The glycine-to-aspartic acid missense mutation at the codon 551(G551D) of the cystic fibrosis transmembrane conductance regulator(CFTR) is one of the five most frequent cystic fibrosis(CF) mutations associated with a ...The glycine-to-aspartic acid missense mutation at the codon 551(G551D) of the cystic fibrosis transmembrane conductance regulator(CFTR) is one of the five most frequent cystic fibrosis(CF) mutations associated with a severe CF phenotype. To explore the feasibility of pharmacological correction of disrupted activation of CFTR chloride channel caused by G551D mutation, we developed a halide-sensitive fluorescence miniassay for G551D-CFTR in Fisher rat thyroid(FRT) epithelial cells for the discovery of novel activators of G551D-CFTR. A class of bicyclooctane small molecule compounds that efficiently stimulate G551D-CFTR chloride channel activity was identified by high throughput screening via the FRT cell-based assay. This class of compounds selectively activates G551D-CFTR with a high affinity, whereas little effect of the compounds on wildtype CFTR can be seen. The discovery of a class of bicyclooctane G551D-CFTR activators will permit the analysis of structure-activity relationship of the compounds to identify ideal leads for in vivo therapeutic studies.展开更多
Xenobiotic drugs and chemicals directly interact with DNA,proteins,or other biomolecules in cells. These direct interactions with molecular targets may trigger a series of downstream effects on metabolic-biochemical a...Xenobiotic drugs and chemicals directly interact with DNA,proteins,or other biomolecules in cells. These direct interactions with molecular targets may trigger a series of downstream effects on metabolic-biochemical and regulatory-signaling networks that can invoke cellular consequences leading to adaptive homeostatic or adverse pathological responses. Regulators for drug and chemicals safety have therefore since long required the testing of toxicity in animal models before drugs and pesticides can enter the market. The US National Research Council of the National Academy of Sciences,in its report,Toxicity Testing in the 21st Century: a Vision and a Strategy [1] ,proposed that toxicity testing should become less reliant on apical endpoints from whole animal tests and eventually rely instead on quantitative,doseresponse models based on information from in vitro assays and in vivo biomarkers,which can be used to screen large numbers of chemicals. The present paper reports about a combination of HTS in vitro assays that can be used to study the potential tumorigenic effect of xenobiotics ( drug targets,environmental chemicals) via a set of"sentinel"genes [2] that are functionally interrelated based on evidence weighted functional linkage network ( FLN ) log-likelihood scores ( Linghu et al [3] ) .展开更多
The P2X 7 receptor (P2X7R) is an important member of the P2X family of ligand-gated ion channels that respond to ATP as the endogenous agonist. Studies suggest that P2X7R plays a potentially pivotal role in a variety ...The P2X 7 receptor (P2X7R) is an important member of the P2X family of ligand-gated ion channels that respond to ATP as the endogenous agonist. Studies suggest that P2X7R plays a potentially pivotal role in a variety of physiological functions, including peripheral and central neuronal transmission, smooth muscle contraction, and inflammation. Thus, P2X7R may be a potential target for drug development. Here, we used a FlexStation to examine the function of recombinant P2X7R stably expressed in human embryonic kidney 293 cells and to compare three high-throughput screening assays: a membrane potential assay, an ethidium bromide uptake assay, and a calcium influx assay. We found that all three assays were suitable for the analysis of P2X7R, but the calcium influx assay was the most robust and is the best choice as a first high-throughput screening assay when embarking on a P2X7R drug discovery project.展开更多
The process of identifying novel human immunodeficiency virus 1 (HIV-1) inhibitors presents a challenge for industrial and scientific research.Virus-cell-based screening approaches offer some advantages in the quest f...The process of identifying novel human immunodeficiency virus 1 (HIV-1) inhibitors presents a challenge for industrial and scientific research.Virus-cell-based screening approaches offer some advantages in the quest for novel inhibitors because they include multiple targets in a single screen and in some cases reveal targets not captured in biochemical assays.In this study,a high-throughput screening (HTS) system for HIV-1 inhibitors was developed,which allows the simultaneous screening of all the HIV-1 targets required for replication in the cell culture.HeLa/cluster of differentiation 4 (CD4)/long terminal repeat (LTR) indicator cells,which stably expressed high levels of HIV receptor CD4 and contained the firefly luciferase reporter gene under the control of the HIV-1 LTR promoter,were generated.The expression of CD4 and LTR function in this cell line was validated by Western blot and luciferase assay.MT2 cells,a human T-cell leukemia cell line that support high levels of HIV-1 replication,were infected with HIV-1,and then the infected MT2 cells were co-cultured with HeLa/CD4/LTR cells.In the optimized assay conditions,HIV-1 replication occurs rapidly in the MT2 cells,resulting in the infection of the HeLa/CD4/LTR cells and a significant induction of luciferase signals through LTR,which is activated by the expression of HIV-1 tat gene.The luciferase signals of HeLa/CD4/LTR cells co-cultured with HIV-1 infected MT2 cells were significantly stronger than the signals of noninfected HeLa/CD4/LTR cells (P < 0.001).The inhibitory effects of HIV-1 inhibitors (3'-Azido-3'-deoxythymidine [AZT],efavirenz [EFV],and nevirapine [NVP]) were evaluated with this assay,and the inhibitory concentration 50% (IC50) values of the above three inhibitors were 58,1.4,and 85 nmol/L,respectively,indicating that the assay provides the necessary sensitivity for identifying antiviral molecules.The Z' factor had a value of 0.563,indicating this is a very robust assay.These results suggested that HIV-1 infection assay represents a novel approach to HIV-1 antiviral screening that allows for the effective execution of HTS campaigns.展开更多
AIM: To establish a novel, sensitive and high-throughput gelatinolytic assay to define new inhibitors and compare domain deletion mutants of gelatinase B/matrix metalloproteinase (MMP)-9. METHODS: Fluorogenic Dye-quen...AIM: To establish a novel, sensitive and high-throughput gelatinolytic assay to define new inhibitors and compare domain deletion mutants of gelatinase B/matrix metalloproteinase (MMP)-9. METHODS: Fluorogenic Dye-quenched (DQ)TM-gelatin was used as a substrate and biochemical parameters (substrate and enzyme concentrations, DMSO solvent concentrations) were optimized to establish a highthroughput assay system. Various small-sized libraries (ChemDiv, InterBioScreen and ChemBridge) of hetero-cyclic, drug-like substances were tested and compared with prototypic inhibitors. RESULTS: First, we designed a test system with gelatin as a natural substrate. Second, the assay was validated by selecting a novel pyrimidine-2,4,6-trione (barbitu- rate) inhibitor. Third, and in line with present structural data on collagenolysis, it was found that deletion of the O-glycosylated region significantly decreased gelatinolytic activity (kcat/kM ± 40% less than full-length MMP-9). CONCLUSION: The DQTM-gelatin assay is useful in high-throughput drug screening and exosite targeting. We demonstrate that flexibility between the catalytic and hemopexin domain is functionally critical for gelatinolysis.展开更多
A variety of technologies continue to develop for high throughput screening This review summarized recent advances of bioassay techniques used in high throughput screening The principles, applications, characteristics...A variety of technologies continue to develop for high throughput screening This review summarized recent advances of bioassay techniques used in high throughput screening The principles, applications, characteristics and other parameters of these methods were described such as homogeneous time resolved fluorescence, fluorescence polarization, fluorescence resonance energy transfer, fluorescence correlation spectroscopy and scintillation proximity展开更多
The experimental models for drug screening are very important points in drug discovery Although the drug screening techniques have been developed, such as high throughput screening (HTS), the screening assay methods (...The experimental models for drug screening are very important points in drug discovery Although the drug screening techniques have been developed, such as high throughput screening (HTS), the screening assay methods (models) still limited drug discovery In present paper, advanced animal models, cell assays and molecular methodology used for drug discovery were reviewed The characteristics and requires of the assay methods used for drug discovery in HTS were also展开更多
目的构建适于高通量筛选的激活素受体样激酶4(ALK4),ALK5和ALK7抑制剂的筛选模型。方法利用Bac to Bac昆虫杆状病毒表达系统,构建ALK4激酶结构域(ALK4kd),ALK5kd和ALK7kd及Smad2和Smad3蛋白的病毒表达系统,转染Sf9昆虫细胞,表达目的蛋白...目的构建适于高通量筛选的激活素受体样激酶4(ALK4),ALK5和ALK7抑制剂的筛选模型。方法利用Bac to Bac昆虫杆状病毒表达系统,构建ALK4激酶结构域(ALK4kd),ALK5kd和ALK7kd及Smad2和Smad3蛋白的病毒表达系统,转染Sf9昆虫细胞,表达目的蛋白;通过谷胱甘肽S-转移酶(GST)亲和柱纯化,获得纯化的目的蛋白;分别以纯化的ALK4,ALK5,ALK7激酶片段与纯化的Smad3和ATP构建ALK4,ALK5,ALK7激酶活性测定体系,优化各反应底物浓度及反应条件,建立适于通量化分析的筛选模型;以已知ALK激酶抑制剂SB431542为工具分子,检测其对激酶的抑制活性,确证筛选模型的可靠性。结果经酶切和PCR鉴定,所得重组Bacmid均正确。转染Sf9昆虫细胞后纯化获得相应目的蛋白。经条件优化,反应体系中激酶和底物蛋白Smad3最佳浓度均为10 mg·L^(-1),ATP最佳初始浓度为10 nmol·L^(-1)。所得ALK4,ALK5和ALK7激酶抑制剂筛选体系的Z′因子分别为0.71,0.51和0.74,符合高通量筛选要求。已知SB431542在所建模型上对ALK4kd,ALK5kd和ALK7kd的抑制活性IC50值分别为22,188和91 nmol·L^(-1)。结论成功构建了ALK4,ALK5和ALK7激酶抑制剂的体外筛选模型。展开更多
基金the Start- up Fund for Returned Overseas Scholars from Northeast Normal U niversity,National ScienceFund for Distinguished Young Scholars(No.30 32 5 0 11) ,Distinguished Young Scholars Fund of Jilin Province(No.2 0 0 30 112 ) ,Excellent Young Teachers Pr
文摘The glycine-to-aspartic acid missense mutation at the codon 551(G551D) of the cystic fibrosis transmembrane conductance regulator(CFTR) is one of the five most frequent cystic fibrosis(CF) mutations associated with a severe CF phenotype. To explore the feasibility of pharmacological correction of disrupted activation of CFTR chloride channel caused by G551D mutation, we developed a halide-sensitive fluorescence miniassay for G551D-CFTR in Fisher rat thyroid(FRT) epithelial cells for the discovery of novel activators of G551D-CFTR. A class of bicyclooctane small molecule compounds that efficiently stimulate G551D-CFTR chloride channel activity was identified by high throughput screening via the FRT cell-based assay. This class of compounds selectively activates G551D-CFTR with a high affinity, whereas little effect of the compounds on wildtype CFTR can be seen. The discovery of a class of bicyclooctane G551D-CFTR activators will permit the analysis of structure-activity relationship of the compounds to identify ideal leads for in vivo therapeutic studies.
文摘Xenobiotic drugs and chemicals directly interact with DNA,proteins,or other biomolecules in cells. These direct interactions with molecular targets may trigger a series of downstream effects on metabolic-biochemical and regulatory-signaling networks that can invoke cellular consequences leading to adaptive homeostatic or adverse pathological responses. Regulators for drug and chemicals safety have therefore since long required the testing of toxicity in animal models before drugs and pesticides can enter the market. The US National Research Council of the National Academy of Sciences,in its report,Toxicity Testing in the 21st Century: a Vision and a Strategy [1] ,proposed that toxicity testing should become less reliant on apical endpoints from whole animal tests and eventually rely instead on quantitative,doseresponse models based on information from in vitro assays and in vivo biomarkers,which can be used to screen large numbers of chemicals. The present paper reports about a combination of HTS in vitro assays that can be used to study the potential tumorigenic effect of xenobiotics ( drug targets,environmental chemicals) via a set of"sentinel"genes [2] that are functionally interrelated based on evidence weighted functional linkage network ( FLN ) log-likelihood scores ( Linghu et al [3] ) .
文摘The P2X 7 receptor (P2X7R) is an important member of the P2X family of ligand-gated ion channels that respond to ATP as the endogenous agonist. Studies suggest that P2X7R plays a potentially pivotal role in a variety of physiological functions, including peripheral and central neuronal transmission, smooth muscle contraction, and inflammation. Thus, P2X7R may be a potential target for drug development. Here, we used a FlexStation to examine the function of recombinant P2X7R stably expressed in human embryonic kidney 293 cells and to compare three high-throughput screening assays: a membrane potential assay, an ethidium bromide uptake assay, and a calcium influx assay. We found that all three assays were suitable for the analysis of P2X7R, but the calcium influx assay was the most robust and is the best choice as a first high-throughput screening assay when embarking on a P2X7R drug discovery project.
文摘The process of identifying novel human immunodeficiency virus 1 (HIV-1) inhibitors presents a challenge for industrial and scientific research.Virus-cell-based screening approaches offer some advantages in the quest for novel inhibitors because they include multiple targets in a single screen and in some cases reveal targets not captured in biochemical assays.In this study,a high-throughput screening (HTS) system for HIV-1 inhibitors was developed,which allows the simultaneous screening of all the HIV-1 targets required for replication in the cell culture.HeLa/cluster of differentiation 4 (CD4)/long terminal repeat (LTR) indicator cells,which stably expressed high levels of HIV receptor CD4 and contained the firefly luciferase reporter gene under the control of the HIV-1 LTR promoter,were generated.The expression of CD4 and LTR function in this cell line was validated by Western blot and luciferase assay.MT2 cells,a human T-cell leukemia cell line that support high levels of HIV-1 replication,were infected with HIV-1,and then the infected MT2 cells were co-cultured with HeLa/CD4/LTR cells.In the optimized assay conditions,HIV-1 replication occurs rapidly in the MT2 cells,resulting in the infection of the HeLa/CD4/LTR cells and a significant induction of luciferase signals through LTR,which is activated by the expression of HIV-1 tat gene.The luciferase signals of HeLa/CD4/LTR cells co-cultured with HIV-1 infected MT2 cells were significantly stronger than the signals of noninfected HeLa/CD4/LTR cells (P < 0.001).The inhibitory effects of HIV-1 inhibitors (3'-Azido-3'-deoxythymidine [AZT],efavirenz [EFV],and nevirapine [NVP]) were evaluated with this assay,and the inhibitory concentration 50% (IC50) values of the above three inhibitors were 58,1.4,and 85 nmol/L,respectively,indicating that the assay provides the necessary sensitivity for identifying antiviral molecules.The Z' factor had a value of 0.563,indicating this is a very robust assay.These results suggested that HIV-1 infection assay represents a novel approach to HIV-1 antiviral screening that allows for the effective execution of HTS campaigns.
基金Supported by A postdoctoral fellowship (Van den Steen PE) and a research assistantship (Geurts N) of the Fund for Scientific Research,Belgium (FWO-Vlaanderen)
文摘AIM: To establish a novel, sensitive and high-throughput gelatinolytic assay to define new inhibitors and compare domain deletion mutants of gelatinase B/matrix metalloproteinase (MMP)-9. METHODS: Fluorogenic Dye-quenched (DQ)TM-gelatin was used as a substrate and biochemical parameters (substrate and enzyme concentrations, DMSO solvent concentrations) were optimized to establish a highthroughput assay system. Various small-sized libraries (ChemDiv, InterBioScreen and ChemBridge) of hetero-cyclic, drug-like substances were tested and compared with prototypic inhibitors. RESULTS: First, we designed a test system with gelatin as a natural substrate. Second, the assay was validated by selecting a novel pyrimidine-2,4,6-trione (barbitu- rate) inhibitor. Third, and in line with present structural data on collagenolysis, it was found that deletion of the O-glycosylated region significantly decreased gelatinolytic activity (kcat/kM ± 40% less than full-length MMP-9). CONCLUSION: The DQTM-gelatin assay is useful in high-throughput drug screening and exosite targeting. We demonstrate that flexibility between the catalytic and hemopexin domain is functionally critical for gelatinolysis.
文摘A variety of technologies continue to develop for high throughput screening This review summarized recent advances of bioassay techniques used in high throughput screening The principles, applications, characteristics and other parameters of these methods were described such as homogeneous time resolved fluorescence, fluorescence polarization, fluorescence resonance energy transfer, fluorescence correlation spectroscopy and scintillation proximity
文摘The experimental models for drug screening are very important points in drug discovery Although the drug screening techniques have been developed, such as high throughput screening (HTS), the screening assay methods (models) still limited drug discovery In present paper, advanced animal models, cell assays and molecular methodology used for drug discovery were reviewed The characteristics and requires of the assay methods used for drug discovery in HTS were also
