A dual-target SPR screening system for simultaneous ligand discovery of SARS-CoV-2 spike protein and its receptor ACE2 from Chinese herbs.

Traditional Chinese Medicine (TCM) is a supremely valuable resource for the development of drug discovery. Few methods are capable of hunting for potential molecule ligands from TCM towards more than one single protein target. In this study, a novel dual-target surface plasmon resonance (SPR) biosensor was developed to perform targeted compound screening of two key proteins involved in the cellular invasion process of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2): the spike (S) protein receptor binding domain (RBD) and the angiotensin-converting enzyme 2 (ACE2). The screening and identification of active compounds from six Chinese herbs were conducted taking into consideration the multi-component and multi-target nature of Traditional Chinese Medicine (TCM). Puerarin from Radix Puerariae Lobatae was discovered to exhibit specific binding affinity to both S protein RBD and ACE2. The results highlight the efficiency of the dual-target SPR system in drug screening and provide a novel approach for exploring the targeted mechanisms of active components from Chinese herbs for disease treatment.

Iron deficiency in hepatocellular carcinoma cells induced sorafenib resistance by upregulating HIF-1α to inhibit apoptosis.

Sorafenib is the first-line therapeutic agent for hepatocellular carcinoma (HCC), but the drug resistance has become a major impediment. Previously we found that the abnormal iron metabolism in HCC led to iron deficiency, whether it induces sorafenib resistance during the treatment of HCC is not yet disclosed. In this study, we observed the effects of iron deficiency on sorafenib resistance and explored the underlying mechanisms. The results revealed that the killing effects of sorafenib on HCC cells were weakened by iron deficiency but effectively restored by iron re-supplementation. The ferroptosis indicators, including the contents of lipid hydroperoxide (LPO) and malondialdehyde (MDA), the level of intracellular reactive oxygen species (ROS), and the expression of glutathione peroxidase 4 (GPX4), were not significantly changed by iron deficiency in sorafenib-treated HCC cells. However, the sorafenib-induced apoptosis of HCC cells was inhibited by iron deficiency. Notably, the expression of anti-apoptotic protein B-cell lymphoma-2 (BCL-2) was elevated, and the expressions of other apoptotic proteins, BCL2-associated X (Bax), caspase-3, and caspase-9, were inhibited by iron deficiency. Mechanistically, iron deficiency upregulated hypoxia-inducible factor 1 alpha (HIF-1α) to increase BCL-2. Inhibition of HIF-1α suppressed the iron deficiency-induced BCL-2 and sorafenib resistance. In summary, iron deficiency in HCC cells generated sorafenib resistance by increasing HIF-1α and BCL-2, which therefore inhibited the sorafenib-induced apoptosis of HCC cells. These results identified iron deficiency as a new factor of sorafenib resistance in HCC cells, which would be an effective target to alleviate sorafenib resistance.

A multi-omics study of the anti-cancer effect of a ferulic acid derivative FA-30.

The active ingredients of Traditional Chinese Medicine are an important source of bioactive molecules and play an important role in the research and development of innovative drugs. FA-30, which is a derivative of natural product ferulic acid, inhibited cervical cancer cell proliferation and induced apoptosis as well. To understand the underlying mechanisms of FA-30, a complementary multi-omics study was conducted. Cysteine and methionine metabolism and aminoacyl-tRNA biosynthesis pathways were significantly changed both at the metabolic level and proteomic level. This may help us to get a better understanding of cervical cancer and FA-30 at the same time.

A strategy of screening and binding analysis of bioactive components from traditional Chinese medicine based on surface plasmon resonance biosensor.

Elucidating the active components of traditional Chinese medicine (TCM) is essential for understanding the mechanisms of TCM and promote its rational use as well as TCM-derived drug development. Recent studies have shown that surface plasmon resonance (SPR) technology is promising in this field. In the present study, we propose an SPR-based integrated strategy to screen and analyze the major active components of TCM. We used Radix Paeoniae Alba (RPA) as an example to identify the compounds that can account for its anti-inflammatory mechanism via tumor necrosis factor receptor type 1 (TNF-R1). First, RPA extraction was analyzed using an SPR-based screening system, and the potential active ingredients were collected, enriched, and identified as paeoniflorin and paeonol. Next, the affinity constants of paeoniflorin and paeonol were determined as 4.9 and 11.8 µM, respectively. Then, SPR-based competition assays and molecular docking were performed to show that the two compounds could compete with tumor necrosis factor-α (TNF-α) while binding to the subdomain 1 site of TNF-R1. Finally, in biological assays, the two compounds suppressed cytotoxicity and apoptosis induced by TNF-α in the L929 cell line. These findings prove that SPR technology is a useful tool for determining the active ingredients of TCM at the molecular level and can be used in various aspects of drug development. The SPR-based integrated strategy is reliable and feasible in TCM studies and will shed light on the elucidation of the pharmacological mechanism of TCM and facilitate its modernization.

A strategy of screening and binding analysis of bioactive components from traditional Chinese medicine based on surface plasmon resonance biosensor / 药物分析学报

Elucidating the active components of traditional Chinese medicine(TCM)is essential for understanding the mechanisms of TCM and promote its rational use as well as TCM-derived drug development.Recent studies have shown that surface plasmon resonance(SPR)technology is promising in this field.In the present study,we propose an SPR-based integrated strategy to screen and analyze the major active components of TCM.We used Radix Paeoniae Alba(RPA)as an example to identify the compounds that can account for its anti-inflammatory mechanism via tumor necrosis factor receptor type 1(TNF-R1).First,RPA extraction was analyzed using an SPR-based screening system,and the potential active in-gredients were collected,enriched,and identified as paeoniflorin and paeonol.Next,the affinity con-stants of paeoniflorin and paeonol were determined as 4.9 and 11.8 μM,respectively.Then,SPR-based competition assays and molecular docking were performed to show that the two compounds could compete with tumor necrosis factor-α(TNF-α)while binding to the subdomain 1 site of TNF-R1.Finally,in biological assays,the two compounds suppressed cytotoxicity and apoptosis induced by TNF-α in the L929 cell line.These findings prove that SPR technology is a useful tool for determining the active in-gredients of TCM at the molecular level and can be used in various aspects of drug development.The SPR-based integrated strategy is reliable and feasible in TCM studies and will shed light on the eluci-dation of the pharmacological mechanism of TCM and facilitate its modernization.

Surface plasmon resonance biosensor combined with lentiviral particle stabilization strategy for rapid and specific screening of P-Glycoprotein ligands.

A novel surface plasmon resonance-based P-gp ligand screening system (SPR-PLSS) combined with lentiviral particle (LVP) stabilization strategy was constructed to screen out potential P-gp inhibitors from natural products. Firstly, we constructed LVPs with high and low expression levels of P-gp. The LVPs can ensure the natural conformation of P-gp based on the principle that LVPs germinated from packaging cells will contain cell membrane fragments and P-gp they carry. Then the LVPs with high P-gp expression for active channel and LVPs with low P-gp expression for reference channel were immobilized on CM5 chip respectively. The affinity detection was thus carried out with the signal reduction on the two channels. The P-gp inhibitors, Valspodar (Val) and cyclosporin (CsA), as positive compounds, were detected to characterize the chip's activity, and the KD of Val and CsA were 14.09 µM and 16.41 µM, respectively. Forty compounds from natural product library were screened using the SPR CM5 chip, and magnolol (Mag), honokiol (Hon), and resveratrol (Res) were screened out as potential P-gp ligands, showing a significant response signal. This work presented a novel P-gp ligand screening system based on LVP-immobilized biosensor to rapidly screen out P-gp ligands from natural product library. Compared with traditional cell experiments which the screening time may take up to several days, our method only takes several hours. Furthermore, this study has also provided solid evidences to support that some complicated membrane proteins would apply to the lentivirus-based SPR screening system.

Mild iron overload induces TRIP12-mediated degradation of YY1 to trigger hepatic inflammation.

Increasing populations are found to bear mild hepatic iron overload (HIO) due to unhealthy lifestyles, metabolic diseases, etc., whether this mild but chronic HIO induces hepatic inflammation is unknown. In the present study, mice receiving a 12-months 0.3% dextran-iron diet show mild HIO with no detectable oxidative damages in the liver but have infiltrated macrophages and increased IL-6, TNFα, AST and ALT since 6-months. The HNF4α/miR-122/CCL2 pathway, identified by our previous studies to induce macrophages infiltration, is initiated by chronic mild HIO. After excluding the role of DNA methylation, a modified transcription factor microarray is applied to find that transcription factor YY1 is responsible for HIO-decreased HNF4α expression. Then the E3 ubiquitin ligase TRIP12 is identified by an immunoprecipitation coupled LC-MS/MS and proved to bind and ubiquitinate YY1, leading to its degradation. The overexpression or silence of YY1 in the liver regulates the HNF4α/miR-122/CCL2 pathway. More importantly, YY1 overexpression alleviates chronic mild HIO induced hepatic inflammatory responses. In conclusion, these results elucidate an oxidative-stress-independent, TRIP12/YY1/HNF4α/miR-122/CCL2 pathway of chronic mild HIO inducing hepatic inflammation, implying that effective measures in addition to antioxidants are needed for individuals at the risk of chronic mild HIO.

Surface Plasmon Resonance-Based Membrane Protein-Targeted Active Ingredients Recognition Strategy: Construction and Implementation in Ligand Screening from Herbal Medicines.

Membrane proteins (MPs) are playing important roles in several biological processes. Screening new candidate compounds targeting MPs is important for drug discovery. However, it remains challenging to characterize the interactions between MPs and small-molecule ligands in a label-free method. In this study, a surface plasmon resonance (SPR)-based membrane protein-targeted active ingredients recognition strategy was constructed. This strategy contains two major modules: affinity detection module and ligand screening module. Through the combination of these two functional modules, it is feasible to screen small molecular ligands targeting MPs from herbal medicines. First, we have constructed high/low comparative C-X-C chemokine receptor type 4 (CXCR4)-expressed lentiviral particles (LVPs) models and characterized the expression levels. Then we immobilized LVPs on CM5 chips and detected the affinity between AMD3100 and CXCR4 by using affinity detection module. The KD of AMD3100 was 32.48 ± 3.17 nM. Furthermore, the suitability and robustness of the ligand screening module were validated by using AMD3100 as a positive compound. Subsequently, this module was applied in the screening of CXCR4 small molecular ligands from herbal medicine extracts. Senkyunolide I was screened out from Chuanxiong extract. The affinity constant between senkyunolide I and CXCR4 was 2.94 ± 0.36 µM. The Boyden chamber assay revealed that senkyunolide I could inhibit cell migration process. In conclusion, an SPR-based small molecular ligand recognition strategy combined with virus-based membrane protein stabilization method was constructed. The SPR-based membrane protein-targeted active ingredients recognition strategy will be an effective tool to screen target components from complex systems acting on MPs.

Identification of eupatilin and ginkgolide B as p38 ligands from medicinal herbs by surface plasmon resonance biosensor-based active ingredients recognition system.

Screening of bioactive ligands for a certain protein target from medicinal herbs is a highly important yet challenging task during drug discovery process. In this study, a surface plasmon resonance biosensor-based active ingredient recognition system (SPR-AIRS) was applied to screen p38 mitogen-activated protein kinase (p38) ligands from herbal extracts. After p38 protein was immobilized on a SPR chip and the suitability of SPR-AIRS was validated, thirty-four p38-related medicinal herbs were selected and pre-screened. Two medicinal herbs having high response signal with p38-immobilized chip, Folium Ginkgo and Herba Artemisiae Scopariae, were injected into SPR system for ligand fishing. Among them, two active compounds, eupatilin (EPT) and ginkgolide B (GKB), were identified as p38 ligands, and then the KD values of EPT and GKB were measured as 21.68 ± 2.21 and 44.71 ± 1.80 µM, respectively. They can inhibit p38 activities significantly and bind to the ATP binding site on p38. Furthermore, EPT and GKB can inhibit cell proliferation (IC50 = 30.31 ± 6.84 and 42.97 ± 0.83 µM), induce apoptosis and G2/M cell cycle arrest against K562 cell line. This is the first time that EPT and GKB are reported as effective p38 binding ligands. These results prove that SPR-AIRS could be an effective method to screen active compounds acting on a specific protein from complex systems.

Simulation Strategies for Characterizing Phosphodiesterase-5 Inhibitors in Botanical Dietary Supplements.

A novel "Prediction and Confirmation" (PC) strategy was proposed for characterizing phosphodiesterase-5 inhibitor (PDE-5) derivatives in botanical dietary supplements (BDSs) for on-site detection. Discovery Studio (DS) and density functional theory (DFT) calculations were used for the "Prediction" step in order to estimate PDE-5 derivative structures and theoretical Raman shifts without synthesizing the derivatives. After 11 potentially bioactive sildenafil derivatives were acquired through DS, 32 common calculated Raman shifts were obtained through DFT. The mean absolute wavenumber deviation (δ, peak range) of the major bands and the minimum number (τ) of Raman spectral peaks matching the calculated common shifts were optimized, so that a positive result of an unknown sample could be reasonably produced. In this study, δ was set at ±10 cm-1 and the corresponding τ was set at 4-5 after optimization. Surface plasmon resonance (SPR) biosensor and surface-enhanced Raman scattering (SERS) detection were the "Confirmation" step to validate the reliability and accuracy of DS and DFT in the "Prediction" step, respectively. The optimized δ and τ criteria were used as indexes for on-site SERS detection after thin-layer chromatographic (TLC) separation of six real-world samples, one of which was preliminarily identified as "suspected positive samples." This strategy allows for a quick determination of the BDSs adulterated with sildenafil or its derivatives, independent of any standard materials.

Biosensor-Based Active Ingredients Recognition System for Screening STAT3 Ligands from Medical Herbs.

A surface plasmon resonance (SPR) biosensor-based active ingredients recognition system (SPR-AIRS) was developed, validated, and applied to screen signal transducer and activator of transcription 3 (STAT3) ligands. First, features of the screening system were investigated in four aspects: (1) specificity of the STAT3-immobilized chip, it shows that the chip could be applied to screen STAT3 ligands from complex mixture; (2) linearity and limit of detection (LOD) of the system, the minimum recovery cycle number was determined as 5 cycles; (3) saturability of the chip, the results indicate that it is necessary to select a proper concentration based on the compound's Kd value; (4) robustness of the system, it indicates that inactive compounds in the matrix could not interfere with active compounds in the process of screening. Next, SPR-AIRS was applied to screen STAT3 ligands from medicinal herbs. Nine candidate compounds were fished out. Then SPR assay and molecular docking were performed to verify the interplay between STAT3 and candidate compounds. Apoptosis assay and luciferase report assay were performed to investigate the drug effect of candidate compounds on STAT3 activity. Western blot results indicated that neobaicalein and polydatin could inhibit the phosphorylation of STAT3. As far as we know, this is the first time that neobaicalein and polydatin are reported as effective STAT3 ligands. In a conclusion, we have systemically demonstrated the feasibility of SPR biosensor-based screening method applying to complex drug systems, and our findings suggest that SPR-AIRS could be a sensitive and effective solution for the discovery of active compounds from a complex matrix.

A method for screening active components from Chinese herbs by cell membrane chromatography-offline-high performance liquid chromatography/mass spectrometry and an online statistical tool for data processing.

Cell membrane chromatography (CMC) has been successfully applied to screen bioactive compounds from Chinese herbs for many years, and some offline and online two-dimensional (2D) CMC-high performance liquid chromatography (HPLC) hyphenated systems have been established to perform screening assays. However, the requirement of sample preparation steps for the second-dimensional analysis in offline systems and the need for an interface device and technical expertise in the online system limit their extensive use. In the present study, an offline 2D CMC-HPLC analysis combined with the XCMS (various forms of chromatography coupled to mass spectrometry) Online statistical tool for data processing was established. First, our previously reported online 2D screening system was used to analyze three Chinese herbs that were reported to have potential anti-inflammatory effects, and two binding components were identified. By contrast, the proposed offline 2D screening method with XCMS Online analysis was applied, and three more ingredients were discovered in addition to the two compounds revealed by the online system. Then, cross-validation of the three compounds was performed, and they were confirmed to be included in the online data as well, but were not identified there because of their low concentrations and lack of credible statistical approaches. Last, pharmacological experiments showed that these five ingredients could inhibit IL-6 release and IL-6 gene expression on LPS-induced RAW cells in a dose-dependent manner. Compared with previous 2D CMC screening systems, this newly developed offline 2D method needs no sample preparation steps for the second-dimensional analysis, and it is sensitive, efficient, and convenient. It will be applicable in identifying active components from Chinese herbs and practical in discovery of lead compounds derived from herbs.

Identification of Annexin A2 as a target protein for plant alkaloid matrine.

Matrine is a plant alkaloid and a major active component in the Chinese medical herb Sophora flavescens. Matrine has shown potent anti-cancer activities but its molecular target(s) and mechanism are still unknown. Using the photo-affinity labeling approach, for the first time, Annexin A2 was identified as a direct-binding target of matrine in cancer cells.

A novel strategy of profiling the mechanism of herbal medicines by combining network pharmacology with plasma concentration determination and affinity constant measurement.

Herbal medicines have long been widely used in the treatment of various complex diseases in China. However, the active constituents and therapeutic mechanisms of many herbal medicines remain undefined. Therefore, the identification of the active components and target proteins in these herbal medicines is a formidable task in herbal medicine research. In this study, we proposed a strategy, which integrates network pharmacology with biomedical analysis and surface plasmon resonance (SPR) to predict the active ingredients and potential targets of herbal medicine Sophora flavescens or Kushen in Chinese, and evaluate its anti-fibrosis activity. First, we applied a virtual HTDocking platform to predict the potential targets of Kushen related to liver fibrosis by selecting five crucial protein targets based on network parameters and text mining. Then, we identified nine components in mice plasma after oral administration of Kushen extract and determined the plasma concentration of each compound. Binding affinities between the nine potential active compounds and five target proteins were detected by SPR assays. Finally, we constructed a multi-parameter network model on the basis of three important parameters to tentatively explain the anti-fibrosis mechanism of Kushen. The results not only provide evidence for the therapeutic mechanism of Kushen but also shed new light on the activity-based analysis of other Chinese herbal medicines.

Cardiovascular Disease Chemogenomics Knowledgebase-guided Target Identification and Drug Synergy Mechanism Study of an Herbal Formula.

Combination therapy is a popular treatment for various diseases in the clinic. Among the successful cases, Traditional Chinese Medicinal (TCM) formulae can achieve synergistic effects in therapeutics and antagonistic effects in toxicity. However, characterizing the underlying molecular synergisms for the combination of drugs remains a challenging task due to high experimental expenses and complication of multicomponent herbal medicines. To understand the rationale of combination therapy, we investigated Sini Decoction, a well-known TCM consisting of three herbs, as a model. We applied our established diseases-specific chemogenomics databases and our systems pharmacology approach TargetHunter to explore synergistic mechanisms of Sini Decoction in the treatment of cardiovascular diseases. (1) We constructed a cardiovascular diseases-specific chemogenomics database, including drugs, target proteins, chemicals, and associated pathways. (2) Using our implemented chemoinformatics tools, we mapped out the interaction networks between active ingredients of Sini Decoction and their targets. (3) We also in silico predicted and experimentally confirmed that the side effects can be alleviated by the combination of the components. Overall, our results demonstrated that our cardiovascular disease-specific database was successfully applied for systems pharmacology analysis of a complicated herbal formula in predicting molecular synergetic mechanisms, and led to better understanding of a combinational therapy.

Development of APTES-Decorated HepG2 Cancer Stem Cell Membrane Chromatography for Screening Active Components from Salvia miltiorrhiza.

Cell membrane chromatography (CMC) is an ideal method for screening potential active components acting on target cell membranes from a complex system, such as herbal medicines. But due to the decay and falling-off of membranes, the CMC column suffers from short life span and low reproducibility. This has greatly limited the application of this model, especially when the cell materials are hard to obtain. To solve this problem, a novel type of (3-aminopropyl)triethoxysilane (APTES)-decorated silica gel was employed. The silica gel was decorated with aldehydes with the help of APTES, which react with the amino groups on cell membranes to form a covalent bond. In this way, cell membranes were immobilized on the surface of silica gel, so it is not easy for membranes to fall off. According to our investigation, the column life of the APTES-decorated group was prolonged to more than 12 days, while the control group showed a sharp decline in column efficiency in the first 3 days. To verify this model, a novel APTES-decorated HepG2 cancer stem cell membrane chromatography (CSCMC) was established and applied in a comprehensive two-dimensional chromatographic system to screen potential active components in Salvia miltiorrhiza. As a result, tanshinone IIA, cryptotanshinone, and dihydrotanshinone I were retained on this model and proved to be effective on HepG2 cancer stem cells by the following cell proliferation and apoptosis assay, with IC50 of 10.30 µM, 17.85 µM, and 2.53 µM, respectively. This improvement of CMC can significantly prolong its column life span and broaden the range of its application, which is very suitable for making invaluable or hard-to-obtain cell materials, such as stem cells, for specific drug screening.

Activity ranking of synthetic analogs targeting vascular endothelial growth factor receptor 2 by an integrated cell membrane chromatography system.

Evaluating the biological activities of small molecules represents an important part of the drug discovery process. Cell membrane chromatography (CMC) is a well-developed biological chromatographic technique. In this study, we have developed combined SMMC-7721/CMC and HepG2/CMC with high-performance liquid chromatography and time-of-flight mass spectrometry to establish an integrated screening platform. These systems was subsequently validated and used for evaluating the activity of quinazoline compounds, which were designed and synthesized to target vascular endothelial growth factor receptor 2. The inhibitory activities of these compounds towards this receptor were also tested using a classical caliper mobility shift assay. The results revealed a significant correlation between these two methods (R(2) = 0.9565 or 0.9420) for evaluating the activities of these compounds. Compared with traditional methods of evaluating the activities analogous compounds, this integrated cell membrane chromatography screening system took less time and was more cost effective, indicating that it could be used as a practical method in drug discovery.