The techniques used in the field of drug discovery have been quickly developed during the past decades High throughput screening (HTS) is a new technique in drug discovery with the characteristics of efficiency, huge ...The techniques used in the field of drug discovery have been quickly developed during the past decades High throughput screening (HTS) is a new technique in drug discovery with the characteristics of efficiency, huge scale and automatically operation The assay methods used in HTS are based on the molecular targets and cells Screening results obtained from these methods indicate not only the bioactivities of compounds (samples) but also the mechanisms of actions of the compounds (samples) HTS has altered the strategy of drug展开更多
Neuraminidase (NA) is one of the most important targets to screen the drugs of anti-influenza virus A and B. After virtual screening approaches were applied to a compound database which possesses more than 10000 compo...Neuraminidase (NA) is one of the most important targets to screen the drugs of anti-influenza virus A and B. After virtual screening approaches were applied to a compound database which possesses more than 10000 compound structures, 160 compounds were selected for bioactivity assay, then a High Throughput Screening (HTS) model established for influenza virus NA inhibitors was applied to detect these compounds. Finally, three compounds among them displayed higher inhibitory activities, the range of their IC5o was from 0.1 μmol/L to 3 μmol/L. Their structural scaffolds are novel and different from those of NA inhibitors approved for influenza treatment, and will be useful for the design and research of new NA inhibitors. The result indicated that the combination of virtual screening with HTS was very significant to drug screening and drug discovery.展开更多
目的：建立糖原磷酸化酶（GPa）抑制剂的体外高通量筛选模型。方法：用梯度离心方法提取大鼠肝脏GPa，以葡萄糖-1-磷酸作为底物，通过测定反应中磷酸根的释放量，间接反映肝脏GPa的活性。通过对反应体系的优化，调整反应条件，建立96孔...目的：建立糖原磷酸化酶（GPa）抑制剂的体外高通量筛选模型。方法：用梯度离心方法提取大鼠肝脏GPa，以葡萄糖-1-磷酸作为底物，通过测定反应中磷酸根的释放量，间接反映肝脏GPa的活性。通过对反应体系的优化，调整反应条件，建立96孔微孔板的高通量筛选模型，用咖啡因对此模型进行验证，并评价高通量技术参数[Z＇-因子（Z＇-factor），信号本底比（signal to background，S／B），信噪比（signal to noise，S／N）]。用此模型对5000个样品（包括合成化合物、天然提取物）组成的随机库进行体外筛选，考察这些样品对GPa的抑制作用。结果：确定反应体系条件是：反应温度25℃，反应时间30min，底物浓度0．5mmol·L^-1，大鼠肝脏GPa的用量为250ng。用此条件测定咖啡因对GPa的抑制曲线，计算其半数抑制浓度（IC50）为（285．3±39．6）μmol·L^-1，与文献报道（IC50=240μmol·L^-1）基本一致；本模型技术参数Z＇-因子=0．79，S／B=0．45，S／N=11．32，表明此模型可以用于高通量筛选。应用此模型筛选得到有活性的化合物9个，其IC50在3．56～45．75μmol·L^-1。结论：建立的体外GPa抑制剂高通量筛选模型具有快速、微量、准确的特点，可以作为研究降糖药的工具。展开更多
The recent advent of robust methods to grow human tissues as 3D organoids allows us to recapitulate the 3D architecture of tumors in an in vitro setting and offers a new orthogonal approach for drug discovery.However,...The recent advent of robust methods to grow human tissues as 3D organoids allows us to recapitulate the 3D architecture of tumors in an in vitro setting and offers a new orthogonal approach for drug discovery.However,organoid culturing with extracellular matrix to support 3D architecture has been challenging for high-throughput screening(HTS)-based drug discovery due to technical difficulties.Using genetically engineered human colon organoids as a model system,here we report our effort to miniaturize such 3D organoid culture with extracellular matrix support in high-density plates to enable HTS.We first established organoid culturing in a 384-well plate format and validated its application in a cell viability HTS assay by screening a 2036-compound library.We further miniaturized the 3D organoid culturing in a 1536-well ultra-HTS format and demonstrated its robust performance for large-scale primary compound screening.Our miniaturized organoid culturing method may be adapted to other types of organoids.By leveraging the power of 3D organoid culture in a high-density plate format,we provide a physiologically relevant screening platform to model tumors to accelerate organoid-based research and drug discovery.展开更多
A stably transfected CHO cell line coexpressing G551D-CFTR and iodide-sensitive yellow fluorescent protein mutant EYFP-H148Q-I152L was successfully established and used as assay model to identify small-molecule activa...A stably transfected CHO cell line coexpressing G551D-CFTR and iodide-sensitive yellow fluorescent protein mutant EYFP-H148Q-I152L was successfully established and used as assay model to identify small-molecule activators of G551D-CFTR chloride channel from 100000 diverse combinatorial compounds by high throughput screening on a customized Beckman robotic system. A bicyclooctane compound was identified to activate G551D-CFTR chloride channel with high-affinity(K d=1.8 μmol/L). The activity of the bicyclooctane compound is G551D-CFTR-specific, reversible and non-toxic. The G551D-CFTR activator may be useful as a tool to study the mutant G551D-CFTR chloride channel structure and transport properties and as a candidate drug to cure cystic fibrosis caused by G551D-CFTR mutation.展开更多
Traditional Chinese medicine(TCM) has been widely used in China and other Asia countries for thousands of years to treat or prevent human diseases. Chinese herbal medicine, one of the most important components of TCM,...Traditional Chinese medicine(TCM) has been widely used in China and other Asia countries for thousands of years to treat or prevent human diseases. Chinese herbal medicine, one of the most important components of TCM, has unique diversities in chemical components, and thus results in a wide range of biological activities. However, pharmaceutical industry is facing a major challenge to develop a large population of novel natural products and drugs, and considerable efforts have not resulted in highvolume of novel drug discovery and productivity. At present, increasing attention has been paid to Chinese herb medicine modernization in combination with the cutting-age technologies of drug discovery, especially the high throughput selection. High content imaging is an image-based high throughput screening method by using automated microscopy and image analysis software to capture and analyze phenotypes at a large scale to investigate multiple biological features simultaneously in the biological complex. Here, we described the pipeline of the state-of-the-art high content imaging technology, summarized the applications of the high content imaging technology in drug discovery from traditional Chinese herbal medicine, and finally discussed the current challenges and future perspectives for development of high throughput image-based screening technology in novel drug research and discovery.展开更多
Hantaviruses,such as Hantaan virus(HTNV)and Seoul virus,are the causative agents of Hantavirus cardiopulmonary syndrome(HCPS)and hemorrhagic fever with renal syndrome(HFRS),and are important zoonotic pathogens.China h...Hantaviruses,such as Hantaan virus(HTNV)and Seoul virus,are the causative agents of Hantavirus cardiopulmonary syndrome(HCPS)and hemorrhagic fever with renal syndrome(HFRS),and are important zoonotic pathogens.China has the highest incidence of HFRS,which is mainly caused by HTNV and Seoul virus.No approved antiviral drugs are available for these hantaviral diseases.Here,a chemiluminescence-based highthroughput-screening(HTS)assay was developed and used to screen HTNV pseudovirus(HTNVpv)inhibitors in a library of 1813 approved drugs and 556 small-molecule compounds from traditional Chinese medicine sources.We identified six compounds with in vitro anti-HTNVpvactivities in the low-micromolar range(EC50values of0.1–2.2μmol/L;selectivity index of 40–900).Among the six selected compounds,cepharanthine not only showed good anti-HTNVpvactivity in vitro but also inhibited HTNVpv-fluc infection in Balb/c mice 5 h after infection by94%(180 mg/kg/d,P<0.01),93%(90 mg/kg/d,P<0.01),or 92%(45 mg/kg/d,P<0.01),respectively,in a bioluminescent imaging mouse model.A time-of-addition analysis suggested that the antiviral mechanism of cepharanthine involves the membrane fusion and entry phases.Overall,we have established a HTS method for antiviral drugs screening,and shown that cepharanthine is a candidate for HCPS and HFRS therapy.These findings may offer a starting point for the treatment of patients infected with hantaviruses.展开更多
文摘The techniques used in the field of drug discovery have been quickly developed during the past decades High throughput screening (HTS) is a new technique in drug discovery with the characteristics of efficiency, huge scale and automatically operation The assay methods used in HTS are based on the molecular targets and cells Screening results obtained from these methods indicate not only the bioactivities of compounds (samples) but also the mechanisms of actions of the compounds (samples) HTS has altered the strategy of drug
文摘Neuraminidase (NA) is one of the most important targets to screen the drugs of anti-influenza virus A and B. After virtual screening approaches were applied to a compound database which possesses more than 10000 compound structures, 160 compounds were selected for bioactivity assay, then a High Throughput Screening (HTS) model established for influenza virus NA inhibitors was applied to detect these compounds. Finally, three compounds among them displayed higher inhibitory activities, the range of their IC5o was from 0.1 μmol/L to 3 μmol/L. Their structural scaffolds are novel and different from those of NA inhibitors approved for influenza treatment, and will be useful for the design and research of new NA inhibitors. The result indicated that the combination of virtual screening with HTS was very significant to drug screening and drug discovery.
文摘目的：建立糖原磷酸化酶（GPa）抑制剂的体外高通量筛选模型。方法：用梯度离心方法提取大鼠肝脏GPa，以葡萄糖-1-磷酸作为底物，通过测定反应中磷酸根的释放量，间接反映肝脏GPa的活性。通过对反应体系的优化，调整反应条件，建立96孔微孔板的高通量筛选模型，用咖啡因对此模型进行验证，并评价高通量技术参数[Z＇-因子（Z＇-factor），信号本底比（signal to background，S／B），信噪比（signal to noise，S／N）]。用此模型对5000个样品（包括合成化合物、天然提取物）组成的随机库进行体外筛选，考察这些样品对GPa的抑制作用。结果：确定反应体系条件是：反应温度25℃，反应时间30min，底物浓度0．5mmol·L^-1，大鼠肝脏GPa的用量为250ng。用此条件测定咖啡因对GPa的抑制曲线，计算其半数抑制浓度（IC50）为（285．3±39．6）μmol·L^-1，与文献报道（IC50=240μmol·L^-1）基本一致；本模型技术参数Z＇-因子=0．79，S／B=0．45，S／N=11．32，表明此模型可以用于高通量筛选。应用此模型筛选得到有活性的化合物9个，其IC50在3．56～45．75μmol·L^-1。结论：建立的体外GPa抑制剂高通量筛选模型具有快速、微量、准确的特点，可以作为研究降糖药的工具。
基金This research was supported by the NCI Cancer TargetDiscovery and Development(CTD^2)Network(1U01CA217875 toH.F.and 1uo1CA217851 to C.J.K.)the RAS Synthetic LethalNetwork(RSLN+4 种基金1UO1CA199241 to C.J.K.)the Emory LungCancer SPORE(NIH P5OCA217691)the Winship Cancerlnstitute(NIH 5P30CA138292)the Emory WHSC 10x SingleCell Sequencing Seed Grant(X.M.and Y.D.)Emory WoodruffHealth Sciences Center Synergy Award,and the lmagine,lnnovate and Impact(3)Funds from the Emory School ofMedicine and through the Georgia CTSA NIH award(UL1-TRO02378).
文摘The recent advent of robust methods to grow human tissues as 3D organoids allows us to recapitulate the 3D architecture of tumors in an in vitro setting and offers a new orthogonal approach for drug discovery.However,organoid culturing with extracellular matrix to support 3D architecture has been challenging for high-throughput screening(HTS)-based drug discovery due to technical difficulties.Using genetically engineered human colon organoids as a model system,here we report our effort to miniaturize such 3D organoid culture with extracellular matrix support in high-density plates to enable HTS.We first established organoid culturing in a 384-well plate format and validated its application in a cell viability HTS assay by screening a 2036-compound library.We further miniaturized the 3D organoid culturing in a 1536-well ultra-HTS format and demonstrated its robust performance for large-scale primary compound screening.Our miniaturized organoid culturing method may be adapted to other types of organoids.By leveraging the power of 3D organoid culture in a high-density plate format,we provide a physiologically relevant screening platform to model tumors to accelerate organoid-based research and drug discovery.
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
文摘A stably transfected CHO cell line coexpressing G551D-CFTR and iodide-sensitive yellow fluorescent protein mutant EYFP-H148Q-I152L was successfully established and used as assay model to identify small-molecule activators of G551D-CFTR chloride channel from 100000 diverse combinatorial compounds by high throughput screening on a customized Beckman robotic system. A bicyclooctane compound was identified to activate G551D-CFTR chloride channel with high-affinity(K d=1.8 μmol/L). The activity of the bicyclooctane compound is G551D-CFTR-specific, reversible and non-toxic. The G551D-CFTR activator may be useful as a tool to study the mutant G551D-CFTR chloride channel structure and transport properties and as a candidate drug to cure cystic fibrosis caused by G551D-CFTR mutation.
文摘Traditional Chinese medicine(TCM) has been widely used in China and other Asia countries for thousands of years to treat or prevent human diseases. Chinese herbal medicine, one of the most important components of TCM, has unique diversities in chemical components, and thus results in a wide range of biological activities. However, pharmaceutical industry is facing a major challenge to develop a large population of novel natural products and drugs, and considerable efforts have not resulted in highvolume of novel drug discovery and productivity. At present, increasing attention has been paid to Chinese herb medicine modernization in combination with the cutting-age technologies of drug discovery, especially the high throughput selection. High content imaging is an image-based high throughput screening method by using automated microscopy and image analysis software to capture and analyze phenotypes at a large scale to investigate multiple biological features simultaneously in the biological complex. Here, we described the pipeline of the state-of-the-art high content imaging technology, summarized the applications of the high content imaging technology in drug discovery from traditional Chinese herbal medicine, and finally discussed the current challenges and future perspectives for development of high throughput image-based screening technology in novel drug research and discovery.
基金National Science and Technology Major Projects of Infectious Disease(grant number 2018ZX10731101)。
文摘Hantaviruses,such as Hantaan virus(HTNV)and Seoul virus,are the causative agents of Hantavirus cardiopulmonary syndrome(HCPS)and hemorrhagic fever with renal syndrome(HFRS),and are important zoonotic pathogens.China has the highest incidence of HFRS,which is mainly caused by HTNV and Seoul virus.No approved antiviral drugs are available for these hantaviral diseases.Here,a chemiluminescence-based highthroughput-screening(HTS)assay was developed and used to screen HTNV pseudovirus(HTNVpv)inhibitors in a library of 1813 approved drugs and 556 small-molecule compounds from traditional Chinese medicine sources.We identified six compounds with in vitro anti-HTNVpvactivities in the low-micromolar range(EC50values of0.1–2.2μmol/L;selectivity index of 40–900).Among the six selected compounds,cepharanthine not only showed good anti-HTNVpvactivity in vitro but also inhibited HTNVpv-fluc infection in Balb/c mice 5 h after infection by94%(180 mg/kg/d,P<0.01),93%(90 mg/kg/d,P<0.01),or 92%(45 mg/kg/d,P<0.01),respectively,in a bioluminescent imaging mouse model.A time-of-addition analysis suggested that the antiviral mechanism of cepharanthine involves the membrane fusion and entry phases.Overall,we have established a HTS method for antiviral drugs screening,and shown that cepharanthine is a candidate for HCPS and HFRS therapy.These findings may offer a starting point for the treatment of patients infected with hantaviruses.
