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
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
Current in vitro assays for the activity of HIV-RT(reverse transcriptase)require radio-labeled or chemically modified nucleotides to detect reaction products.However,these assays are inherently end-point measurements ...Current in vitro assays for the activity of HIV-RT(reverse transcriptase)require radio-labeled or chemically modified nucleotides to detect reaction products.However,these assays are inherently end-point measurements and labor intensive.Here we describe a novel non-radioactive assay based on the principle of pyrosequencing coupledenzyme system to monitor the activity of HIV-RT by indirectly measuring the release of pyrophosphate(PPi),which is generated during nascent strand synthesis.The results show that our assay could monitor HIV-RT activity with high sensitivity and is suitable for rapid highthroughput drug screening targeting anti-HIV therapies due to its high speed and convenience.Moreover,this assay can be used to measure primase activity in an easy and sensitive manner,which suggests that this novel approach could be wildly used to analyze the activity of PPi-generated and ATP-free enzyme reactions.展开更多
While the human genome is pervasively transcribed,<2%of the human genome is transcribed into protein-coding mRNAs,leaving most of the transcripts as noncoding RNAs,such as microRNAs and long-noncoding RNAs(lncRNAs)...While the human genome is pervasively transcribed,<2%of the human genome is transcribed into protein-coding mRNAs,leaving most of the transcripts as noncoding RNAs,such as microRNAs and long-noncoding RNAs(lncRNAs),which are critical components of epigenetic regulation.lncRNAs are emerging as critical regulators of gene expression and genomic stability.However,it remains largely unknown about how lncRNAs are regulated.Here,we develop a highly sensitive and dynamic reporter that allows us to identify and/or monitor negative modulators of lncRNA transcript levels in a high throughput fashion.Specifically,we engineer a fluorescent fusion protein by fusing three copies of the PEST destruction domain of mouse ornithine decarboxylase(MODC)to the C-terminal end of the codon-optimized bilirubin-inducible fluorescent protein,designated as dBiFP,and show that the dBiFP protein is highly destabilized,compared with the commonly-used eGFP protein.We further demonstrate that the dBiFP signal is effectively down-regulated when the dBiFP and mouse lncRNA H19 chimeric transcript is silenced by mouse H19-specific siRNAs.Therefore,our results strongly suggest that the dBiFP fusion protein may serve as a sensitive and dynamic transcript reporter to monitor the inhibition of lncRNAs by microRNAs,synthetic regulatory RNA molecules,RNA binding proteins,and/or small molecule inhibitors so that novel and efficacious inhibitors targeting the epigenetic circuit can be discovered to treat human diseases such as cancer and other chronic disorders.展开更多
Since the discovery of protein phosphorylation as an important modulator of many cellular processes, the involvement of protein kinases in diseases, such as cancer, diabetes, cardiovascular diseases, and central nervo...Since the discovery of protein phosphorylation as an important modulator of many cellular processes, the involvement of protein kinases in diseases, such as cancer, diabetes, cardiovascular diseases, and central nervous system pathologies, has been extensively documented. Our understanding of many disease pathologies at the molecular level, therefore, requires the comprehensive identification of substrates targeted by protein kinases. In this review, we focus on recent techniques for kinase substrate identification in high throughput, in particular on genetic and proteomic approaches. Each method with its inherent advantages and limitations is discussed.展开更多
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.
基金the National Natural Science Foundation of China(Grant Nos.30221003,30720022)the Ministry of Science and Technology 973 Project(Grant No.2006CB806503)+2 种基金the Ministry of Science and Technology National High Technology Research and Development Program("863"Program)(Grant No.2006AA02A322)the Ministry of Science and Technology International Cooperation Project(Grant No.2006DFB32420)the Chinese Academy of Sciences Knowledge Innovation Project(Grant No.KSCX1-YW-R-05)。
文摘Current in vitro assays for the activity of HIV-RT(reverse transcriptase)require radio-labeled or chemically modified nucleotides to detect reaction products.However,these assays are inherently end-point measurements and labor intensive.Here we describe a novel non-radioactive assay based on the principle of pyrosequencing coupledenzyme system to monitor the activity of HIV-RT by indirectly measuring the release of pyrophosphate(PPi),which is generated during nascent strand synthesis.The results show that our assay could monitor HIV-RT activity with high sensitivity and is suitable for rapid highthroughput drug screening targeting anti-HIV therapies due to its high speed and convenience.Moreover,this assay can be used to measure primase activity in an easy and sensitive manner,which suggests that this novel approach could be wildly used to analyze the activity of PPi-generated and ATP-free enzyme reactions.
基金The reported work was supported in part by research grants from the National Institutes of Health(AT004418,DE020140 to TCH and RRR)the US Department of Defense(OR130096 to JMW)+5 种基金the Scoliosis Research Society(TCH and MJL)the National Key Research and Development Program of China(2016YFC1000803 and 2011CB707906 to TCH)the National Natural Science Foundation of China(#81201916 to XW)ZZ was a recipient of protectorate fellowship from China Scholarship CouncilThis project was also supported in part by The University of Chicago Cancer Center Support Grant(P30CA014599)the National Center for Advancing Translational Sciences of the National Institutes of Health through Grant Number UL1 TR000430.
文摘While the human genome is pervasively transcribed,<2%of the human genome is transcribed into protein-coding mRNAs,leaving most of the transcripts as noncoding RNAs,such as microRNAs and long-noncoding RNAs(lncRNAs),which are critical components of epigenetic regulation.lncRNAs are emerging as critical regulators of gene expression and genomic stability.However,it remains largely unknown about how lncRNAs are regulated.Here,we develop a highly sensitive and dynamic reporter that allows us to identify and/or monitor negative modulators of lncRNA transcript levels in a high throughput fashion.Specifically,we engineer a fluorescent fusion protein by fusing three copies of the PEST destruction domain of mouse ornithine decarboxylase(MODC)to the C-terminal end of the codon-optimized bilirubin-inducible fluorescent protein,designated as dBiFP,and show that the dBiFP protein is highly destabilized,compared with the commonly-used eGFP protein.We further demonstrate that the dBiFP signal is effectively down-regulated when the dBiFP and mouse lncRNA H19 chimeric transcript is silenced by mouse H19-specific siRNAs.Therefore,our results strongly suggest that the dBiFP fusion protein may serve as a sensitive and dynamic transcript reporter to monitor the inhibition of lncRNAs by microRNAs,synthetic regulatory RNA molecules,RNA binding proteins,and/or small molecule inhibitors so that novel and efficacious inhibitors targeting the epigenetic circuit can be discovered to treat human diseases such as cancer and other chronic disorders.
文摘Since the discovery of protein phosphorylation as an important modulator of many cellular processes, the involvement of protein kinases in diseases, such as cancer, diabetes, cardiovascular diseases, and central nervous system pathologies, has been extensively documented. Our understanding of many disease pathologies at the molecular level, therefore, requires the comprehensive identification of substrates targeted by protein kinases. In this review, we focus on recent techniques for kinase substrate identification in high throughput, in particular on genetic and proteomic approaches. Each method with its inherent advantages and limitations is discussed.
