Screening Method For Inhibitors Research Articles

Fast identification of lipase inhibitors in oolong tea by using lipase functionalised Fe3O4 magnetic nanoparticles coupled with UPLC–MS/MS

Oolong tea is an important member in tea family, which claims for various health benefits such as preventing obesity and improving lipid metabolism. In this work, using pancreatic lipase (PL) functionalised magnetic nanoparticles (PL-MNPs) as solid phase extraction absorbent in combination with ultra-high performance liquid chromatography–mass spectrometry (UPLC–MS), we developed a method for rapid screening and identification of lipase inhibitors from oolong tea. Three PL ligands were selectively extracted and identified as (−)-epigallocatechin-3-O-gallate (EGCG), (−)-gallocatechin-3-O-gallate (GCG) and (−)-epicatechin-3-O-gallate (ECG). Their lipase inhibitory activities were significantly higher than those non-ligands. Structure–activity analysis revealed that the presence of a galloyl moiety in the structure was required for binding to PL-MNPs, and therefore, exhibiting a strong inhibition on the enzyme. Taking advantages of the specificity in enzyme binding and the convenience of magnetic separation, this method has great potential for fast screening of lipase inhibitors from natural resources.

Ultrahigh-performance liquid chromatography/tandem mass spectrometry method for evaluating enzyme activity and screening inhibitors of cyclooxygenase-2.

Prostaglandin E2 is an important biomarker in many biological systems. The development of sensitive and reliable analytical methods for monitoring PGE2 contents in various samples is of great interest. Here we developed an improved method for evaluating the enzyme activity and screening COX-2 inhibitors using ultrahigh-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) combined with PGE2 derivatization. Girard's reagent-T was used as the derivatization reagent and the reaction conditions were optimized. The established method was performed to screen the COX-2 inhibitors from effective constituents of herbs and detect the concentration of PGE2 in biological tissue samples (liver and kidney). The IC50 values of celecoxib, rofecoxib, sinomenine, bulleyaconitine A, tetrandrine, fangchinoline, berberine hydrochloride and sophocarpidine towards COX-2 were determined. This method improves the quantitative ability for PGE2 , including the linearly dependent coefficient, linearity range and limit of detection. After derivatization, the derivatized PGE2 could be detected in positive ion mode of electrospray ionization (ESI), which improves the detection sensitivity 10-fold compared to that of the direct detection of underivatized PGE2 in negative ESI mode. Besides the positive control, sinomenine (IC50 =113 μM) and bulleyaconitine A (IC50=53 μM) were found to be potent COX-2 inhibitors. All the results indicate that the present derivatization quantification method of PGE2 could be used as the detection method of COX-2 enzyme activity and as the screening method for COX-2 inhibitors.

A comparison of protein kinases inhibitor screening methods using both enzymatic activity and binding affinity determination.

Binding assays are increasingly used as a screening method for protein kinase inhibitors; however, as yet only a weak correlation with enzymatic activity-based assays has been demonstrated. We show that the correlation between the two types of assays can be improved using more precise screening conditions. Furthermore a marked improvement in the correlation was found by using kinase constructs containing the catalytic domain in presence of additional domains or subunits.

Development of an LC-MS/MS method for aromatase inhibitor screening

Aromatase (CYP 19A1) is a key steroidogenic enzyme that catalyzes the conversion of androgen to estrogen. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for aromatase inhibitor screening was developed and validated. The substrate androstenedione was incubated with human CYP 19A1 supersomes in the presence of NADPH for 30 min, and estrone formation was determined by LC-MS/MS analysis. Cortisone was used as internal standard. The incubation mixture was extracted using a liquid-liquid extraction method with ethyl acetate. Chromatographic separation was achieved using a C18 column (3.0 × 50 mm, 2.7 μm) with a mobile phase consisting of 0.1% formic acid/acetonitrile adopting gradient elution at a flow rate of 0.4 mL/min. The mass spectrometer was operated in positive electrospray ionization mode. The precursor-product ion pairs used for multiple reaction monitoring were m/z 287→97 (androstenedione), m/z 271 → 159 (estrone), and m/z 361 → 163 (IS, cortisone). The developed method met the required criteria for the validation of bioanalytical methods. The validated method was successfully applied to evaluate aromatase inhibitory activity of plants extracts of Simaroubaceae.

DNA Methylation Detection and Inhibitor Screening Based on the Discrimination of the Aggregation of Long and Short DNA on a Negatively Charged Indium Tin Oxide Microelectrode

On the basis of the different diffusivity and existence of electrostatic repulsion between long and short DNA on the negatively changed indium tin oxide (ITO) microelectrode, a simple but sensitive immobilization free solution-phase electrochemical method for DNA methylation detection and inhibitor screening has been developed. Electroactive substance (methylene blue) tagged at the penultimate base T close to the 3'-terminal first, in the absence of DNA methylation, methylene blue-labeled electroactive fragments cannot be generated by Dpn I and results in a weak electrochemical response being detected on the ITO electrode. On the contrary, a remarkable electrochemical response can be achieved by the cleavage in the presence of DNA methylation since methylene blue-labeled electroactive fragments can be generated and aggregate on the ITO electrode. The proposed system does not need complex operation procedures such as bisulfite treatment, PCR amplification, and electrode immobilization. Six ITO microelectrodes had been assembled on the same microchip, which can achieve the parallel detection of the same sample and improve the experimental efficiency of drug screening. The system was used to conveniently and specifically monitor the change of the DNA methylation level with high sensitivity and selectivity. The proposed system has the potential application to screen the drugs as inhibitors on the activity of methyltransferase in the clinic.

High-throughput HDR inhibitor screening

Isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), biochemical precursors of the isoprenoids, are biosynthesized by mevalonate pathway and methylerythritol phosphate (MEP) pathway. Because these two pathways are mutually exclusive in most organisms, inhibition of MEP pathway has become a target for the development of new bioactive materials, including antibiotics, antimalarial drugs, and herbicides. In the final step of MEP pathway, (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HMBPP reductase, HDR) catalyzes reduction of HMBPP to IPP and DMAPP. HDR requires electron transfers to the Fe/S cluster in the active site for the catalysis, and methyl viologen has been used as a common redox mediator for in vitro studies. We developed a high throughput colorimetric HDR inhibitor screening method by utilizing microtiter-plate screening method. This method was applied to various plant extracts to measure HDR inhibition activity, and potent HDR inhibitor was successfully screened.

Assessment of the Inhibitory Effect of Rifampicin on Amyloid Formation of Hen Egg White Lysozyme: Thioflavin T Fluorescence Assay versus FTIR Difference Spectroscopy

The inhibitory effect of rifampicin on the amyloid formation of hen egg white lysozyme was assessed with both Thioflavin T (ThT) fluorescence assay and Fourier transform infrared (FTIR) difference spectroscopy. We reveal that ThT fluorescence assay gives a false positive result due to rifampicin interference, while FTIR difference spectroscopy provides a reliable assessment. With FTIR, we show that rifampicin only has marginally inhibitory effect. We then propose that FTIR difference spectroscopy can potentially be a convenient method for inhibitor screening in amyloid study.

A sensitive electrochemical method for DNA methyltransferase assay and inhibitor screening based on DNA methylation-sensitive cleavage

A sensitive electrochemical method for detection of DNA methylation and assay of DNA methyltransferase (Dam) was investigated based on methylation-sensitive restriction endonuclease, DNA-Au bio bar code and enzymatic signal amplification. After the DNA probe was hybridized with complementary DNA sequence, and the obtained double-strand DNA (dsDNA) was further methylated by Dam MTase, it could be successively digested by Dpn I at the methylated sites. As a result, the bio bar code and streptavidin-horseradish peroxidase (S-HRP) could not be conjugated on the electrode surface through the specific interaction between biotin and S-HRP, where biotin was labeled at the DNA-Au bio bar code. Thus, only a low electrochemical reduction signal was obtained. However, the hybridized probe could hybrid with DNA-Au bio bar code, and then S-HRP could be conjugated on the electrode surface in the absence of Dam MTase and Dpn I, so a larger reduction current of enzymatic product was obtained. The change of the reduction current of enzymatic product was used to detect DNA methylation and Dam MTase activity. The detection limit of Dam MTase was 0.02unit/mL (S/N=3). Moreover, the inhibitions of mitomycin C, paclitaxel, chlorogenic acid and epicatechin were also investigated. Therefore, this method provided a sensitive platform for monitoring DNA methylation and DNA MTase activity.

Development of a Small-Molecule Screening Method for Inhibitors of Cellular Response to Myostatin and Activin A

Myostatin, a member of the transforming growth factor (TGF)-β family of secreted ligands, is a strong negative regulator of muscle growth. As such, therapeutic inhibitors of myostatin are actively being investigated for their potential in the treatment of muscle-wasting diseases such as muscular dystrophy and sarcopenia. Here, we sought to develop a high-throughput screening (HTS) method for small-molecule inhibitors that target myostatin. We created a HEK293 stable cell line that expresses the (CAGA)12-luciferase reporter construct and robustly responds to signaling of certain classes of TGF-β family ligands. After optimization and miniaturization of the assay to a 384-well format, we successfully screened a library of compounds for inhibition of myostatin and the closely related activin A. Selection of some of the tested compounds was directed by in silico screening against myostatin, which led to an enrichment of target hits as compared with random selection. Altogether, we present an HTS method that will be useful for screening potential inhibitors of not only myostatin but also many other ligands of the TGF-β family.

Tagging of Genomic STAT3 and STAT1 with Fluorescent Proteins and Insertion of a Luciferase Reporter in the Cyclin D1 Gene Provides a Modified A549 Cell Line to Screen for Selective STAT3 Inhibitors

Signal transducer and activator of transcription 3 (STAT3) is an oncogenic protein that is constitutively activated in numerous cancer cell lines and human cancers. Another STAT family member, STAT1, possesses cancer-inhibitory properties and can promote apoptosis in tumor cells upon activation. To better characterize these important cancer related genes, we tagged STAT3 and STAT1 loci with fluorescent protein (FP) sequences (RFP and GFP respectively) by targeted integration via zinc finger nuclease (ZFN) - mediated homologous recombination in A549 cells that express aberrantly activated STAT3. We inserted the FP transgenes at the N-terminus of the STAT3 locus and at the C-terminus of the STAT1 locus. The integration resulted in endogenous expression of fluorescent STAT3 and STAT1 chimeric fusion proteins. When stimulated with IL-6 or IFN-γ, the cells showed robust nuclear translocation of RFP-STAT3 or STAT1-GFP, respectively. Pre-incubation of cells with a known specific STAT3 inhibitor showed that IFN-γ-induced translocation of STAT1-GFP was not impaired. STAT3 activates multiple downstream targets such as genes involved in cell cycle progression - e.g. cyclin D1. To detect changes in expression of endogenous cyclin D1, we used ZFN technology to insert a secreted luciferase reporter behind the cyclin D1 promoter and separated the luciferase and cyclin D1 coding regions by a 2A sequence to induce a translational skip. The luciferase insertion was made in the RFP-STAT3/STAT1-GFP cell line to have all three reporters in a single cell line. Addition of a STAT3 inhibitor led to suppression of cyclin D1 promoter activity and cell growth arrest. The triple-modified cell line provides a simple and convenient method for high-content screening and pre-clinical testing of potential STAT3 inhibitors in live cells while ensuring that the STAT1 pathway is not affected. This approach of reporting endogenous gene activities using ZFN technology could be applied to other cancer targets.

An antibody‐free strategy for screening putative HDM2 inhibitors using crude bacterial lysates expressing GST‐HDM2 recombinant protein

Targeting the interaction of p53 with its natural inhibitor MDM2 by the use of small synthetic molecules has emerged as a promising pharmacological approach to restore p53 oncosuppressor function in cancers retaining wild-type p53. The first critical step in the experimental validation of newly synthesized small molecules developed to inhibit MDM2-p53 interaction is represented by the evaluation of their efficacy in preventing the formation of the MDM2-p53 complex. This can be achieved using the in vitro reconstructed recombinant MDM2-p53 complex in cell-free assays. A number of possible approaches have been proposed, which are however not suitable for screening large chemical libraries, due to the high costs of reagents and instrumentations, or the need of large amounts of highly pure recombinant proteins. Here we describe a rapid and cheap method for high-throughput screening of putative inhibitors of MDM2-p53 complex formation--based on the use of GST-recombinant proteins--that does not require antibodies and recombinant protein purification steps from bacterial cell lysates.

Graphene Oxide–Peptide Nanocomplex as a Versatile Fluorescence Probe of Protein Kinase Activity Based on Phosphorylation Protection against Carboxypeptidase Digestion

The research on complicated kinomics and kinase-target drug discovery requires the development of simple, cost-effective, and multiplex kinase assays. Herein, we propose a novel and versatile biosensing platform for the detection of protein kinase activity based on graphene oxide (GO)-peptide nanocomplex and phosphorylation-induced suppression of carboxypeptidase Y (CPY) cleavage. Kinase-catalyzed phosphorylation protects the fluorophore-labeled peptide probe against CPY digestion and induces the formation of a GO/peptide nanocomplex resulting in fluorescence quenching, while the nonphosphopeptide is degraded by CPY to release free fluorophore as well as restore fluorescence. This GO-based nanosensor has been successfully applied to sensitively detect two model kinases, casein kinase (CKII) and cAMP-dependent protein kinase (PKA) with low detection limits of 0.0833 mU/μL and 0.134 mU/μL, respectively. The feasibility of this GO-based sensor was further demonstrated by the assessment of kinase inhibition by staurosporine and H-89, in vitro kinase assay in cell lysates, and simultaneous detection of CKII and PKA activity. Moreover, the GO-based fluorescence anisotropy (FA) kinase assay has been also developed using GO as a FA signal amplifier. The proposed sensor is homogeneous, facile, universal, label-free, and applicable for multiplexed kinase assay, presenting a promising method for kinase-related biochemical fundamental research and inhibitor screening.

Immobilized capillary tyrosinase microreactor for inhibitor screening in natural extracts by capillary electrophoresis

A method for creating an immobilized capillary tyrosinase (TRS) reactor based on a layer-by-layer (LBL) assembly for inhibitor screening is described. Tyrosinase was immobilized on the surface of fused-silica capillary via ionic binding technique with cationic polyelectrolyte hexadimethrine bromide (HDB). Then, HDB solution with the same plug length as the TRS was injected again into the capillary to cover the immobilized enzyme by forming HDB–TRS–HDB sandwich-like structure. Then, the substrate of l-tyrosine was introduced into the capillary and on-line enzyme inhibition study was performed by capillary electrophoresis (CE). The enzyme activity was determined by the quantification of peak area of the product of l-DOPA. Enzyme inhibition can be read out directly from the reduced peak area of the product in comparison with a reference electropherogram obtained in the absence of any inhibitor. The immobilized enzyme could withstand 25 consecutive assays by only losing 12% activity. A known TRS inhibitor, kojic acid was employed as a model compound for the validation of the inhibitor screening method. Finally, screening 19 natural extracts of traditional Chinese drugs was demonstrated. The results indicated that inhibition activity could be straightforwardly identified with the system.

Exploring Kinetic Hydrate Inhibitor Test Methods and Conditions Using a Multicell Steel Rocker Rig

High pressure rocking cells have overtaken the use of stirred autoclaves as the standard industry method for the first stage laboratory screening of kinetic hydrate inhibitors (KHIs). This study in...

High-throughput fluorescent-based NKCC functional assay in adherent epithelial cells.

BackgroundThe kidney-specific NKCC cotransporter isoform NKCC2 is involved in the Na+ reabsorption in the Thich Ascending Limb (TAL) cells and in the regulation of body fluid volume. In contrast, the isoform NKCC1 represents the major pathway for Cl- entry in endothelial cells, playing a crucial role in cell volume regulation and vascular tone. Importantly, both NKCC isoforms are involved in the regulation of blood pressure and represent important potential drug targets for the treatment of hypertension.ResultsTaking advantage of an existing Thallium (Tl+)-based kit, we set up a Tl+ influx-based fluorescent assay, that can accurately and rapidly measure NKCC transporter activity in adherent epithelial cells using the high-throughput Flex station device. We assessed the feasibility of this assay in the renal epithelial LLC-PK1 cells stably transfected with a previously characterized chimeric NKCC2 construct (c-NKCC2). We demonstrated that the assay is highly reproducible, offers high temporal resolution of NKCC-mediated ion flux profiles and, importantly, being a continuous assay, it offers improved sensitivity over previous endpoint NKCC functional assays.ConclusionsSo far the screening of NKCC transporters activity has been done by 86Rb+ influx assays. Indeed, a fluorescence-based high-throughput screening method for testing NKCC inhibitors would be extremely useful in the development and characterization of new anti-hypertensive drugs.

A straightforward ninhydrin-based method for collagenase activity and inhibitor screening of collagenase using spectrophotometry

Currently protease assay kits, requiring substrate that is either radiolabeled or fluorescence labeled and specialized instruments, are all expensive. A simple, reliable assay of protease activity and its inhibitor screening for general laboratory is rare. Here we demonstrated a straightforward ninhydrin-based method for assay of collagenase activity and its inhibitor screening using spectrophotometry. In the method, without multistep sample treatments and substrate labeling, the hydrolytic products were directly traced by ninhydrin. The method is expected to be suitable for not only the assay of collagenase activity but also the others matrix metalloproteinases activities, and can be used for kinetic study.

Screening of the active ingredients in natural products by capillary electrophoresis and high performance liquid chromatography-mass spectrometry

A new strategy for screening the crude natural extracts and quickly identifying the bioactive compounds was developed. In combination with high performance liquid chromatography-mass spectrometry (HPLC-MS) , the biologically active compounds, such as the enzyme i n the crude natural extract ca n be quickly identified by capillary electrophoresis (CE) -based activity assay. The crude natural extracts were assayed by a CE-based enzyme inhibitor screening method, and the active extract was isolated by HPLC-MS/MS with a semipreparative column. Then, each eluted component was assayed again with the CE-based assay method. Finally, the structures of the identified active compounds were elucidated by MS/MS analysis. Acetylcholinesterase ( ACHE), its substrate acetylthiocholine chloride ( AThCh), as well as the crude extract of Rhizoma coptidis were utilized for the proof of the methodology. Seven isoquinoline alkaloids, namely jatrorrhizine, epiberberine, columbamine, coptisine, corysamine, palmatine and berberine were identified to be active as the inhibitors of ACHE. Their IC50 values were 40, 442, 38, 182, 419, 54 and 16 micromol/L, respectively. Compared with the traditional screening methods, the method is characterized with several advantages, such as extremely low sample and reagent consumption, high speed of analysis, high sensitivity of detection, high throughput in terms of preparation of the natural products by HPLC. Overall, the results demonstrate that the method is valuable for the screening of the bioactive compounds in the crude natural extracts.

Study of Effects of Cigarette Smoke Condensates on Acetylcholinesterase Activity in Human Lung Epithelial Cells by Matrix-Assisted Laser Desorption/Ionization-Fourier Transform Mass Spectrometry

Matrix-Assisted Laser Desorption/Ionization-Fourier Transform Mass Spectrometry (MALDI-FTMS) is an emerging method for kinetic measurements and inhibitor screening of acetylcholinesterase (AChE). This method is accurate, reproducible, and was successfully applied to the analysis of various biosamples. Recent studies showed the important biological functions of acetylcholinesterase in many kinds of tumors caused by smoking; however, the effect of cigarette smoke condensates (CSCs) on AChE was unclear. Herein, the effects of CSCs on AChE activity in human lung epithelial cells were studied by MALDI-FTMS. The results showed that AChE activity in human lung epithelial cells could be inhibited by CSCs, and inhibition degree on AChE activity depended on the amount of CSCs. Owing to the close relationship of AChE to lung cancer caused by smoking, such findings provide information regarding the relation between the aggressiveness of lung tumor and cigarette smoking.

Evaluation of a High-Throughput Screening Method for the Detection of the Excipient-Mediated Precipitation Inhibition of Poorly Soluble Drugs

The objective of the present study was to develop and evaluate a high-throughput (HT) precipitation inhibitor screening method that can be used for the identification of the excipient-mediated precipitation inhibition of poorly soluble drugs. The impact of incubation temperature, shaking intensity, phase separation, inter- and intraday variability, cosolvent, and plate selection on the HT screening method performance was investigated. Additionally, the pipetting quality of the automated workstation, the correlation with the classical laboratory approach, and the practical implementation of the developed HT screening method using two model compounds are disclosed. Investigation of the HT method resulted in optimized experimental conditions, which showed low inter- and intraday variability (relative standard deviation [RSD]<5.88%). Higher shaking intensity (7 Hz) and incubation temperature (37°C) resulted in a lower likelihood of obtaining false-negative results. The acceptable dimethyl sulfoxide concentration in the precipitation inhibitor screening assay was set to ≤1% (v/v). All liquid dispensing steps resulted in an RSD of <3.4%, and an excellent correlation (R(2)=0.96, P<0.01) with the classical laboratory method was obtained. The practical implementation of the developed HT method was demonstrated by investigating the impact of 23 diverse excipients on the precipitation inhibition of two poorly soluble drugs (fenofibrate and carbamazepine). The screen resulted in the identification of hit excipients, which were not identical for fenofibrate and carbamazepine. This outcome emphasized that the HT screening approach is a reasonable starting point for searching for effective precipitation inhibitors, especially because the excipient-mediated precipitation inhibition effect is case specific and cannot be predicted in a straightforward manner.

Screen of Monoamine Oxidase Inhibitors by Protein-Liposome Conjugate Capillary Electrophoresis

Abstract Based on the interaction between enzyme and substrate, a simple and elegant method was developed to screen the inhibitors of monoamine oxidase (MAO). Four kinds of N-propargyl amine compounds with gradient concentrations were added in the CE running buffer solution containing protein-liposome conjugate to inhibit the activity of MAO to some extent. The relative migration time ratio (RMTR) of Kyn was investigated in running buffer solution containing different compounds in gradient concentrations. The results indicate that R-N-(2-heptyl)-N-methylpropargylamine (R-2-HMP) and N-propargyl-R-2-heptylamine (R-2-HPA) could inhibit the activity of MAO obviously, such that the interaction between MAO and Kyn was weakened, RMTR of Kyn increased with the increase of R-2-HMP and R-2-HPA. The inhibition activities of N-dipropargyl-R-2-hexylamine and N,N-dipropargyl-R-2-octylamine were not obvious; RMTR of Kyn changed little with the increase of the two compounds. This result was consistent with that of activity assay of compounds incubation offline. In comparison with the traditional screening method for MAO inhibitors, this method is rapid, low cost, less consumption of enzyme and independent of voltage and other interference factors.