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.

High-throughput BCRP inhibitors screening system based on styrene maleic acid polymer membrane protein stabilization strategy and surface plasmon resonance biosensor.

Multidrug resistance (MDR) is a dominant challenge in cancer chemotherapy failure. The over-expression of breast cancer resistance protein (BCRP) in tumorous cells, along with its extensive substrate profile, is a leading cause of tumor MDR. Herein, on the basis of styrene maleic acid (SMA) polymer membrane protein stabilization strategy and surface plasmon resonance (SPR) biosensor, a novel high-throughput screening (HTS) system for BCRP inhibitors has been established. Firstly, LLC-PK1 and LLC-PK1/BCRP cell membranes were co-incubated with SMA polymers to construct SMA lipid particles (SMALPs). PK1-SMALPs were thus immobilized in channel 1 of the L1 chip as the reference channel, and BCRP-SMALPs were immobilized in channel 2 as the detection channel to establish the BCRP-SMALPs-SPR screening system. The methodological investigation demonstrated that the screening system was highly specific and stable. Three active compounds were screened out from 26 natural products and their affinity constants with BCRP were determined. The KD of xanthotoxin, bergapten, and naringenin were 5.14 µM, 4.57 µM, and 3.72 µM, respectively. The in vitro cell verification experiments demonstrated that xanthotoxin, bergapten, and naringenin all significantly increased the sensitivity of LLC-PK1/BCRP cells to mitoxantrone with possessing reversal BCRP-mediated MDR activity. Collectively, the developed BCRP-SMALPs-SPR screening system in this study has the advantages of rapidity, efficiency, and specificity, providing a novel strategy for the in-depth screening of BCRP inhibitors with less side effects and higher efficacy.

Effect of P-Glycoprotein on the Blood-Brain Barrier Transport of the Major Active Constituents of Salvia miltiorrhiza Based on the MDCK-MDR1 Cell Model.

Salvia miltiorrhiza Bunge (S. miltiorrhiza) is a traditional Chinese medicine that has been widely used in the treatment of various central nervous system (CNS) diseases. However, the mechanism of active components of S. miltiorrhiza crossing the blood-brain barrier (BBB) stays unclear. The purpose of this study was to clarify the mechanism of four ingredients of S. miltiorrhiza, i.e., cryptotanshinone (CTS), dihydrotanshinone I (DTS I), tanshinone IIA (TS IIA), and protocatechuic acid (PCTA) crossing the BBB using the in vitro model. The bidirectional transport of detectable components was tested using the MDCK-MDR1 monolayers. High performance liquid chromatography coupled to triple-quadrupole mass spectrometry (HPLC-QQQ/MS) was used to detect the content changes of S. miltiorrhiza monomer components transported through the BBB. Papp of CTS, DTS I, and TS IIA in the absorption direction were lower than 1.0 × 10-6 cm/s, suggesting that these components were poorly absorbed, while PCTA was moderately absorbed through the BBB. The efflux ratio (ER) of CTS, DTS I, TS IIA, and PCTA were 1.65, 0.92, 4.27, and 1.48, respectively. After treatment with P-gp inhibitor tariquidar, the efflux ratio (ER) of CTS, DTS I, and TS IIA significantly decreased from 1.65 to 1.27, 0.92 to 0.36, and 4.27 to 0.86 (P < 0.05), respectively, while the efflux ratio of PCTA decreased without significance from 1.48 to 0.80. This indicated that the transport of CTS, DTS I, and TS IIA might be related to P-gp. TS IIA and CTS were verified as the substrates of P-gp among the four components since the ER of TS IIA and CTS is greater than 1.5. For PCTA and DTS I, their transport mechanism may be related to other transport proteins or passive transport. The results were confirmed by molecular docking in our current work. In this study, an in vitro BBB model was established and applied to the trans-BBB study of active components in S. miltiorrhiza for the first time, which may provide a basis for further research on the mechanisms of other TCMs in treating CNS diseases and is of great significance in promoting the rational and effective use of TCMs.

Construction of a novel blood brain barrier-glioma microfluidic chip model: Applications in the evaluation of permeability and anti-glioma activity of traditional Chinese medicine components.

Since most anti-glioma drug candidates hardly permeate through the blood-brain barrier (BBB), preclinical models that can integrate the complexity of the tumor microenvironment and the structure and function of the BBB is urgently needed for the treatment of glioma. Herein, we constructed an in vitro BBB-glioma microfluidic chip model lined by primary human brain microvascular endothelial cells, pericytes, astrocytes and glioma cells, which could recapitulate the high level of barrier function of the in vivo human BBB and glioma microenvironment. The BBB unit in BBB-glioma microfluidic chip (BBB-U251 chip) displayed selective permeability to fluorescein isothiocyanate isomer-dextran (FITC-dextran) with different molecular weights and three model drugs with different permeability behavior across BBB, which indicated that this glioma model included a functional barrier. Six potential anti-glioma components in traditional Chinese medicine (TCM) were delivered into the blood channel and the permeated amount was quantified by high-performance liquid chromatography combined with ultraviolet (HPLC-UV). The permeated drugs then directly acted on 3D cultured glioma cells (U251) to evaluate the drug efficacy. The results of permeability coefficients of drugs showed that the data were closer to the in vivo data of traditional Transwell model. The effect of the drugs on U251 cells in the BBB-U251 chip was significantly lower due to the existence of BBB. Drug responses on glioma demonstrated the necessity to take BBB into account during the development of anti-glioma new drugs. Therefore, this 3D glioma microfluidic models integrating the BBB functionality can be a useful platform for screening the anticancer drug for brain tumors.

In Vitro Evaluation of the Interaction of Seven Biologically Active Components in Anemarrhenae rhizoma with P-gp.

The efficacy and pharmacokinetics of the biologically active components in Anemarrhenae rhizoma (AR) would be affected by the interaction of P-glycoprotein(P-gp) and effective components in AR. However, little is known about the interaction between them. The goal of this research was to examine the transmembrane absorption of timosaponin AIII(TAIII), timosaponin BII(TBII), sarsasapogenin (SSG), mangiferin(MGF), neomangiferin(NMGF), isomangiferin(IMGF), and baohuosideI(BHI) in AR and their interaction with P-gp. Seven effective components in AR(TAIII, TBII, SSG, MGF, NMGF, IMGF, and BHI) were investigated, and MDCK-MDR1 cells were used as the transport cell model. CCK-8 assays, bidirectional transport assays, and Rhodamine-123 (Rh-123) transport assays were determined in the MDCK-MDR1 cells. LC/MS was applied to the quantitative analysis of TAIII, TBII, MGF, NMGF, IMGF, SSG, and BHI in transport samples. The efflux ratio of MGF, TAIII, TBII, and BHI was greater than 2 and significantly descended with the co-administration of Verapamil, indicating MGF, TAIII, TBII, and BHI as the substrates of P-gp. The efflux ratio of the seven effective components in the extracts (10 mg/mL) of AR decreased from 3.00~1.08 to 1.92~0.48. Compared to the efflux ratio of Rh-123 in the control group (2.46), the efflux ratios of Rh-123 were 1.22, 1.27, 1.25, 1.09, 1.31, and 1.47 by the addition of TAIII, TBII, MGF, IMGF, NMGF, and BHI, respectively, while the efflux ratio of Rh-123 with the co-administration of SSG had no statistical difference compared to the control group. These results indicated that MGF, TAIII, TBII, and BHI could be the substrates of P-gp. TAIII, TBII, MGF, IMGF, NMGF, and BHI show the effect of inhibiting P-gp function, respectively. These findings provide important basic pharmacological data to assist the therapeutic development of AR constituents and extracts.

Screening of immune cell activators from Astragali Radix using a comprehensive two-dimensional NK-92MI cell membrane chromatography/C18 column/time-of-flight mass spectrometry system.

Astragali Radix (AR) is a clinically used herbal medicine with multiple immunomodulatory activities that can strengthen the activity and cytotoxicity of natural killer (NK) cells. However, owing to the complexity of its composition, the specific active ingredients in AR that act on NK cells are not clear yet. Cell membrane chromatography (CMC) is mainly used to screen the active ingredients in a complex system of herbal medicines. In this study, a new comprehensive two-dimensional (2D) NK-92MI CMC/C18 column/time-of-flight mass spectrometry (TOFMS) system was established to screen for potential NK cell activators. To obtain a higher column efficiency, 3-mercaptopropyltrimethoxysilane-modified silica was synthesized to prepare the NK-92MI CMC column. In total, nine components in AR were screened from this system, which could be washed out from the NK-92MI/CMC column after 10 min, and they showed good affinity for NK-92MI/CMC column. Two representative active compounds of AR, isoastragaloside I and astragaloside IV, promoted the killing effect of NK cells on K562 cells in a dose-dependent manner. It can thus suggest that isoastragaloside I and astragaloside IV are the main immunomodulatory components of AR. This comprehensive 2D NK-92MI CMC analytical system is a practical method for screening immune cell activators from other herbal medicines with immunomodulatory effects.

Novel transketolase inhibitor oroxylin A suppresses the non-oxidative pentose phosphate pathway and hepatocellular carcinoma tumour growth in mice and patient-derived organoids.

BACKGROUND: Transketolase (TKT), a key rate-limiting enzyme in the non-oxidative branch of the pentose phosphate pathway (PPP), provides more than 85% of the ribose required for de novo nucleotide biosynthesis and promotes the development of hepatocellular carcinoma (HCC). Pharmacologic inhibition of TKT could impede HCC development and enhance treatment efficacy. However, no safe and effective TKT inhibitor has been approved. METHODS: An online two-dimensional TKT protein immobilised biochromatographic system was established for high-throughput screening of TKT ligands. Oroxylin A was found to specifically bind TKT. Drug affinity responsive target stability, cellular thermal shift assay, surface plasmon resonance, molecular docking, competitive displacement assay, and site mutation were performed to identify the binding of oroxylin A with TKT. Antitumour effects of oroxylin A were evaluated in vitro, in human xenograft mice, diethylnitrosamine (DEN)-induced HCC mice, and patient-derived organoids (PDOs). Metabolomic analysis was applied to detect the enzyme activity. Transcriptome profiling was conducted to illustrate the anti-HCC mechanism of oroxylin A. TKT knocking-down HCC cell lines and PDOs were established to evaluate the role of TKT in oroxylin A-induced HCC suppression. RESULTS: By targeting TKT, oroxylin A stabilised the protein to proteases and temperature extremes, decreased its activity and expression, resulted in accumulation of non-oxidative PPP substrates, and activated p53 signalling. In addition, oroxylin A suppressed cell proliferation, induced apoptosis and cell-cycle arrest, and inhibited the growth of human xenograft tumours and DEN-induced HCC in mice. Crucially, TKT depletion exerted identical effects to oroxylin A, and the promising inhibitor also exhibited excellent therapeutic efficacy against clinically relevant HCC PDOs. CONCLUSIONS: These results uncover a unique role for oroxylin A in TKT inhibition, which directly targets TKT and suppresses the non-oxidative PPP. Our findings will facilitate the development of small-molecule inhibitors of TKT and novel therapeutics for HCC.

I n situ synthesis and unidirectional insertion of membrane proteins in liposome-immobilized silica stationary phase for rapid preparation of microaffinity chromatography.

Cell membrane affinity chromatography has been widely applied in membrane protein (MP)-targeted drug screening and interaction analysis. However, in current methods, the MP sources are derived from cell lines or recombinant protein expression, which are time-consuming for cell culture or purification, and also difficult to ensure the purity and consistent orientation of MPs in the chromatographic stationary phase. In this study, a novel in situ synthesis membrane protein affinity chromatography (iSMAC) method was developed utilizing cell-free protein expression (CFE) and covalent immobilized affinity chromatography, which achieved efficient in situ synthesis and unidirectional insertion of MPs into liposomes in the stationary phase. The advantages of iSMAC are: 1) There is no need to culture cells or prepare recombinant proteins; 2) Specific and purified MPs with stable and controllable content can be obtained within 2 h; 3) MPs maintain the transmembrane structure and a consistent orientation in the chromatographic stationary phase; 4) The flexible and personalized construction of cDNAs makes it possible to analyze drug binding sites. iSMAC was successfully applied to screen PDGFRß inhibitors from Salvia miltiorrhiza and Schisandra chinensis. Micro columns prepared by in-situ synthesis maintain satisfactory analysis activity within 72 h. Two new PDGFRß inhibitors, salvianolic acid B and gomisin D, were screened out with K D values of 13.44 and 7.39 µmol/L, respectively. In vitro experiments confirmed that the two compounds decreased α-SMA and collagen Ӏ mRNA levels raised by TGF-ß in HSC-T6 cells through regulating the phosphorylation of p38, AKT and ERK. In vivo, Sal B could also attenuate CCl4-induced liver fibrosis by downregulating PDGFRß downstream related protein levels. The iSMAC method can be applied to other general MPs, and provides a practical approach for the rapid preparation of MP-immobilized or other biological solid-phase materials.

Therapeutic effect and mechanism of Anemarrhenae Rhizoma on Alzheimer's disease based on multi-platform metabolomics analyses.

Anemarrhenae Rhizoma (AR) has multiple pharmacological activities to prevent and treat Alzheimer's disease (AD). However, the effect and its molecular mechanism are not elucidated clear. This study aims to evaluate AR's therapeutic effect and mechanism on AD model rats induced by D-galactose and AlCl3 with serum metabolomics. Behavior study, histopathological observations, and biochemical analyses were applied in the AD model assessment. Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-QTOF/MS) were combined with multivariate statistical analysis to identify potential biomarkers of AD and evaluate the therapeutic effect of AR on AD from the perspective of metabolomics. A total of 49 biomarkers associated with the AD model were identified by metabolomics, and pathway analysis was performed to obtain the metabolic pathways closely related to the model. With the pre-treatment of AR, 32 metabolites in the serum of AD model rats were significantly affected by AR compared with the AD model group. The regulated metabolites affected by AR were involved in the pathway of arginine biosynthesis, arginine and proline metabolism, ether lipid metabolism, glutathione metabolism, primary bile acid biosynthesis, and steroid biosynthesis. These multi-platform metabolomics analyses were in accord with the results of behavior study, histopathological observations, and biochemical analyses. This study explored the therapeutic mechanism of AR based on multi-platform metabolomics analyses and provided a scientific basis for the application of AR in the prevention and treatment of AD.

Screening potential P-glycoprotein inhibitors by combination of a detergent-free membrane protein extraction with surface plasmon resonance biosensor.

P-glycoprotein (P-gp) highly expressed in cancer cells can lead to multidrug resistance (MDR) and the combination of anti-cancer drugs with P-gp inhibitor has been a promising strategy to reverse MDR in cancer treatment. In this study, we established a label-free and detergent-free system combining surface plasmon resonance (SPR) biosensor with styrene maleic acid (SMA) polymer membrane proteins (MPs) stabilization technology to screen potential P-gp inhibitors. First, P-gp was extracted from MCF-7/ADR cells using SMA polymer to form SMA liposomes (SMALPs). Following that, SMALPs were immobilized on an SPR biosensor chip to establish a P-gp inhibitor screening system, and the affinity between P-gp and small molecule ligand was determined. The methodological investigation proved that the screening system had good specificity and stability. Nine P-gp ligands were screened out from 50 natural products, and their affinity constants with P-gp were also determined. The in vitro cell verification experiments demonstrated that tetrandrine, fangchinoline, praeruptorin B, neobaicalein, and icariin could significantly increase the sensitivity of MCF-7/ADR cells to Adriamycin (Adr). Moreover, tetrandrine, praeruptorin B, and neobaicalein could reverse MDR in MCF-7/ADR cells by inhibiting the function of P-gp. This is the first time that SMALPs-based stabilization strategy was applied to SPR analysis system. SMA polymer can retain P-gp in the environment of natural lipid bilayer and thus maintain the correct conformation and physiological functions of P-gp. The developed system can quickly and accurately screen small molecule ligands of complex MPs and obtain affinity between complex MPs and small molecule ligands without protein purification.

Simultaneous Determination of Seven Lipophilic and Hydrophilic Components in Salvia miltiorrhiza Bunge by LC-MS/MS Method and Its Application to a Transport Study in a Blood-Brain-Barrier Cell Model.

Salvia miltiorrhiza Bunge (SM) has been extensively used in Alzheimer's disease treatment, the permeability through the blood-brain barrier (BBB) determining its efficacy. However, the transport mechanism of SM components across the BBB remains to be clarified. A simple, precise, and sensitive method using LC-MS/MS was developed for simultaneous quantification of tanshinone I (TS I), dihydrotanshinone I (DTS I), tanshinone IIA (TS IIA), cryptotanshinone (CTS), protocatechuic aldehyde (PAL), protocatechuic acid (PCTA), and caffeic acid (CFA) in transport samples. The analytes were separated on a C18 column by gradient elution. Multiple reaction monitoring mode via electrospray ionization source was used to quantify the analytes in positive mode for TS I, DTS I, TS IIA, CTS, and negative mode for PAL, PCTA, and CFA. The linearity ranges were 0.1-8 ng/mL for TS I and DTS I, 0.2-8 ng/mL for TS IIA, 1-80 ng/mL for CTS, 20-800 ng/mL for PAL and CFA, and 10-4000 ng/mL for PCTA. The developed method was accurate and precise for the compounds. The relative matrix effect was less than 15%, and the analytes were stable for analysis. The established method was successfully applied for transport experiments on a BBB cell model to evaluate the apparent permeability of the seven components.

Development of a surface plasmon resonance biosensor for accurate and sensitive quantitation of small molecules in blood samples.

Therapeutic drug monitoring (TDM) has played an important role in clinical medicine for precise dosing. Currently, chromatographic technology and immunoassay detection are widely used in TDM and have met most of the needs of clinical drug therapy. However, some problems still exist in practical applications, such as complicated operation and the influence of endogenous substances. Surface plasmon resonance (SPR) has been applied to detect the concentrations of small molecules, including pesticide residues in crops and antibiotics in milk, which indicates its potential for in vivo drug detection. In this study, a new SPR-based biosensor for detecting chloramphenicol (CAP) in blood samples was developed and validated using methodological verification, including precision, accuracy, matrix effect, and extraction recovery rate, and compared with the classic ultra-performance liquid chromatography-ultraviolet (UPLC-UV) method. The detection range of SPR was 0.1-50 ng/mL and the limit of detection was 0.099 ± 0.023 ng/mL, which was lower than that of UPLC-UV. The intra-day and inter-day accuracies of SPR were 98%-114% and 110%-122%, which met the analysis requirement. The results show that the SPR biosensor is identical to UPLC-UV in the detection of CAP in rat blood samples; moreover, the SPR biosensor has better sensitivity. Therefore, the present study shows that SPR technology can be used for the detection of small molecules in the blood samples and has the potential to become a method for therapeutic drug monitoring.

High-throughput LC-MS/MS Method for Simultaneous Determination of Pantoprazole and Atorvastatin in Rat Plasma: Application to a Pharmacokinetic Interaction Study.

BACKGROUND: Pantoprazole and atorvastatin are often used jointly in the clinic. The drug-drug interaction of pantoprazole and atorvastatin is worthy of being investigated. OBJECTIVE: A highly rapid, sensitive, and selective LC-MS/MS method was developed for simultaneous quantification of pantoprazole and atorvastatin in rat plasma. METHODS: Omeprazole and atorvastatin-d5 were used as the internal standards (ISs) of pantoprazole and atorvastatin, respectively. Simple protein precipitation was used to extract analytes from 50.0 µL plasma samples. RESULTS: The chromatographic separation was achieved on a C18 column and the total chromatographic run time was 3.2 min. Acquisition of mass spectrometric data was performed on a triple-quadrupole mass spectrometer in multiple- reaction-monitoring (MRM) mode with an ESI source using the transition m/z 384→ 200 for pantoprazole and m/z 559.4→ 440.2 for atorvastatin, respectively. The method was validated over the concentration range of 20.0 ∼ 5000 ng/mL for pantoprazole and 1.00 ∼ 250 ng/mL for atorvastatin. All the validation results, including linearity, specificity, precision, accuracy, extraction recovery, matrix effect, and stability, met the acceptance criteria as per FDA guidelines. CONCLUSION: This method was successfully applied to a pharmacokinetic interaction study in Wistar rats. The results revealed significant evidence for the drug-drug interaction between pantoprazole and atorvastatin.

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.

A stop-flow comprehensive two-dimensional HK-2 and HK-2/CIKI cell membrane chromatography comparative analysis system for screening the active ingredients from Pyrrosia calvata (Bak.) Ching against crystal-induced kidney injury.

Crystal-induced kidney injury (CIKI) is the fundamental pathological change during nephrolithiasis, although the molecular mechanism is still unclear. Pyrrosia calvata (Bak.) Ching has been used in folk medicine to treat urolithiasis for years. To clarify the pharmacodynamic substances and the mechanism of its antiurolithiasis effects, in this study, a novel, stop-flow, comprehensive, two-dimensional (2D) HK-2 and HK-2/CIKI cell membrane chromatography (CMC) comparative analysis system was developed to screen for the potential active ingredients from Pyrrosia calvata (Bak.) Ching against CIKI. The comprehensive 2D CMC comparative analysis system showed satisfactory selectivity, and eight ingredients were screened and identified by this system. Among them, mangiferin exhibited higher affinity for the HK-2/CIKI CMC column than the HK-2 CMC column and was selected for further efficacy verification. Cell proliferation assays showed that mangiferin could protect HK-2 cell viability after stimulation with sodium oxalate (NaOX). Additionally, in a rodent model of CIKI, mangiferin decreased the deposition of calcium oxalate (CaOX) crystals in mouse kidneys, alleviated the pathological damage to kidney tissue, and inhibited the upregulation of OPN, MCP1, and CD44 expression caused by CaOX crystals. The established comprehensive 2D CMC comparative analysis system can be applied to screen active ingredients with disease specificity from traditional Chinese medicine (TCM) and is suitable for other cell models.

The Effects of Traditional Chinese Medicine on P-Glycoprotein-Mediated Multidrug Resistance and Approaches for Studying the Herb-P-Glycoprotein Interactions.

As a member of the ATP-dependent membrane transport proteins, P-Glycoprotein (P-gp) is known to pump substrates out of cells using an ATP-dependent mechanism. The overexpression of P-gp in tumor cells reduces the intracellular drug concentrations, which decreases the efficacy of extensive antitumor drugs and leads to multidrug resistance (MDR) clinically. The combination of anticancer drugs with P-gp inhibitor has been an attractive and promising strategy to reverse MDR in cancer treatment. However, nonspecific or nonselective distribution of P-gp inhibitors to nontarget organs is one of the most fatal shortcomings in clinical application. Thus, there is an urgent need for effective and nontoxic MDR reversal agents, particularly in P-gp-mediated MDR. Traditional Chinese medicine (TCM) natural products may prove less toxic for use in P-gp inhibition to promote MDR reversal. P-gp modulatory effects have been previously demonstrated using selected TCM, including the flavonoid, alkaloid, terpenoid, coumarin, and quinonoid compounds, and some Chinese medicine extracts. Moreover, the approaches for screening active components from TCM are necessary, and these approaches face challenges. At present, the approaches to study the interaction between TCM and P-gp are divided into in vitro, in vivo, and in silico methods. This review will provide an overview and update on the role of TCM in overcoming P-gp-mediated MDR and the approaches to study the interaction between TCM and P-gp. SIGNIFICANCE STATEMENT: This review summarized some traditional Chinese medicines identified to have a modulatory effect on P-gp, including flavonoids, alkaloids, terpenoids, coumarins, quinonoid compounds, and some Chinese medicine extracts, and it introduced possible mechanisms. The approaches to study the interaction between TCM and P-gp are divided into in vitro, in vivo, and in silico methods.

Nuclear magnetic resonance-based plasma metabolomics revealed the protective effect of tea polyphenols on sulfur mustard-induced injury in rats.

Tea polyphenols (TP) are the major antioxidant components from tea, which could be beneficial to oxidative stress injury, such as sulfur mustard (SM) exposure. However, the holistic efficacy of TP on SM poisoning remains unexplored and needs further investigation. In this study, Nuclear magnetic resonance(NMR)-based metabolomics along with multivariate statistical analysis was used to explore the metabolic alteration after SM injury and the protective mechanism of TP. Thirteen potential plasma biomarkers of SM injury were identified, which primarily related to synthesis of ketone bodies, arginine and proline metabolism, butanoate metabolism and alanine aspartate and glutamate metabolism. After TP pre-treatment, the biomarkers were mostly restored to normal levels, which suggested that TP provided effective protection against SM injury and might play its role by rebalancing disordered metabolism pathways. This work enhanced our comprehension of the metabolic profiling of SM injury and revealed the protective mechanism of TP.

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.

[Advances in dispersive liquid-liquid microextraction and its application to analysis of biological samples].

Dispersive liquid-liquid microextraction (DLLME) is a mini-extraction technology with merits of low cost, environment friendliness, and high extraction efficiency; moreover, this method is easy to operate and requires only a short operation time. In this paper, several extraction models, including normal dispersive liquid-liquid microextraction, ionic liquid dispersive liquid-liquid microextraction, and ultrasound-assisted dispersive liquid-liquid microextraction, are reviewed according to the type of extraction solvents and dispersive methods. This paper also summarizes the application of DLLME to the analysis of biological samples in the last five years.

Covalent Design of Cell Membrane Stationary Phase with Enhanced Stability for Fast Screening P-Glycoprotein Inhibitors.

Cell membrane chromatography (CMC) has been widely used for characterizing the interaction between drugs and membrane receptors to screen target components from herbal medicines. However, the column life, stability, and the efficiency cannot meet the needs of high-throughput screening purpose. In this study, a P-glycoprotein immobilized cell membrane stationary phase (P-gp/CMSP) was prepared with a simple and mild two-step aldehyde modification, realizing the covalent bonding between cell membrane and stationary phase. The column life and stability were significantly enhanced compared with the unmodified columns. The P-gp/CMC column was equipped into a comprehensive 2D P-gp/CMC/Capcell-C18/TOFMS system, which actualizes the automated and high-throughput analytical process and rapid identification of complex chemical samples with no data loss. Five compounds with significant retention were screened out and unambiguously identified by the comprehensive 2D analytical system. Baicalin was confirmed as a P-gp inhibitor with ATP depletion inhibition ratio of 83.4%. Moreover, the reversal index of baicalin on DOX significantly increased to 11.13 when its concentration reached 25 µM, revealing that baicalin could effectively reverse the MDR cell model induced by DOX. The integrated system is a practical drug discovery platform and could be applied to other transmembrane protein models.