Screening Method For Inhibitors Research Articles

Human and rat precision-cut intestinal slices as ex vivo models to study bile acid uptake by the apical sodium-dependent bile acid transporter

The apical sodium-dependent bile acid transporter (ASBT) is the primary transporter for the uptake of bile acids in the small intestine. It is localized on the apical membrane of the ileal enterocytes and is known for its high capacity and affinity for taurocholic acid in vitro. However, less is known about its activity towards taurocholic acid and other bile acids in vivo due to the lack of a suitable model. The aim of this study was to validate precision-cut intestinal slices (PCIS) of rat and human intestine as ex vivo model to study the activity of ASBT-mediated transport, and subsequently to study regional and interspecies differences in ASBT function. PCIS maintain the natural cell polarization and communication, expression of transporters and metabolizing enzymes. Therefore this model is expected to be more relevant to the in vivo situation than in vitro cell culture models. PCIS of human and rat ileum were prepared and incubated with 0.04–2.00 mM taurocholic acid, deoxycholic acid and cholic acid, respectively at 4 and 37 °C. In this study, the respective contribution of active uptake appeared to be higher for taurocholic acid whereas the passive diffusion was higher for deoxycholic acid and cholic acid. Furthermore, the rank order of calculated (apparent) Km and Vmax for these bile acids is in line with literature reports. The active uptake of taurocholic acid in rat PCIS could be inhibited partly by simvastatin and fluvastatin and was fully inhibited by the specific inhibitor GSK2299027B, supporting the involvement of rat ASBT as uptake transporter. In both species, the ASBT-mediated active uptake of bile acids was observed only in the ileum whereas only passive diffusion was observed in the jejunum and colon. In addition, ASBT activity was higher in the human ileum compared to the rat ileum. In the future rat and human PCIS can be used to study the uptake of new ASBT substrates and as a screening method for potential ASBT inhibitors.

A label-free fluorescent biosensor for the detection of protein kinase activity based on gold nanoclusters/graphene oxide hybrid materials

Protein kinase (PKA) can regulate many cellular biological processes by phosphorylation substrate peptide or protein. A new fluorescent biosensing method for the detection of PKA activity was developed by using 11-mercaptoundecanoic acid-capped gold nanoclusters (MUA-Au NCs) and graphene oxide (GO) with low background noise. In this strategy, the special designed peptide could be anchored on the surface of MUA-Au NCs by the Au-S bond and also adsorbed on the surface of GO owing to the electrostatic interaction. As a result, the fluorescence of MUA-Au NCs was quenched leading to low background fluorescence due to the forster resonance energy transfer (FRET) between MUA-Au NCs and GO via peptide as a bridge. However, when the substrate peptide was phosphorylated by PKA, the FRET between GO and MUA-Au NCs was disrupted because of the weakened interaction between the phosphorylated peptide and the GO, resulting in recovery of the fluorescence intensity. The developed label-free fluorescence “turn-off-on” method can detect protein kinase activity in the range of 0.6–2.0 U mL−1 with a detection limit of 0.17 U mL−1 (3σ). The feasibility of this present method for kinase inhibitor screening was also studied by assessment of H-89 kinase inhibition with an IC50 value of 0.049 μmol L−1.

Analysis of Xanthine Oxidase Inhibitors from Clerodendranthus spicatus with Xanthine Oxidase Immobilized Silica Coated Fe3O4 Nanoparticles

In this study, xanthine oxidase immobilized silica coated Fe3O4 nanoparticles (Fe3O4@SiO2-XO) were successfully prepared and characterized by transmission electron microscope, X-ray powder diffraction, Fourier transform infrared spectroscopy and vibrating sample magnetometer. The average diameter of the Fe3O4 nanoparticles was about 300 nm to 350 nm with a shell thickness of 60 nm. The maximum saturation magnetization of the Fe3O4@SiO2-XO nanoparticles was 44.9 emu/g, which ensured the separation from the medium within one minute by using an ordinary magnet. A xanthine oxidase (XO) inhibitor screening method using Fe3O4@SiO2-XO nanoparticles was established and utilized in the extract of Clerodendranthus spicatus. Under the optimized conditions, two compounds were screened out and identified as gardenin B and eupatorin. The half maximal inhibitory concentration (IC50) values of these two compounds were 1.488 μg/mL and 11.197 μg/mL, respectively. The interactions between these two compounds and XO were investigated by the fluorescence spectroscopic method. The results suggested that the quenching effects of gardenin B and eupatorin were due to a static quenching mechanism. Furthermore, gardenin B showed stronger binding capacity than that of eupatorin. In conclusion, this screening method exhibited efficiency and reusability in screening, identification and analysis of enzyme inhibitors from complex mixtures.

Temperature Augmented Visual Method for Initial Screening of Hydrate Inhibitors

The formation of gas hydrates in oil & gas pipelines and drilling fluid flow lines is a major issue in the petroleum industry. Gas hydrate inhibitors are normally used to inhibit the formation of gas hydrates in the pipelines/flowlines. Initial screening of hydrate inhibitors and AntiAgglomerants (AA) requires a safe and economical experimental setup/method. Conventional visual method was used for initial screening of hydrate inhibitors in many researches. Some researchers also suggested modified visual methods, but all of them lacks accurate measurement of induction time and found to be inappropriate for experimental solutions like drilling mud, etc. In this work, a temperature augmented visual method was presented which can be used in academic research laboratories for study and initial screening of hydrate inhibitors. This method is capable of parallel screening of inhibitors and determines hydrate induction time precisely. Experiments were conducted to determine the hydrate induction time of different inhibitors using augmented method and compared with conventional visual method. The developed method found to be more precise in determining the induction time of hydrates in all types of experimental solutions.

A High-Throughput Chemical Screening Method for Inhibitors and Potentiators of Hypersensitive Cell Death Using Suspension Cell Culture of Arabidopsis thaliana.

Chemical biology provides an alternative way to identify genes involved in a particular biological process. It has the potential to overcome issues such as redundancy or lethality often found in genetic approaches, since the chemical compounds can simultaneously target all homologous proteins that function at the same step, and chemicals can be applied conditionally. Even with a variety of genetic approaches, the molecular mechanisms of plant hypersensitive cell death that occurs during disease resistance responses remain unclear. Therefore, application of chemical biology should provide new insights into this phenomenon. Here we describe a high-throughput chemical screening procedure to detect hypersensitive cell death quantitatively, using a suspension cell culture of Arabidopsis thaliana and a well-studied avirulent bacterial pathogen, Pseudomonas syringae pv. tomato DC3000 avrRpm1.

Facile and Sensitive Method for Protein Kinase A Activity Assay Based on Fluorescent Off-On PolyU-peptide Assembly.

Phosphorylation mediated by protein kinases plays a pivotal role in metabolic and cell-signaling processes, and the dysfunction of protein kinases such as protein kinase A (PKA) may induce several human diseases. Therefore, it is of great significance to develop a facile and effective method for PKA activity assay and high-throughput screening of inhibitors. Herein, we develop a new fluorescent off-on method for PKA assay based on the assembly of anionic polyuridylic acid (polyU) and cationic fluorescent peptide. The phosphorylation of the peptide disrupts its electrostatic binding with polyU, suppresses the concentration quenching effect of polyU, and thus causes fluorescence recovery. The recovered fluorescence intensity at 585 nm is directly proportional to the PKA activity in the range of 0.1-3.2 U/mL with a detection limit of 0.05 U/mL. Using our method, the PKA activity in HeLa cell lysate is determined to be 58.2 ± 5.1 U/mg protein. The method has also been employed to evaluate the inhibitory effect of PKA inhibitors with satisfactory results and may be expected to be a promising candidate for facile and cost-effective assay of kinase activity and high-throughput inhibitor screening.

An Overview on Screening Methods for Lysine Specific Demethylase 1 (LSD1) Inhibitors.

In the past few years, great of attention has been paid to the identification and characterization of selective and potent inhibitors of the first identified histone demethylase LSD1, which may erase mono- and di-methylated histone 3 lysine 4 and 9. As the aberrant overexpression of LSD1 is involved in various pathological processes, especially cancer, obtaining selective and potent LSD1 inhibitors has emerged as a crucial issue in medicinal chemistry research. Until now, several LSD1 inhibitor screening models have been established, including enzyme coupled assay, LC-MS based assay, and FRET based assay. Nevertheless, due to some special instrument requirement and additional costs of LC-MS and FRET, the enzyme coupled assay is the most widely applied method for LSD1 inhibitor screening. We summarized and compared several reported in vitro LSD1 inhibitor screening models. Each of them has distinct advantages and disadvantages, and none of these methods is perfect. In order to exclude the false positive results, at least one additional method should be applied to screen LSD1 inhibitors.

A physicochemical descriptor based method for effective and rapid screening of dual inhibitors against BACE-1 and GSK-3β as targets for Alzheimer’s disease

Due to multifactorial nature of Alzheimer’s disease one target-one ligand hypothesis often looks insufficient. BACE-1 and GSK-3β are well established therapeutic drug targets and interaction between BACE-1 and GSK-3β pathways has also been established. Thus, designing of dual inhibitor for these two targets seems rational and may provide effective therapeutic strategies against AD. Recent studies revealed that only two scaffolds i.e. triazinone and curcumin act as a dual inhibitor against BACE-1 and GSK-3β. Thus, this discovery set the path to screen new chemical entities from a vast chemical space (∼1060 compounds) that inhibit both the targets. However, small part of the large chemical space will only show biological activity for specific targets. Virtual screening of large libraries is impractical and computational expensive especially in case of dual inhibitor design. In the case of dual or multi target inhibitor designing, we screened the database for each target that further increases time and resources. In this study we have done physicochemical descriptor based profiling to know the biological relevant chemical space for BACE-1 and GSK-3β inhibitors and proposed the suitable range of important physicochemical properties, occurrence of functional groups. We generated scaffolds tree of known inhibitors of BACE-1 and GSK-3β suggesting the common structure/fragment that can be used to design dual inhibitors. This approach can filter the potential dual inhibitor candidates of BACE-1 and GSK-3β from non inhibitors.

Gold nanocluster-based fluorescent assay for label-free detection of protein kinase and its inhibitors

A sensitive fluorometric assay is reported for label-free detection of the activity of protein kinase (PKA) and of its inhibitors. It is based on the europium(III)-modulated fluorescence of peptide-stabilized gold nanoclusters (peptide-AuNCs). Both adenosine-5′-triphosphate (ATP) and adenosine-5′-diphosphate (ADP) (formed from ATP by PKA-catalyzed hydrolysis) enhance the fluorescence of peptide-AuNCs with its excitation/emission peaks of 330/405 nm. The addition of Eu(III) quenches the fluorescence of the ATP/peptide-AuNC system to the original fluorescence level of peptide-AuNCs, while addition of Eu(III) has no effect on the fluorescence of the ATP/PKA/peptide-AuNC system. Based on this “turn-on” method, the activity of PKA can be detected with high sensitivity in 0.05–1.6 U⋅mL−1 activity range, with a detection limit of 0.02 U⋅mL−1 (3σ). The feasibility of this method for screening of inhibitors was also studied. The IC50 value for the commercially available inhibitor H-89 was found to be 43 nM. Moreover, the detection scheme was also applied to monitoring the drug-stimulated activation of PKA in HepG-2 cell lysates.

High-Throughput Dual Screening Method for Ras Activities and Inhibitors.

Ras GTPases act as "molecular switches", alternating between inactive GDP-bound and active GTP-bound conformation. Ras-oncogenes were discovered over three decades ago, but there are still no effective therapies for Ras-driven cancers. So far, drug discovery strategies have been unsuccessful, because of a lack of suitable screening methodologies and well-defined binding pockets on the Ras proteins. Here, we addressed the former by introducing a homogeneous quenching resonance energy transfer (QRET) technique-based screening strategy for Ras interfacial and competitive inhibitors. We demonstrate that using a unique GTP-specific antibody fragment to monitor GTPase cycling in the presence of a guanine nucleotide exchange factor (GEF) and a GTPase activating protein (GAP) is an efficient method for Ras inhibitor high-throughput screening. When compared to a conventional GEF-stimulated nucleotide exchange assay in a proof-of-concept screen, we identified an overlapping set of potential inhibitor compounds but also compounds found exclusively with the new GTP hydrolysis monitoring-based GTPase cycling assay.

Rapid Screening of Potential Phosphodiesterase Inhibitors from the Roots of Ilex pubescens Hook. et Arn. Using a Combination of Ultrafiltration and LC-MS.

The cyclic nucleotide phosphodiesterase (PDE) plays an important role in regulating the levels of second messenger molecules cAMP and cGMP. Various PDE inhibitors have been successfully developed into drugs for targeted diseases. In addition, PDE inhibitors can also be found in different foods and natural medicines. In this study, ultrafiltration liquid chromatography–diode-array detector–electrospray ionization–ion-trap–time-of-flight–mass spectrometry (ultrafiltration LC–DAD–ESI–IT–TOF–MS) was applied to screen PDE inhibitors from the roots of Ilex pubescens Hook. et Arn. As a result, 11 major compounds were identified in I. pubescens roots, with nine compounds as potential PDE inhibitors, among which five were further confirmed to be active against PDEI and PDE5A dose-dependently in vitro, with ilexsaponin A1 and ilexsaponin B2 being the strongest. HPLC quantification of these bioactive compounds suggested that they are major components in the plant. The results demonstrate that ultrafiltration LC–DAD–ESI–IT–TOF–MS is an efficient method for rapid screening of PDE inhibitors from natural medicines.

A single electrochemical biosensor for detecting the activity and inhibition of both protein kinase and alkaline phosphatase based on phosphate ions induced deposition of redox precipitates

Protein kinase (PKA) and alkaline phosphatase (ALP) are clinically relevant enzymes for a number of diseases. In this work, we developed a new simple electrochemical biosensor for the detection of the activity and inhibition of both PKA and ALP. One common feature of the PKA and ALP catalyzing process is that PKA can hydrolysis adenosine-5′-triphosphate (ATP) and ALP can hydrolysis pyrophosphate, both reactions produce phosphate ions, and the amount of phosphate ion produced is proportional to enzyme activity. Our assay is based on the principle that phosphate ions react with molybdate to form redox molybdophosphate precipitates on the electrode surface, thus generating electrochemical current. The detection limit for PKA and ALP were much lower than existing assays. The biosensor has good specificity and was used to measure drug-stimulated PKA from lysates of HeLa cells. We also evaluated the use of the biosensor as a screening tool for enzyme inhibitors. To the best of our knowledge, this is the first report of a biosensor capable of detecting the activity of both PKA and ALP. This tool has the potential to simplify PKA and ALP clinical measurement, thereby improving diagnostics of relevant diseases. It also may serve as the basis for a simple screening method for new enzyme inhibitors for disease treatment.

A 96-well microtiter plate assay for high-throughput screening of Mycobacterium tuberculosis dTDP-d-glucose 4,6-dehydratase inhibitors

Mycobacterium tuberculosis dTDP-d-glucose 4,6-dehydratase (RmlB) is the second enzyme for the biosynthesis of dTDP-l-rhamnose, which is a sugar donor to the synthesis of the cell wall linker, d-N-acetylglucosamine-l-rhamnose. RmlB is essential to mycobacterial growth and is not found in humans; therefore, it is a potential target for developing new anti-tuberculosis drugs. So far, there has been no suitable method for high-throughput screening of RmlB inhibitors. Here, the recombinant M. tuberculosis RmlB was purified and an absorbance-based microtiter plate assay was developed for RmlB activity. It could be used for high-throughput screening of RmlB inhibitors. The kinetic properties of M. tuberculosis RmlB, including optimal pH, optimal temperature, the effect of metal ions, and the kinetic parameters, were determined with this assay. The inhibitory effects of dTTP and dTDP on M. tuberculosis RmlB were also studied with the assay.

Selective Sensing of Tyrosine Phosphorylation in Peptides Using Terbium(III) Complexes.

Phosphorylation of tyrosine residues in proteins, as well as their dephosphorylation, is closely related to various diseases. However, this phosphorylation is usually accompanied by more abundant phosphorylation of serine and threonine residues in the proteins and covers only 0.05% of the total phosphorylation. Accordingly, highly selective detection of phosphorylated tyrosine in proteins is an urgent subject. In this review, recent developments in this field are described. Monomeric and binuclear TbIII complexes, which emit notable luminescence only in the presence of phosphotyrosine (pTyr), have been developed. There, the benzene ring of pTyr functions as an antenna and transfers its photoexcitation energy to the TbIII ion as the emission center. Even in the coexistence of phosphoserine (pSer) and phosphothreonine (pThr), pTyr can be efficintly detected with high selectivity. Simply by adding these TbIII complexes to the solutions, phosphorylation of tyrosine in peptides by protein tyrosine kinases and dephosphorylation by protein tyrosine phosphatases can be successfully visualized in a real-time fashion. Furthermore, the activities of various inhibitors on these enzymes are quantitatively evaluated, indicating a strong potential of the method for efficient screening of eminent inhibitors from a number of candidates.

Screening of inhibitors of glycogen synthase kinase-3β from traditional Chinese medicines using enzyme-immobilized magnetic beads combined with high-performance liquid chromatography

Glycogen synthase kinase-3β (GSK-3β) was immobilized on magnetic beads (MBs) by affinity method for the first time. The enzyme-immobilized MBs were coupled with high-performance liquid chromatography-ultraviolet (HPLC-UV) technique to establish a cost-effective and reliable method for screening of inhibitors of GSK-3β. A peptide substrate of GSK-3β containing a tyrosine residue was employed since it can be sensitively detected by UV detector at 214nm. The substrate and its phosphorylated product were separated by baseline within 10min. The enzyme activity was determined by the quantification of peak area of the product. Parameters including enzyme immobilization, enzyme reaction and the performance of immobilized-enzyme were investigated. The immobilized enzyme can be reused for 10 times and remain stable for 4 days at 4°C. The inhibitory activities of extracts of 15 traditional Chinese medicines (TCMs) were screened. As a result, three of them including Euonymus fortunei, Amygdalus communis and Garcinia xanthochymus were found possessing high inhibitory activities (inhibition rate >90%). From G. xanthochymus, a new inhibitor of GSK-3β, fukugetin, was discovered with an IC50 value of 3.18±0.07μM. Enzyme kinetics and molecular docking experiments further revealed the inhibitory mechanism, indicating fukugetin was a non-ATP competitive inhibitor interacting with the phosphate recognizing substrate binding site of GSK-3β.

Practical method for the rapid screening of xanthine oxidase inhibitors in herbal extracts by high-performance liquid chromatography based on on-line precolumn enzymatic reaction.

A high-performance liquid chromatography method with on-line precolumn enzymatic reaction for the screening of xanthine oxidase inhibitors in natural extracts was developed. In this method, the enzymatic reaction occurred at the capillary inlet during a predetermined waiting period, after which the reaction product, uric acid, was separated and detected by liquid chromatography using ultraviolet absorption at 295 nm. Enzyme inhibition can be read out directly from the reduced peak area of uric acid in comparison to a reference chromatogram obtained in the absence of any inhibitor. In the present study, the availability of on-line precolumn enzymatic reaction with ultraviolet detection was firstly evaluated by determining the inhibitory mechanism and IC50 values of allopurinol, a commercially available positive drug. Then, the newly developed method was applied to screening of ten natural extracts from traditional Chinese medicine and as a result, the extract of Epimedium sagittatum (Sieb. et Zucc.) Maxim was found to be most positive for xanthine oxidase inhibition. The results obtained were compared with those obtained by offline enzyme assay and the effectiveness of the present method was confirmed. A rapid, low-cost, and fully automated method for xanthine oxidase inhibitor screening was proposed.

Sensitive fluorimetric assays for α-glucosidase activity and inhibitor screening based on β-cyclodextrin-coated quantum dots.

A fluorescence method was established for a α-glucosidase activity assay and inhibitor screening based on β-cyclodextrin-coated quantum dots. p-Nitrophenol, the hydrolysis product of the α-glucosidase reaction, could quench the fluorescence of β-cyclodextrin-coated quantum dots via an electron transfer process, leading to fluorescence turn-off, whereas the fluorescence of the system turned on in the presence of α-glucosidase inhibitors. Taking advantage of the excellent properties of quantum dots, this method provided a very simple, rapid and sensitive screening method for α-glucosidase inhibitors. Two α-glucosidase inhibitors, 2,4,6-tribromophenol and acarbose, were used to evaluate the feasibility of this screening model, and IC50 values of 24 μM and 0.55 mM were obtained respectively, which were lower than those previously reported. The method may have potential application in screening α-glucosidase inhibitors.

Screening of epidermal growth factor receptor inhibitors in natural products by capillary electrophoresis combined with high performance liquid chromatography–tandem mass spectrometry

A method for screening of inhibitors to epidermal growth factor receptor (EGFR) in natural product extracts with capillary electrophoresis (CE) in conjunction with high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) is reported. The method was established by employing 5-carboxyfluorescein labeled substrate peptide, two commercially available EGFR inhibitors OSI-744 and ZD1839, and a small chemical library consisted of 39 natural product extracts derived from the Traditional Chinese Medicines. Biochemical assay of crude natural product extracts was carried out by using CE equipped with a laser induced fluorescence detector. The CE separation allowed an accurately quantitative measurement of the phosphorylated product, hence the measurement of the enzymatic activity as well as the inhibition kinetics. The hits are identified if the peak area of the phosphorylated product is reduced in comparison with the negative control. The active constituents in the natural product extract were then identified by an assay-guided isolation with HPLC–MS/MS system. With the method, the flavonoids component of the Lycopus lucidus extract, namely quercetin and rutin were identified to be the active ingredients. Their IC50 values were determined as 0.88μM and 10.1μM, respectively. This result demonstrated a significant merit of our method in the identification of the bioactive compounds in natural products.

Development of new methods for determining the heparanase enzymatic activity

IntroductionHeparanase is a mammalian endo-β-glucuronidase. Notwithstanding its importance in various pathological and non-pathological events few straightforward methods for heparanase enzymatic activity has been stated. The aim of this study was to develop two heparanase activity assays to cover a whole range of applications. First, a fast and easy method based on commercial homogenous substrate, fondaparinux, was described. The other method is a quantitative assay based on biotinylated heparan sulfate that uses an easier technique to immobilize the substrate in a 96-well plate. Methods: 1)The heparanase recombinant enzyme and fondaparinux were incubated overnight. After incubation, a fluorescent redox marker, resazurin, was added. The reduction of resazurin depends on the amount of glucuronic acid released by heparanase digestion. Fluorescence measurements were done using excitation and emission wavelengths of 560 nm and 590 nm, respectively. Methods: 2)The 96-well plate was incubated with protamine sulfate. Subsequently, biotinylated heparan sulfate was immobilized. The enzymatic assay was performed using chimeric recombinant heparanase at different concentrations. In sequence, the immobilized biotinylated heparan sulfate that was not digested by recombinant heparanase was bound to streptavidin conjugated with europium. Fluorescence was measured using a time-resolved fluorometer. ConclusionBoth methods have high sensitivity and can be used to detect heparanase activity. Fondaparinux assay is a quick and easy method for screening of heparanase inhibitors using recombinant enzyme or bacterial crude extract. Biotinylated heparan sulfate assay can be used for quantitative analysis in biological samples and protamine sulfate showed been capable to immobilized heparan sulfate.

Determination of α-glucosidase inhibitors from ScutScutellaria baicalensis using liquid chromatography with quadrupole time of flight tandem mass spectrometry coupled with centrifugal ultrafiltration

The present study aimed at identifying potential lead compounds for diabetes mellitus drug discovery. We developed a novel method involving centrifugal ultrafiltration separation subsequent liquid chromatography with quadrupole time of flight tandem mass spectrometry (LC-Q/TOF-MS/MS) determination to screen α-glucosidase inhibitors in complex Scutellaria baicalensis Georgi (SBG) extract. By adding a second filter to the screening process, the level of non-specific binding of Compounds 1, 3, 10 and 11 was significantly decreased, and the level of non-specific binding of Compounds 5 and 15 also was reduced. As a result, five flavonoids identified as baicalein, baicalein, wogonin, chrysin, and oroxylin A, were rapidly found to interact with α-glucosidase and possess potent anti-α-glucosidase activity in vitro. Specific binding of ligands to α-glucosidase was demonstrated though the proposed method and the ligands could be ranked in order of affinity for α-glucosidase, which were corresponded to the order of inhibitory activity in vitro. In conclusion, our results indicated that the developed method is a rapid and effective screening method for rat intestinal α-glucosidase inhibitors from complex herbal medicines such as SBG.