mobility spectrometry. With the additional separation provided by ion mobility, SCM-induced structural changes can be probed both in theory and in practice, although the structural resolution for low-abundance SCMs still requires further improvement to enable accurate quantification or unambiguous identification of stereoisomers. Herein, we present a multi-component-enabled multidimensional ion mobility-mass spectrometry (3M-IM-MS) analytical workflow, based upon the metal-enhanced chiral amplification strategy we proposed previously (Nat. Commun., 2019, 5038). Notably, the 3M-IM-MS strategy comprises and features the powerful mathematical tools of continuous wavelet transform and Gaussian fitting-enabled peak splitting. Consequently, the resolving capability of ion mobility spectrometry for SCM analysis has been significantly enhanced, providing mobility profiles with baseline separation and more than fivefold improvement in resolving power and overall resolution. This study represents an alternative toward ultrahigh-resolution structural interrogation of mixtures with very small differences, featuring an important and long-lasting topic in chemical measurement.

Expedited Evaluation of Conformational Stability-Heterogeneity Associations for Crude Polyclonal Antibodies in Response to Conjugate Vaccines.

Conjugate vaccines have been demonstrated to be a promising strategy for immunotherapeutic intervention in substance use disorder, wherein a hapten structurally similar to the target drug is conjugated to an immunogenic carrier protein. The antibodies generated following immunization with these species can provide long-lasting protection against overdose through sequestration of the abused drug in the periphery, which mitigates its ability to cross the blood-brain barrier. However, these antibodies exhibit a high degree of heterogeneity in structure. The resultant variations in chemical and structural compositions have not yet been clearly linked to the stability that directly affects their in vivo functional performance. In this work, we describe a rapid mass-spectrometry-based analytical workflow capable of simultaneous and comprehensive interrogation of the carrier protein-dependent heterogeneity and stability of crude polyclonal antibodies in response to conjugate vaccines. Quantitative collision-induced unfolding-ion mobility-mass spectrometry with an all-ion mode is adapted to rapidly assess the conformational heterogeneity and stability of crude serum antibodies collected from four different vaccine conditions, in an unprecedented manner. A series of bottom-up glycoproteomic experiments was performed to reveal the driving force underlying these observed heterogeneities. Overall, this study not only presents a generally applicable workflow for fast assessment of crude antibody conformational stability and heterogeneity at the intact protein level but also leverages carrier protein optimization as a simple solution to antibody quality control.

Isolation and Reactivity of Homoleptic Diphosphene Lead Complexes.

Due to their limited capacity for π-backdonation, isolation of π-complexes of main-group elements remains a great challenge. We report herein the synthesis of a homoleptic diphosphene lead complex (2) from the degradation of P4 with a bis(germylene)-stabilized Pb(0) complex. Structural and computational studies showed that 2 possesses significant π bonding interactions between Pb atom and diphosphene ligands, which is reminiscent of transition-metal diphosphene complexes. Consistent with its unique electronic structure, complex 2 can deliver Pb(0) atoms to perform redox reaction with an iminoquinone to produce a cyclic plumbylene (4) and perform 2,5-dimethyl-3,4-dimethylimidazol-1-ylidene (IMe2 Me2 ) induced phosphorus cation abstraction to give an anionic PbP3 complex (6).

Artificial Peptide-Protein Necrosomes Promote Cell Death.

The presence of disordered region or large interacting surface within proteins significantly challenges the development of targeted drugs, commonly known as the "undruggable" issue. Here, we report a heterogeneous peptide-protein assembling strategy to selectively phosphorylate proteins, thereby activating the necroptotic signaling pathway and promoting cell necroptosis. Inspired by the structures of natural necrosomes formed by receptor interacting protein kinases (RIPK) 1 and 3, the kinase-biomimetic peptides are rationally designed by incorporating natural or D -amino acids, or connecting D -amino acids in a retro-inverso (DRI) manner, leading to one RIPK3-biomimetic peptide PR3 and three RIPK1-biomimetic peptides. Individual peptides undergo self-assembly into nanofibrils, whereas mixing RIPK1-biomimetic peptides with PR3 accelerates and enhances assembly of PR3. In particular, RIPK1-biomimetic peptide DRI-PR1 exhibits reliable binding affinity with protein RIPK3, resulting in specific cytotoxicity to colon cancer cells that overexpress RIPK3. Mechanistic studies reveal the increased phosphorylation of RIPK3 induced by RIPK1-biomimetic peptides, elucidating the activation of the necroptotic signaling pathway responsible for cell death without an obvious increase in secretion of inflammatory cytokines. Our findings highlight the potential of peptide-protein hybrid aggregation as a promising approach to address the "undruggable" issue and provide alternative strategies for overcoming cancer resistance in the future.

Nanosecond Photochemical Reaction for Enhanced Identification, Quantification, and Visualization of Primary Amine-Containing Metabolites by MALDI-Mass Spectrometry.

Many metabolites, including amino acids, neurotransmitters, and pharmaceuticals, contain primary amine functional groups. The analysis of these molecules by mass spectrometry (MS) plays an important role in the study of cancers and psychogenic diseases. However, the MS-based detection and visualization of these bioactive metabolites directly from real biological systems still suffer from challenges such as low ionization efficiency and/or matrix interference effects. Here, we introduce a simple and efficient strategy, the nanosecond photochemical reaction (nsPCR)-enabled fast chemical derivatization, enabling direct MS analysis of primary amine-containing metabolites, with enhanced detection sensitivity for numerous metabolites from cell culture medium and rat brain sections. Furthermore, this nsPCR-based chemical derivatization strategy was demonstrated to be a useful visualizing tool that could provide improved spatial information for these metabolites, potentially offering alternative tools for gaining novel insights into metabolic events.

Native Ion Mobility-Mass Spectrometry-Enabled Fast Structural Interrogation of Labile Protein Surface Modifications at the Intact Protein Level.

Protein sialylation has been closely linked to many diseases including Alzheimer's disease (AD). It is also broadly implicated in therapeutics operating in a pattern-dependent (e.g., Neu5Ac vs Neu5Gc) manner. However, how the sialylation pattern affects the AD-associated, transferrin-assisted iron/Aß cellular uptake process remains largely ill-defined. Herein, we report the use of native ion mobility-mass spectrometry (IM-MS)-based fast structural probing methodology, enabling well-controlled, synergistic, and in situ manipulation of mature glycoproteins and attached sialic acids. IM-MS-centered experiments enable the combinatorial interrogation of sialylation effects on Aß cytotoxicity and the chemical, conformational, and topological stabilities of transferrin. Cell viability experiments suggest that Neu5Gc replacement enhances the transferrin-assisted, iron loading-associated Aß cytotoxicity. Native gel electrophoresis and IM-MS reveal that sialylation stabilizes transferrin conformation but inhibits its dimerization. Collectively, IM-MS is adapted to capture key sialylation intermediates involved in fine-tuning AD-associated glycoprotein structural microheterogeneity. Our results provide the molecular basis for the importance of sustaining moderate TF sialylation levels, especially Neu5Ac, in promoting iron cellular transportation and rescuing iron-enhanced Aß cytotoxicity.

[Mechanism of Wuwei Ganlu in treatment of knee osteoarthritis:a study based on network pharmacology and molecular docking].

Wuwei Ganlu, a formula for medicated bath, consists of medicinal materials of Ephedra sinica, Platycladus orientalis, Myricaria squamosa, Artemisia carvifolia, and Rhododendron anthopogonoides, which is effective in inducing perspiration, resisting inflammation, relieving pain, regulating yellow water disease, and activating blood circulation. On this basis, a variety of formulas for Tibetan medicated bath have been derived for the treatment of diseases in internal organs, joints, nerves, etc. Modern studies have confirmed that Wuwei Ganlu has a good therapeutic efficacy on knee osteoarthritis(KOA). The present study explored the mechanism of Wuwei Ganlu in treating KOA based on network pharmacology and molecular docking. Firstly, the chemical components of Wuwei Ganlu were obtained through literature mining and database retrieval, and corresponding potential targets were predicted according to the BATMAN-TCM database. The protein-protein interaction(PPI) network was obtained after the potential targets were input into the STRING database. The network function modules were analyzed by the Molecular Complex Detection(MCODE) algorithm, and the functions of the modules were annotated to analyze the action mode of Wuwei Ganlu. Secondly, the related targets of KOA were collected through the DisGeNET database, and the overlapping targets were confirmed to analyze the mechanism of Wuwei Ganlu in treating KOA. Finally, the key targets were selected for molecular docking with the main components of Wuwei Ganlu to verify the component-target interaction. A total of 550 chemical components and 1 365 potential targets of Wuwei Ganlu were obtained. PPI analysis indicated that this formula could exert the effects of oxidation-reduction, inflammation resistance, bone absorption, bone mineralization, etc. Nineteen common targets were obtained from the intersection of potential targets of Wuwei Ganlu and KOA disease targets. It was found that the Wuwei Ganlu mainly acts on nuclear factor-κB(NF-κB), interleukin-1 beta(IL1ß), tumor necrosis factor(TNF), IL6, IL1 receptor antagonist(IL1 RN), and prostaglandin-endoperoxide synthase-2(PTGS2) to treat KOA. Among the 550 chemical components of Wuwei Ganlu, 252 potential active components were docked with TNF and 163 with PTGS2, indicating good binding of the components with potential key targets. The study preliminarily explored the mechanism of Wuwei Ganlu in treating KOA to provide a reference for the further development and utilization of Tibetan medicated bath that has been included in the UN Intangible Cultural Heritage.

On-Tissue Derivatization with Girard's Reagent P Enhances N-Glycan Signals for Formalin-Fixed Paraffin-Embedded Tissue Sections in MALDI Mass Spectrometry Imaging.

Glycosylation is a major protein post-translational modification whose dysregulation has been associated with many diseases. Herein, an on-tissue chemical derivatization strategy based on positively charged hydrazine reagent (Girard's reagent P) coupled with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was developed for analysis of N-glycans from FFPE treated tissue sections. The performance of the proposed approach was evaluated by analysis of monosaccharides, oligosaccharides, N-glycans released from glycoproteins, as well as MS imaging of N-glycans from human cancer tissue sections. The results demonstrated that the signal-to-noise ratios for target saccharides were notably improved after chemical derivatization, in which signals were enhanced by 230-fold for glucose and over 28-fold for maltooctaose. Improved glycome coverage was obtained for N-glycans derived from glycoproteins and tissue samples after chemical derivatization. Furthermore, on-tissue derivatization was applied for MALDI-MSI of N-glycans from human laryngeal cancer and ovarian cancer tissues. Differentially expressed N-glycans among the tumor region, adjacent normal tissue region, and tumor proximal collagen stroma region were imaged, revealing that high-mannose type N-glycans were predominantly expressed in the tumor region. Overall, our results indicate that the on-tissue labeling strategy coupled with MALDI-MSI shows great potential to spatially characterize N-glycan expression within heterogeneous tissue samples with enhanced sensitivity. This study provides a promising approach to better understand the pathogenesis of cancer related aberrant glycosylation, which is beneficial to the design of improved clinical diagnosis and therapeutic strategies.

Subresidue-Resolution Footprinting of Ligand-Protein Interactions by Carbene Chemistry and Ion Mobility-Mass Spectrometry.

The knowledge of ligand-protein interactions is essential for understanding fundamental biological processes and for the rational design of drugs that target such processes. Carbene footprinting efficiently labels proteinaceous residues and has been used with mass spectrometry (MS) to map ligand-protein interactions. Nevertheless, previous footprinting studies are typically performed at the residue level, and therefore, the resolution may not be high enough to couple with conventional crystallography techniques. Herein we developed a subresidue footprinting strategy based on the discovery that carbene labeling produces subresidue peptide isomers and the intensity changes of these isomers in response to ligand binding can be exploited to delineate ligand-protein topography at the subresidue level. The established workflow combines carbene footprinting, extended liquid chromatographic separation, and ion mobility (IM)-MS for efficient separation and identification of subresidue isomers. Analysis of representative subresidue isomers located within the binding cleft of lysozyme and those produced from an amyloid-ß segment have both uncovered structural information heretofore unavailable by residue-level footprinting. Lastly, a "real-world" application shows that the reactivity changes of subresidue isomers at Phe399 can identify the interactive nuances between estrogen-related receptor α, a potential drug target for cancer and metabolic diseases, with its three ligands. These findings have significant implications for drug design. Taken together, we envision the subresidue-level resolution enabled by IM-MS-coupled carbene footprinting can bridge the gap between structural MS and the more-established biophysical tools and ultimately facilitate diverse applications for fundamental research and pharmaceutical development.

Ultrafast Microelectrophoresis: Behind Direct Mass Spectrometry Measurements of Proteins and Metabolites in Living Cell/Cells.

Direct chemical profiling and protein identification from living single cells using mass spectrometry (MS) have been demonstrated to further our understanding of biological variability and differential susceptibility to several diseases and treatments. Despite the great challenge from extremely complicated cytoplasm, we recently proposed a versatile MS strategy to achieve direct mass spectrometric characterization of both proteins and metabolite-like small molecules directly from living cells or single cells. Although the capability to directly handle cell cytoplasm was presumably attributed to microelectrophoresis in our previous studies, the assumption had only been partially explored by some preliminary experiments. To better understand the mechanism, herein, we systematically characterized its separation behavior with a series of model compounds covering a wide range of molecular size. With the merit of in situ separation, microelectrophoresis herein has been further demonstrated as an attractive and alternative tool, which can potentially contribute to direct MS measurements of more protein interactions or metabolic pathways in living single cells or a few cells.

Characterizing and alleviating ion suppression effects in atmospheric pressure matrix-assisted laser desorption/ionization.

RATIONALE: As a powerful ambient ion source, atmospheric pressure (AP) matrix-assisted laser desorption/ionization (MALDI) enables direct analysis at atmospheric pressure/temperature and minimal sample preparation. With the increasing usage of AP-MALDI sources with Orbitrap instruments, systematic characterization of the extent of ion suppression effect (ISE) in AP-MALDI-Orbitrap mass spectrometry imaging (MSI) is desirable. Recently, a new low-pressure MALDI platform has been introduced that reportedly provided better sensitivity. While extensive research efforts have been devoted to improving spatial resolution, fewer studies focused on the characterization and sensitivity improvement of these MALDI platforms that, coupled with high-resolution Orbitraps, provide powerful strategy for MSI. METHODS: We compared the analytical performance of AP and low-pressure (subatmospheric) MALDI sources to study the effect of pressure control in the ion source. Using a model peptide/protein mixture, we systematically evaluated the factors influencing ISE. Furthermore, the effect of laser spot size was evaluated through tissue imaging analysis of lipids and neuropeptides. The effects of ion suppression and laser spot size have also been examined by comparing the number of identified molecular species during MSI analysis. RESULTS: Several key operating parameters including source pressure, laser energy, laser repetition rate, and microscopic slide coating materials were optimized to minimize the ISE. Under the optimal conditions, the subatmospheric AP-MALDI-Orbitrap platform with high spatial and mass spectral resolution enabled significantly improved coverage of several lipid and neuropeptide families in the MS analysis of mouse brain tissue sections. CONCLUSIONS: The new SubAP-MALDI source coupled with an Orbitrap mass spectrometer was established as a viable platform for in situ endogenous biomolecular analysis with increased sensitivity compared with conventional AP-MALDI sources as evidenced by the confident identification of neuropeptides from mouse brain imaging analyses. The alleviated ISE was key to substantial performance improvement due to optimized intermediate pressure conditions and better ion collection by the ion funnel.

Charge-dependent modulation of specific and nonspecific protein-metal ion interactions in nanoelectrospray ionization mass spectrometry.

RATIONALE: Previous studies found that charge state could affect both specific and nonspecific binding of protein-metal ion interactions in nanoelectrospray ionization mass spectrometry (nESI-MS). However, the two kinds of interactions have been studied individually in spite of the problem that they often coexist in the same system. Thus, it is necessary to study the effects of charge state on specific and nonspecific protein-metal ion interactions in one system to reveal more accurate binding state. METHODS: The HIV-1 nucleocapsid protein (NCp7(31-55)) which can bind specifically and nonspecifically to Zn2+ served as the model to show the charge-dependent protein-metal ion interactions. Hydrogen/deuterium exchange (HDX) and photodissociation (PD) were used to demonstrate that specific binding state was correlated with protein structure. In addition to NCp7(31-55), three other model proteins were used to investigate the reason for the charge-dependent nonspecific binding. RESULTS: For specific binding, we proposed that protein ions with different charge states had different conformations. The HDX results showed that labile protons in the NCp7(31-55)-Zn complex were exchanged in a charge-state-dependent way. The PD experiments revealed differential fragment yields for different charge states. For nonspecific binding, higher charge states had more Zn2+ additions, but less SO4 2- additions. The effects of charge states on nonspecific binding levels were entirely the opposite for Zn2+ and SO4 2- . These results could reveal that the nonspecific binding was caused by electrostatic interaction. CONCLUSIONS: For specific binding, NCp7(31-55) with lower charge states have folding and undenatured structures. The binding states of lower charge states can better reflect more native binding states. For nonspecific binding, when multiple metal ions adduct to proteins, the proteins have more net positive charges, which tend to generate higher charge ions during electrospray.

In Situ Living Cell Protein Analysis by Single-Step Mass Spectrometry.

In situ living cell protein analysis would enable the structural identification and functional interrogation of intracellular proteins in native cellular environments. Previously, we have presented an in situ mass spectrometry (MS) strategy to identify protein and protein/metal ion complex with relatively small molecular weight ( Anal. Chem. 2016, 88, 10860-10866). However, it is still challenging to directly identify larger proteins and protein/ligand complexes in cell, due to numerous nonspecific bindings of ligands, solvents, and other cellular constituents. Here we present a versatile single-step mass spectrometric strategy, "in-cell" mass spectrometry ("in-cell" MS), for in situ protein identification and dynamic protein-ligand interaction monitoring directly from living cells. "In-cell" MS combined all-ion-fragmentation mode with our previous method; thus, on a high-resolution MS instrument, we can greatly improve the signal/noise ratio of the larger proteins and protein/ligand complexes. Meanwhile, we also achieved a much wider mass range for protein complex and detection of 17 proteins with molecular weight ranging from 4 to 44 kDa. In addition, "in-cell" MS could also monitor dynamic protein interactions in living cells. Calcium-regulated calmodulin-melittin interaction was tested to demonstrate the proof of concept. "In-cell" MS provides an alternative for in situ analysis of living cells, which might contribute to rapid protein analysis and quality control in biochemistry laboratories, protein engineering, and even protein industry.

Reliable Tracking In-Solution Protein Unfolding via Ultrafast Thermal Unfolding/Ion Mobility-Mass Spectrometry.

Sequential unfolding of monomeric proteins is important for the global understanding of local conformational elements (e.g., secondary structures and domain connections) within those protein assemblies. Ion mobility-mass spectrometry (IM-MS) is an emerging and promising technique for probing gradual protein structural perturbations in the gas phase. However, it is still challenging to track sequential unfolding in the solution phase. Here, we extended IM-MS to track in-solution sequential unfolding of monomeric proteins having single and/or multidomains. The present method combines ultrafast local heating effect (LHE)-driven sequential unfolding with IM-MS identification. Protein sequential unfolding in solution is demonstrated by the rapid and controllable IM-MS data switch between native and gradually unfolded states. Our results show that LHE induces gradual protein conformational transitions associated with biological functions, where IM-MS tracks the sequential unfolding of monomeric proteins.

In Silico Analysis of the Association Relationship between Neuroprotection and Flavors of Traditional Chinese Medicine Based on the mGluRs.

The metabotropic glutamate receptors (mGluRs) are known as both synaptic receptors and taste receptors. This feature is highly similar to the Property and Flavor theory of Traditional Chinese medicine (TCM), which has the pharmacological effect and flavor. In this study, six ligand based pharmacophore (LBP) models, seven homology modeling models, and fourteen molecular docking models of mGluRs were built based on orthosteric and allosteric sites to screening potential compounds from Traditional Chinese Medicine Database (TCMD). Based on the Pharmacopoeia of the People's Republic of China, TCMs of compounds and their flavors were traced and listed. According to the tracing result, we found that the TCMs of the compounds which bound to orthosteric sites of mGluRs are highly correlated to a sweet flavor, while the allosteric site corresponds to a bitter flavor. Meanwhile, the pharmacological effects of TCMs with highly frequent flavors were further analyzed. We found that those TCMs play a neuroprotective role through the efficiencies of detumescence, promoting blood circulation, analgesic effect, and so on. This study provides a guide for developing new neuroprotective drugs from TCMs which target mGluRs. Moreover, it is the first study to present a novel approach to discuss the association relationship between flavor and the neuroprotective mechanism of TCM based on mGluRs.

Discovery of Potential Inhibitors of Squalene Synthase from Traditional Chinese Medicine Based on Virtual Screening and In Vitro Evaluation of Lipid-Lowering Effect.

Squalene synthase (SQS), a key downstream enzyme involved in the cholesterol biosynthetic pathway, plays an important role in treating hyperlipidemia. Compared to statins, SQS inhibitors have shown a very significant lipid-lowering effect and do not cause myotoxicity. Thus, the paper aims to discover potential SQS inhibitors from Traditional Chinese Medicine (TCM) by the combination of molecular modeling methods and biological assays. In this study, cynarin was selected as a potential SQS inhibitor candidate compound based on its pharmacophoric properties, molecular docking studies and molecular dynamics (MD) simulations. Cynarin could form hydrophobic interactions with PHE54, LEU211, LEU183 and PRO292, which are regarded as important interactions for the SQS inhibitors. In addition, the lipid-lowering effect of cynarin was tested in sodium oleate-induced HepG2 cells by decreasing the lipidemic parameter triglyceride (TG) level by 22.50%. Finally. cynarin was reversely screened against other anti-hyperlipidemia targets which existed in HepG2 cells and cynarin was unable to map with the pharmacophore of these targets, which indicated that the lipid-lowering effects of cynarin might be due to the inhibition of SQS. This study discovered cynarin is a potential SQS inhibitor from TCM, which could be further clinically explored for the treatment of hyperlipidemia.

[Synergistic mechanism of traditional Chinese medicine based on target combination of PepT1 and PPARα].

Synergistic effect is main pharmacological mechanism of traditional Chinese medicine(TCM). The research method based on the key targets combination is an important method to explore the synergistic effect of TCM. Peptide transporter 1 (PepT1) is an essential target for drug uptake into the bloodstream, accounting for about 50% of the total transporter protein content from the small intestine. Peroxisome proliferator-activated receptor α(PPARα) is the lipid-lowering target of fibrates, which have a good hypolipidemic effect by activating PPARα. It has been reported that PPARα could activate the gene expression of PepT1s, and PPARα agonists can promote the uptake of PepT1 substrates, indicating their synergistic effect. In this paper, PepT1 substrates and PPARα agonists from TCM were discovered, and their synergistic mechanism was also been discussed based on the target combination of PepT1 and PPARα. The support vector machine(SVM) model of PepT1 substrates was first constructed and utilized to predict potential TCM components. Meanwhile, merged pharmacophore and docking model of PPARα agonists was used to screen the potential active ingredients from TCM. According to the analysis results of two groups, the TCM combination of Panax notoginseng and Ganoderma lucidum, as well as TCM combination of P. notoginseng and Salvia miltiorrhiza were identified to have the synergistic mechanism based on target combination of PepT1 and PPARα. In this study, synergistic mechanism of TCM was analyzed for absorption and hypolipidemic effect based on target combination, which provides a new way to explore the synergetic mechanism of TCM related to pharmacokinetics.

[Prediction of ETA oligopeptides antagonists from Glycine max based on in silico proteolysis].

Oligopeptides are one of the the key pharmaceutical effective constituents of traditional Chinese medicine(TCM). Systematic study on composition and efficacy of TCM oligopeptides is essential for the analysis of material basis and mechanism of TCM. In this study, the potential anti-hypertensive oligopeptides from Glycine max and their endothelin receptor A (ETA) antagonistic activity were discovered and predicted based on in silico technologies.Main protein sequences of G. max were collected and oligopeptides were obtained using in silico gastrointestinal tract proteolysis. Then, the pharmacophore of ETA antagonistic peptides was constructed and included one hydrophobic feature, one ionizable negative feature, one ring aromatic feature and five excluded volumes. Meanwhile, three-dimensional structure of ETA was developed by homology modeling methods for further docking studies. According to docking analysis and consensus score, the key amino acid of GLN165 was identified for ETA antagonistic activity. And 27 oligopeptides from G. max were predicted as the potential ETA antagonists by pharmacophore and docking studies.In silico proteolysis could be used to analyze the protein sequences from TCM. According to combination of in silico proteolysis and molecular simulation, the biological activities of oligopeptides could be predicted rapidly based on the known TCM protein sequence. It might provide the methodology basis for rapidly and efficiently implementing the mechanism analysis of TCM oligopeptides.