Immobilized enzyme reactors in HPLC and its application in inhibitor screening:A review

This paper sets out to summarize the literatures based on immobilized enzyme bio-chromatography and its application in inhibitors screening in the last decade.In order to screen enzyme inhibitors from a mass of compounds in preliminary screening,multi-pore materials with good biocompatibility are used for the supports of immobilizing enzymes,and then the immobilized enzyme reactor applied as the immobilized enzyme stationary phase in HPLC.Therefore,a technology platform of high throughput screening is gradually established to screen the enzyme inhibitors as new anti-tumor drugs.Here,we briefly summarize the selective methods of supports,immobilization techniques,co-immobilized enzymes system and the screening model.

Recent advances in screening of enzymes inhibitors based on capillary electrophoresis

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.

Preparation of Magnetic Microsphere-Gold Nanoparticle- Immobilized Enzyme Batch Reactor and Its Application to Enzyme Inhibitor Screening in Natural Extracts by Capillary Electrophoresis

A novel strategy for screening the enzyme inhibitors from the natural products by capillary electrophoresis （CE） with a pre column enzyme batch reactor prepared by magnetic microsphere （MB）-gold nanoparticles （AuNPs） is reported. The enzyme batch reactor was prepared by immobilizing the enzymes on the MB conjugated AuNPs （MB-AuNPs）. To demonstrate this strategy, xanthine oxidase （XOD） was employed as a model for the activity of the enzyme, inhibition study, and inhibitor screening. With the developed CE method, the enzyme activity was de- termined by the quantification of the peak area of the product. Enzyme inhibition can be read out directly from the reduced peak area of product in comparison to a reference electropherogram obtained in the absence of any inhibitor A statistical parameter Z＇ factor was recommended for evaluation of the accuracy of a drug screening system. In the present study, it was calculated to be 0.7, implying a good accuracy. The screening of two natural extracts from Cortex Phellodendri and Rhizoma Galangae showed that they were positive for XOD inhibition by the present method. Using this immobilized enzyme technology combined with CE separation not only provides the advantages such as convenience, rapidity and low cost, but also provides a new platform for discovering enzyme-inhibitor drug lead compounds.

High-throughput fluorescent screening of thioredoxin reductase inhibitors to inhibit Mycobacterium tuberculosis

Tuberculosis(TB)is a chronic infectious disease,which is caused by the pathogen Mycobacterium tuberculosis(Mtb)and reemerged as a global health risk with a significant proportion of multi-drug resistant and extensively drug resistant TB cases.It is very urgent to find some novel high-confidence drug targets in Mtb for discovering the effective anti-TB agents.Thioredoxin reductase(TrxR)has been identified to be a highly viable target for anti-TB drugs for its important role in protecting the pathogen from thiol-specific oxidizing stress,regulating intracellular dithiol/disulfide homeostasis and DNA replication and repair.In the present work,a near-infrared(NIR)fluorescent probe DDAT was developed for the detection of TrxR activity and used to high-throughput screen the TrxR inhibitors from natural products.Two screened TrxR inhibitors from Sappan Lignum and microbial metabolites that were further used to inhibit Mycobacterium tuberculosis.All the results indicate that DDAT is a practical fluorescent molecular tool for the discovery of potential anti-TB drugs.

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.

Paper-based fluorescent devices for multifunctional assays:Biomarkers detection,inhibitors screening and chiral recognition

The development of a single analytical platform with different functions is highly desirable but remains a challenge at present.Here,a paper-based device based on fluorescent carbon dots(CDs)functionalized paper/MnO_(2)nanosheets(MnO_(2)NS)hybrid devices(PCD/NS)was proposed for single-device multi-function applications.MnO_(2)NS functioned as a fluorescence quencher of CDs and recognizer of H_(2)O_(2)released from the oxidase catalyzed system.Fluorescence recovery would occur after the decomposition of MnO_(2)NS induced by H_(2)O_(2),by which a simple and effective strategy could be developed for fluorescence monitoring multiplex biological events.Xanthine(XA)sensing,xanthine oxidase(XOD)inhibitors screening analysis and chiral recognition of glucose enantiomers were performed on PCD/NS to investigate the multifunctional application of the paper-based device.By means of PCD/NS,XA could be determined in the range of 0.1–40μmol/L with a low detection of limit of 0.06μmol/L.The IC_(50)value of allopurinol,the model inhibitor of XOD,was sensitively detected to be 7.4μmol/L.Glucose enantiomers were also recognized in terms of the specific fluorescence response to d-glucose.This work firstly presented a paper-based device capable of biomarkers detection,inhibitors screening and chiral recognition,which enlightened a promising strategy for the construction of multifunctional devices and hold the great potential application in clinical diagnosis and drug discovery.

Rapid screening of SARS-CoV-2 inhibitors via ratiometric fluorescence of RBD–ACE2 complexes in living cells by competitive binding

To the Editor:The current coronavirus disease-19(COVID-19)pandemic spurs the development of antiviral drugs for SARS-CoV-2,as the number of patients with viral infections continues to rise globally in the context of widespread vaccination.Targeting the interaction between the receptor binding domain(RBD)of SARS-CoV-2 spike protein and the host cell ACE2 is a promising therapeutic strategy to effectively inhibit viral entry.