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.

Recent advances in enzyme inhibitor screening based on mass spectrometry / 药学学报

Enzymes play crucial functional roles in all biological processes. Enzymatic inhibitors can regulate enzyme activity and may become the starting point for drug discovery. Mass spectrometry (MS) has the advantage for rapid qualitative and quantitative analyses of compounds and enzyme reactions, emerging as an important analytical tool in enzyme inhibitor screening assay for drug discovery. This review will highlight recent advances in the inhibitor screening assay using MS and related techniques, including frontier affinity chromatography, immobilized enzyme beads, ultrafiltration, surface plasmon resonance, capillary electrophoresis and microfluidic chips. The existing MS methods for screening enzyme inhibitor were divided into two types: affinity screening and activity screening.

Recent advances in screening of enzymes inhibitors based on capillary electrophoresis / 药物分析学报

Capillary electrophoresis with many advantages plays an important role in pharmaceutical analysis and drug screening. This review gives an overview on the recent advances in the developments and applications of capillary electrophoresis in the field of enzyme inhibitor screening. The period covers 2013 to 2017. Both the pre-capillary enzyme assays and in-capillary enzyme assays which include electrophoretically mediated microanalysis (EMMA) and immobilized enzyme microreactor (IMER) are summarized in this article.

Development of a method to study the activity and selectivity of SGLT2 inhibitors / 药学学报

The purpose of this study was to develop a screening method to determine the activity and selectivity of SGLT2 inhibitor. Human SGLT1/SGLT2 cDNA was inserted into the pMSCVpuro mammalian expression vector and the plasmid was transfected into HEK293 cells. Stably transfected clones were selected in puromycin containing medium. To evaluate the expression of human SGLT1 and SGLT2 in stable transfected cells, RT-PCR, Western blot and immunofluorescence analysis were performed. 1-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-1-deoxy-D-glucose (1-NBDG) was used as a substrate in the uptake assay to evaluate the Na+ dependent glucose transport activities of SGLT1/2. The inhibitory activity and selectivity of dapagliflozin/phloridzin were also determined, respectively. The hypoglycemic efficacy of dapagliflozin was evaluated in mice with normal blood glucose and mice with alloxan-induced T1DM. The result showed that SGLT1 was overexpressed in pMSCVpuro-SGLT1 transfected HEK293 cells. SGLT2 protein was overexpressed in pMSCVpuro-SGLT2 transfected HEK293 cells and located in both cytoplasm and membrane. The Na+ dependent 1-NBDG uptake was significantly increased in pMSCVpuro-SGLT1/SGLT2 transfected cells compared to that in pMSCVpuro-null transfected cells. The selectivity of dapagliflozin, whose half maximal inhibitory concentration (IC50) for SGLT2 (2.24×10-10 mol·L-1) was far lower than that for SGLT1 (6.20×10-7 mol·L-1), was better than that of phloridzin. The oral glucose tolerance was elevated after a single dose of dapagliflozin in normal mice. In T1DM mice, compared with model group, no-fasting glucose level was decreased at 1 h after administration and maintained at a lower level for 24 h in a dose-dependent manner. A 20-day administration with dapagliflozin dose-dependently improved the hyperglycemia status. Taken together, a system to evaluate the activity and selectivity of SGLT2 inhibitors was established using 1-NBDG in vitro and the hypoglycemic efficacy in vivo in this study. The advantages of this system include non-radioactivity, high efficiency, and good stability which may provide a technique platform for development of novel SGLT2 inhibitors.

Strategies for Antiviral Screening Targeting Early Steps of Virus Infection / 中国病毒学·英文版

Viral infection begins with the entry of the virus into the host target cell and initiates replication.For this reason,the virus entry machinery is an excellent target for antiviral therapeutics.In general,a virus life cycle includes several major steps: cell-surface attachment,entry,replication,assembly,and egress,while some viruses involve another stage called latency.The early steps of the virus life cycle include virus attachment,receptor binding,and entry.These steps involve the initial interactions between a virus and the host cell and thus are major determinants of the tropism of the virus infection,the nature of the virus replication,and the diseases resulting from the infection.Owing to the pathological importance of these early steps in the progress of viral infectious diseases,the development of inhibitors against these steps has been the focus of the pharmaceutical industry.In this review,Herpes Simplex Virus(HSV),Hepatitis C Virus(HCV),and Human Enterovirus 71(EV71)were used as representatives of enveloped DNA,enveloped RNA,and non-enveloped viruses,respectively.The current mechanistic understanding of their attachment and entry,and the strategies for antagonist screenings are summarized herein.