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1.
Acta Pharmaceutica Sinica ; (12): 600-607, 2024.
Article in Chinese | WPRIM | ID: wpr-1016634

ABSTRACT

The coronavirus disease 2019 (COVID-19) is an acute infectious disease caused by the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, which has led to serious worldwide economic burden. Due to the continuous emergence of variants, vaccines and monoclonal antibodies are only partial effective against infections caused by distinct strains of SARS-CoV-2. Therefore, it is still of great importance to call for the development of broad-spectrum and effective small molecule drugs to combat both current and future outbreaks triggered by SARS-CoV-2. Cathepsin L (CatL) cleaves the spike glycoprotein (S) of SARS-CoV-2, playing an indispensable role in enhancing virus entry into host cells. Therefore CatL is one of the ideal targets for the development of pan-coronavirus inhibitor-based drugs. In this study, a CatL enzyme inhibitor screening model was established based on fluorescein labeled substrate. Two CatL inhibitors IMB 6290 and IMB 8014 with low cytotoxicity were obtained through high-throughput screening, the half inhibition concentrations (IC50) of which were 11.53 ± 0.68 and 1.56 ± 1.10 μmol·L-1, respectively. SDS-PAGE and cell-cell fusion experiments confirmed that the compounds inhibited the hydrolysis of S protein by CatL in a concentration-dependent manner. Surface plasmon resonance (SPR) detection showed that both compounds exhibited moderate binding affinity with CatL. Molecular docking revealed the binding mode between the compound and the CatL active pocket. The pseudovirus experiment further confirmed the inhibitory effects of IMB 8014 on the S protein mediated entry process. In vitro pharmacokinetic evaluation indicated that the compounds had relatively good drug-likeness properties. Our research suggested that these two compounds have the potential to be further developed as antiviral drugs for COVID-19 treatment.

2.
Journal of Pharmaceutical Practice ; (6): 114-120, 2024.
Article in Chinese | WPRIM | ID: wpr-1012791

ABSTRACT

Objective Alpha-1-acid glycoprotein (ORM) was a new target for the development of weight loss drugs. To search for potential weight loss drugs that could target ORM from the compound library of already marketed drugs based on drug repurposing. Methods The pGL4.20-ORM1 promoter recombinant plasmid was contructed and validated, and then a lentiviral vector was utilized to establish stable AML12 cell lines expressing ORM1 promoter-LUC-PURO. This cell line was employed for high-throughput screening of compounds from the marketed drug library, and the luminescence value of the cells was characterized by enzyme marker. Results Primary screening and secondary screening of 1 470 compounds identified 42 compounds that increased ORM1 promoter expression and could be used for further weight loss effect assessment. Conclusion This study successfully constructed LV-AML12-ORM1 promoter-LUC-PURO stable expression cell lines using lentiviral vectors, laying a foundation for efficient and stable screening of weight loss drugs targeting ORM.

3.
Acta Pharmaceutica Sinica B ; (6): 223-240, 2024.
Article in English | WPRIM | ID: wpr-1011235

ABSTRACT

Lenvatinib, a second-generation multi-receptor tyrosine kinase inhibitor approved by the FDA for first-line treatment of advanced liver cancer, facing limitations due to drug resistance. Here, we applied a multidimensional, high-throughput screening platform comprising patient-derived resistant liver tumor cells (PDCs), organoids (PDOs), and xenografts (PDXs) to identify drug susceptibilities for conquering lenvatinib resistance in clinically relevant settings. Expansion and passaging of PDCs and PDOs from resistant patient liver tumors retained functional fidelity to lenvatinib treatment, expediting drug repurposing screens. Pharmacological screening identified romidepsin, YM155, apitolisib, NVP-TAE684 and dasatinib as potential antitumor agents in lenvatinib-resistant PDC and PDO models. Notably, romidepsin treatment enhanced antitumor response in syngeneic mouse models by triggering immunogenic tumor cell death and blocking the EGFR signaling pathway. A combination of romidepsin and immunotherapy achieved robust and synergistic antitumor effects against lenvatinib resistance in humanized immunocompetent PDX models. Collectively, our findings suggest that patient-derived liver cancer models effectively recapitulate lenvatinib resistance observed in clinical settings and expedite drug discovery for advanced liver cancer, providing a feasible multidimensional platform for personalized medicine.

4.
Acta Pharmaceutica Sinica B ; (6): 4253-4272, 2023.
Article in English | WPRIM | ID: wpr-1011162

ABSTRACT

It is discovered that activated caspase-3 tends to induce apoptosis in gasdermin E (GSDME)-deficient cells, but pyroptosis in GSDME-sufficient cells. The high GSDME expression and apoptosis resistance of pancreatic ductal adenocarcinoma (PDAC) cells shed light on another attractive strategy for PDAC treatment by promoting pyroptosis. Here we report a hGLuc-hGSDME-PCA system for high-throughput screening of potential GSDME activators against PDAC. This screening system neatly quantifies the oligomerization of GSDME-N to characterize whether pyroptosis occurs under the stimulation of chemotherapy drugs. Based on this system, ponatinib and perifosine are screened out from the FDA-approved anti-cancer drug library containing 106 compounds. Concretely, they exhibit the most potent luminescent activity and cause drastic pyroptosis in PDAC cells. Further, we demonstrate that perifosine suppresses pancreatic cancer by promoting pyroptosis via caspase-3/GSDME pathway both in vitro and in vivo. Collectively, this study reveals the great significance of hGLuc-hGSDME-PCA in identifying compounds triggering GSDME-dependent pyroptosis and developing promising therapeutic agents for PDAC.

5.
Chinese Journal of Laboratory Medicine ; (12): 597-603, 2023.
Article in Chinese | WPRIM | ID: wpr-995765

ABSTRACT

Objective:High-throughput screening to obtain small molecular compounds against Gram-negative bacilli by targeting BamA outer membrane protein.Methods:The sybyl-X2.1 software was used to perform high-throughput virtual screening of small molecular compounds in Chemdiv compound library based on the molecular docking. The top 150 hits by high-throughput screening were re-screened through in vitro biological experiments. The top 4 small molecules with obvious antibacterial activity were selected for in-depth molecular docking analysis, and the small molecule 8308-0401 with the highest docking score was selected for further experiments. The antibacterial effect of 8308-0401 combined with rifampicin was tested by checkerboard assay. Finally, the affinity between 8308-0401 and BamA was tested by plasma surface resonance assay. Results:The docking score of the top 150 hits calculated by high-throughput virtual screening had a mean value of 5.63. In vitro biological experiments showed that small molecules 8308-0401, 8365-1335, C066-2507 and L582-0346 exhibited strong antibacterial activity. Among those molecules, 8308-0401 showed the highest molecular docking score, and synergistic antibacterial activity against both types of strains and clinical isolates when combined with rifampicin. 8308-0401 has a strong affinity to BamA with binding a constant of 182 μmol/L. Conclusion:The small molecule 8308-0401 exerts antibacterial activity against Gram negative bacilli by targeting the outer membrane protein BamA.

6.
Chinese Journal of Biotechnology ; (12): 2485-2501, 2023.
Article in Chinese | WPRIM | ID: wpr-981213

ABSTRACT

Amino acids are the basic building blocks of protein that are very important to the nutrition and health of humans and animals, and widely used in feed, food, medicine and daily chemicals. At present, amino acids are mainly produced from renewable raw materials by microbial fermentation, forming one of the important pillar industries of biomanufacturing in China. Amino acid-producing strains are mostly developed through random mutagenesis- and metabolic engineering-enabled strain breeding combined with strain screening. One of the key limitations to further improvement of production level is the lack of efficient, rapid, and accurate strain screening methods. Therefore, the development of high-throughput screening methods for amino acid strains is very important for the mining of key functional elements and the creation and screening of hyper-producing strains. This paper reviews the design of amino acid biosensors and their applications in the high-throughput evolution and screening of functional elements and hyper-producing strains, and the dynamic regulation of metabolic pathways. The challenges of existing amino acid biosensors and strategies for biosensor optimization are discussed. Finally, the importance of developing biosensors for amino acid derivatives is prospected.


Subject(s)
Animals , Humans , Amino Acids , Biosensing Techniques , Metabolic Engineering , High-Throughput Screening Assays , China
7.
Acta Pharmaceutica Sinica ; (12): 1528-1539, 2023.
Article in Chinese | WPRIM | ID: wpr-978716

ABSTRACT

COVID-19 epidemic continues to spread around the world till these days, and it is urgent to develop more safe and effective new drugs. Due to the limited P3 biosafety laboratories for directly screening inhibitors of virulent viruses with high infectivity, it is necessary to develop rapid and efficient screening methods for viral proteases and other related targets. The main protease (Mpro), which plays a key role in the replication cycle of SARS-CoV-2, is highly conserved and has no homologous proteases in humans, making it an ideal target for drug development. From two different levels, namely, molecular level and cellular level, this paper summarizes the reported screening methods of SARS-CoV-2 Mpro inhibitors through a variety of representative examples, expecting to provide references for further development of SARS-CoV-2 Mpro inhibitors.

8.
Protein & Cell ; (12): 17-27, 2023.
Article in English | WPRIM | ID: wpr-971604

ABSTRACT

The global COVID-19 coronavirus pandemic has infected over 109 million people, leading to over 2 million deaths up to date and still lacking of effective drugs for patient treatment. Here, we screened about 1.8 million small molecules against the main protease (Mpro) and papain like protease (PLpro), two major proteases in severe acute respiratory syndrome-coronavirus 2 genome, and identified 1851Mpro inhibitors and 205 PLpro inhibitors with low nmol/l activity of the best hits. Among these inhibitors, eight small molecules showed dual inhibition effects on both Mpro and PLpro, exhibiting potential as better candidates for COVID-19 treatment. The best inhibitors of each protease were tested in antiviral assay, with over 40% of Mpro inhibitors and over 20% of PLpro inhibitors showing high potency in viral inhibition with low cytotoxicity. The X-ray crystal structure of SARS-CoV-2 Mpro in complex with its potent inhibitor 4a was determined at 1.8 Å resolution. Together with docking assays, our results provide a comprehensive resource for future research on anti-SARS-CoV-2 drug development.


Subject(s)
Humans , Antiviral Agents/chemistry , COVID-19 , COVID-19 Drug Treatment , High-Throughput Screening Assays , Molecular Docking Simulation , Protease Inhibitors/chemistry , SARS-CoV-2/enzymology , Viral Nonstructural Proteins
9.
Chinese Journal of Biotechnology ; (12): 359-371, 2023.
Article in Chinese | WPRIM | ID: wpr-970380

ABSTRACT

This study aims to develop an improved cell screening system for farnesoid X receptor (FXR) agonists based on a dual luciferase reporter gene system. FXR response element (FXRE) fragments from FXR target genes were cloned and inserted into upstream of firefly luciferase (Luc) gene in the plasmid pGL4-luc2P-Hygro. In combination with the internal reference plasmid containing renilla luciferase, a dual luciferase reporter gene system was developed and used for high throughput screening of FXR agonists. After studying the effects of over-expression of RXR, mouse or human FXR, various FXRE fragments, and different ratio of FXR plasmid amount to reporter gene plasmid, induction efficiency of the screening system was optimized by the known FXR agonist GW4064, and Z factor for the system reached 0.83 under optimized conditions. In summary, an improved cell screening system based on double luciferase reporter gene detection system was developed to facilitate the discovery of FXR agonists, where a new enhanced FXRE element was formed by a superposition of multiple FXRE fragments from FXR target genes, instead of a superposition of traditional IR-1 (inverted repeats-1) fragments.


Subject(s)
Humans , Mice , Animals , Transcription Factors/genetics , DNA-Binding Proteins/genetics , Receptors, Cytoplasmic and Nuclear/genetics , Genes, Reporter , Luciferases/genetics
10.
Acta Pharmaceutica Sinica B ; (6): 1068-1099, 2022.
Article in English | WPRIM | ID: wpr-929361

ABSTRACT

Drug-metabolizing enzymes (DMEs), a diverse group of enzymes responsible for the metabolic elimination of drugs and other xenobiotics, have been recognized as the critical determinants to drug safety and efficacy. Deciphering and understanding the key roles of individual DMEs in drug metabolism and toxicity, as well as characterizing the interactions of central DMEs with xenobiotics require reliable, practical and highly specific tools for sensing the activities of these enzymes in biological systems. In the last few decades, the scientists have developed a variety of optical substrates for sensing human DMEs, parts of them have been successfully used for studying target enzyme(s) in tissue preparations and living systems. Herein, molecular design principals and recent advances in the development and applications of optical substrates for human DMEs have been reviewed systematically. Furthermore, the challenges and future perspectives in this field are also highlighted. The presented information offers a group of practical approaches and imaging tools for sensing DMEs activities in complex biological systems, which strongly facilitates high-throughput screening the modulators of target DMEs and studies on drug/herb‒drug interactions, as well as promotes the fundamental researches for exploring the relevance of DMEs to human diseases and drug treatment outcomes.

11.
Journal of Pharmaceutical Analysis ; (6): 15-27, 2021.
Article in Chinese | WPRIM | ID: wpr-883495

ABSTRACT

Mammalian catechol-O-methyltransferases(COMT)are an important class of conjugative enzymes,which play a key role in the metabolism and inactivation of catechol neurotransmitters,catechol es-trogens and a wide range of endobiotics and xenobiotics that bear the catechol group.Currently,COMT inhibitors are used in combination with levodopa for the treatment of Parkinson's disease in clinical practice.The crucial role of COMT in human health has raised great interest in the development of more practical assays for highly selective and sensitive detection of COMT activity in real samples,as well as for rapid screening and characterization of COMT inhibitors as drug candidates.This review summarizes recent advances in analytical methodologies for sensing COMT activity and their applications.Several lists of biochemical assays for measuring COMT activity,including the probe substrates,along with their analytical conditions and kinetic parameters,are presented.Finally,the challenges and future perspec-tives in the field,such as visualization of COMT activity in vivo and in situ,are highlighted.Collectively,this review article overviews the practical assays for measuring COMT activities in complex biological samples,which will strongly facilitate the investigations on the relevance of COMT to human diseases and promote the discovery of COMT inhibitors via high-throughput screening.

12.
Chinese Journal of Biotechnology ; (12): 2878-2889, 2021.
Article in Chinese | WPRIM | ID: wpr-887850

ABSTRACT

In canonical Wnt/β-catenin signaling pathway, β-catenin/TCF4 (T-cell factor 4) interaction plays an important role in the pathogenesis and development of non-small cell lung cancer (NSCLC), and it is tightly associated with the proliferation, chemoresistance, recurrence and metastasis of NSCLC. Therefore, suppressing β-catenin/TCF4 interaction in Wnt/β-catenin signaling pathway would be a new therapeutic avenue against NSCLC metastasis. In this study, considering the principle of enzyme-linked immunosorbent assay (ELISA), an optimized high-throughput screening (HTS) assay was developed for the discovery of β-catenin/TCF4 interaction antagonists. Subsequently, this ELISA-like screening assay was performed using 2 μg/mL GST-TCF4 βBD and 0.5 μg/mL β-catenin, then a high Z' factor of 0.83 was achieved. A pilot screening of a natural product library using this ELISA-like screening assay identified plumbagin as a potential β-catenin/TCF4 interaction antagonist. Plumbagin remarkably inhibited the proliferation of A549, H1299, MCF7 and SW480 cell lines. More importantly, plumbagin significantly suppressed the β-catenin-responsive transcription in TOPFlash assay. In short, this newly developed ELISA-like screening assay will be vital for the rapid screening of novel Wnt inhibitors targeting β-catenin/TCF4 interaction, and this interaction is a potential anticancer target of plumbagin in vitro.


Subject(s)
Humans , Carcinoma, Non-Small-Cell Lung , Cell Line, Tumor , Enzyme-Linked Immunosorbent Assay , High-Throughput Screening Assays , Lung Neoplasms , Transcription Factor 4/genetics , beta Catenin/genetics
13.
Chinese Journal of Biotechnology ; (12): 2197-2210, 2021.
Article in Chinese | WPRIM | ID: wpr-887789

ABSTRACT

Enzymes and cell factories are the core of industrial biotechnology. They play important roles in various fields such as medicine, chemical industry, food, agriculture, and energy. Usually, natural enzymes and cells need to be engineered to improve the catalytic efficiency, stability and enantioselectivity. Directed evolution makes it possible to rapidly improve the properties of enzymes and cell factories. Sensitive and reliable high-throughput screening approaches are the key for successful and efficient engineering of enzymes and cell factories. In this review, we first summarize the advantages and disadvantages of different screening methods and signal generation strategies as well as their application scope; we then describe the latest advances of ultra-high throughput screening technology applied in the directed evolution of enzymes and cell factories in the past three years. On this basis, we discuss the limiting factors that need to be further improved for high-throughput screening systems and forecast the future development trends of high-throughput screening methods, hoping that researchers in various fields including biotechnology and instrument development can cooperate closely to enhance the reliability and applicability of the high-throughput screening techniques.


Subject(s)
Biotechnology , Directed Molecular Evolution , Enzymes , High-Throughput Screening Assays , Reproducibility of Results
14.
Chinese Journal of Biotechnology ; (12): 939-949, 2021.
Article in Chinese | WPRIM | ID: wpr-878605

ABSTRACT

Pichia pastoris is one of the most widely used recombinant protein expression systems. In this study, a novel method for rapid screening of P. pastoris strains capable of efficiently expressing recombinant proteins was developed. Firstly, the ability to express recombinant proteins of the modified strain GS115-E in which a functional Sec63-EGFP (Enhanced green fluorescent protein) fusion protein replaced the endogenous endoplasmic reticulum transmembrane protein Sec63 was tested. Next, the plasmids carrying different copy numbers of phytase (phy) gene or xylanase (xyn) gene were transformed into GS115-E to obtain recombinant strains with different expression levels of phytase or xylanase, and the expression levels of EGFP and recombinant proteins in different strains were tested. Finally, a flow cytometer sorter was used to separate a mixture of cells with different phytase expression levels into sub-populations according to green fluorescence intensity. A good linear correlation was found between the fluorescence intensities of EGFP and the expression levels of the recombinant proteins in the recombinant strains (0.8<|R|<1). By using the flow cytometer, high-yielding P. pastoris cells were efficiently screened from a mixture of cells. The expression level of phytase of the selected high-fluorescence strains was 4.09 times higher than that of the low-fluorescence strains after 120 h of methanol induction. By detecting the EGFP fluorescence intensity instead of detecting the expression level and activity of the recombinant proteins in the recombinant strains, the method developed by the present study possesses the greatly improved performance of convenience and versatility in screening high-yielding P. pastoris strains. Combining the method with high-throughput screening instruments and technologies, such as flow cytometer and droplet microfluidics, the speed and throughput of this method will be further increased. This method will provide a simple and rapid approach for screening and obtaining P. pastoris with high abilities to express recombinant proteins.


Subject(s)
6-Phytase/genetics , Pichia/genetics , Plasmids , Recombinant Proteins/genetics , Saccharomycetales
15.
Chinese Journal of Biotechnology ; (12): 163-177, 2021.
Article in Chinese | WPRIM | ID: wpr-878551

ABSTRACT

Directed evolution is a cyclic process that alternates between constructing different genes and screening functional gene variants. It has been widely used in optimization and analysis of DNA sequence, gene function and protein structure. It includes random gene libraries construction, gene expression in suitable hosts and mutant libraries screening. The key to construct gene library is the storage capacity and mutation diversity, to screen is high sensitivity and high throughput. This review discusses the latest advances in directed evolution. These new technologies greatly accelerate and simplify the traditional directional evolution process and promote the development of directed evolution.


Subject(s)
Base Sequence , Directed Molecular Evolution , Gene Library , Mutation , Proteins/genetics
16.
Journal of Pharmaceutical Analysis ; (6): 808-814, 2021.
Article in Chinese | WPRIM | ID: wpr-931226

ABSTRACT

Suppression of cellular O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) can repress prolifera-tion and migration of various cancer cells,which opens a new avenue for cancer therapy.Based on the regulation of insulin gene transcription,we designed a cell-based fluorescent reporter capable of sensing cellular O-GlcNAcylation in HEK293T cells.The fluorescent reporter mainly consists of a reporter (green fluorescent protein (GFP)),an internal reference (red fluorescent protein),and an operator (neuronal differentiation 1),which serves as a "sweet switch" to control GFP expression in response to cellular O-GlcNAcylation changes.The fluorescent reporter can efficiently sense reduced levels of cellular O-GlcNAcylation in several cell lines.Using the fluorescent reporter,we screened 120 natural products and obtained one compound,sesamin,which could markedly inhibit protein O-GlcNAcylation in HeLa and human colorectal carcinoma-116 cells and repress their migration in vitro.Altogether,the present study demonstrated the development of a novel strategy for anti-tumor drug screening,as well as for con-ducting gene transcription studies.

17.
Chinese Journal of Biotechnology ; (12): 3348-3360, 2021.
Article in Chinese | WPRIM | ID: wpr-921430

ABSTRACT

Tyrosine is an important aromatic amino acid. Besides its nutritional value, tyrosine is also an important precursor for the synthesis of coumarins and flavonoids. Previously, our laboratory constructed a Saccharomyces cerevisiae strain LTH0 (ARO4K229L, ARO7G141S, Δaro10, Δzwf1, Δura3) where tyrosine feedback inhibition was released. In the present study, heterologous expression of betaxanthins synthesis genes DOD (from Mirabilis jalapa) and CYP76AD1 (from sugar beet B. vulgaris) in strain LTH0 enabled production of yellow fluorescence. The engineered strain LTH0-DOD-CYP76AD1 was subjected to UV combined with ARTP mutagenesis, followed by flow cytometry screening. Among the mutants screened, the fluorescence intensity of the mutant strain LTH2-5-DOD-CYP76AD1 at the excitation wavelength of 485 nm and emission wavelength of 505 nm was (5 941±435) AU/OD, which was 8.37 times higher than that of strain LTH0-DOD-CYP76AD1. Fourteen mutant strains were subjected to fermentation to evaluate their tyrosine producing ability. The highest extracellular tyrosine titer reached 26.8 mg/L, which was 3.96 times higher than that of strain LTH0-DOD-CYP76AD1. Heterologous expression of the tyrosine ammonia lyase FjTAL derived from Flavobacterium johnsoniae further increased the titer of coumaric acid to 119.8 mg/L, which was 1.02 times higher than that of the original strain LTH0-FjTAL.


Subject(s)
Flavobacterium , High-Throughput Screening Assays , Mirabilis , Saccharomyces cerevisiae/genetics , Tyrosine
18.
Shanghai Journal of Preventive Medicine ; (12): 78-2020.
Article in Chinese | WPRIM | ID: wpr-876342

ABSTRACT

Objective To investigate the feasibility of genotoxicity assessment for chemicals via flow cytometry (FCM) and high-content screening (HCS) based on high-throughput screening in vitro micronucleus assays. Methods In reference to the methodology of OECD TG487, the typical positive controls, cyclophosphamide (CP) and mitomycin C (MMC), were selected.And no serum MEM medium was treated as negative control.Dose range of CP was 5-20 mg/L and MMC was 0.25-1.0 mg/L.CHL cells were treated with three concentrations of each chemical for 4 h.High-throughput screening in vitro micronucleus assays based on FCM and HCS were established.The results of the frequency of micronuclei were compared to traditional cytokinesis blocking micronucleus assay in each group with or without metabolic activation. Results The frequencies of micronuclei induced by CP and MMC (ascending rank) were separately 1.9%, 7.6%, 10.4% and 5.9%, 11.4%, 16.7%, which were obtained by conventional microscopic scoring.The frequencies of micronuclei induced by CP and MMC (ascending rank) were separately 2.8%, 2.6%, 7.8% and 3.2%, 3.7%, 5.1%, which were obtained by flow cytometry screening.The frequencies of micronuclei induced by CP and MMC (ascending rank) were separately2.8%, 6.2%, 9.1% and 7.9%, 10.1%, 10.2%, which were obtained by high-content screening.Compared with negative controls, the differences of the results were statistically significant(P < 0.05), and there was a dose-response relationship. Conclusion In this study, the results of high-throughput screening assays of FCM and HCS are in accordance to the results of traditional cytokinesis blocking micronucleus assay, indicating that high-throughput screening in vitro micronucleus assays could detect micronucleus formation automatically and improve the efficiency.Therefore, the method could provide data support for using high-throughput screening in vitro micronucleus assays into genotoxicity assessment of chemicals.

19.
Shanghai Journal of Preventive Medicine ; (12): 78-2020.
Article in Chinese | WPRIM | ID: wpr-876325

ABSTRACT

Objective To investigate the feasibility of genotoxicity assessment for chemicals via flow cytometry (FCM) and high-content screening (HCS) based on high-throughput screening in vitro micronucleus assays. Methods In reference to the methodology of OECD TG487, the typical positive controls, cyclophosphamide (CP) and mitomycin C (MMC), were selected.And no serum MEM medium was treated as negative control.Dose range of CP was 5-20 mg/L and MMC was 0.25-1.0 mg/L.CHL cells were treated with three concentrations of each chemical for 4 h.High-throughput screening in vitro micronucleus assays based on FCM and HCS were established.The results of the frequency of micronuclei were compared to traditional cytokinesis blocking micronucleus assay in each group with or without metabolic activation. Results The frequencies of micronuclei induced by CP and MMC (ascending rank) were separately 1.9%, 7.6%, 10.4% and 5.9%, 11.4%, 16.7%, which were obtained by conventional microscopic scoring.The frequencies of micronuclei induced by CP and MMC (ascending rank) were separately 2.8%, 2.6%, 7.8% and 3.2%, 3.7%, 5.1%, which were obtained by flow cytometry screening.The frequencies of micronuclei induced by CP and MMC (ascending rank) were separately2.8%, 6.2%, 9.1% and 7.9%, 10.1%, 10.2%, which were obtained by high-content screening.Compared with negative controls, the differences of the results were statistically significant(P < 0.05), and there was a dose-response relationship. Conclusion In this study, the results of high-throughput screening assays of FCM and HCS are in accordance to the results of traditional cytokinesis blocking micronucleus assay, indicating that high-throughput screening in vitro micronucleus assays could detect micronucleus formation automatically and improve the efficiency.Therefore, the method could provide data support for using high-throughput screening in vitro micronucleus assays into genotoxicity assessment of chemicals.

20.
Medical Journal of Chinese People's Liberation Army ; (12): 1032-1039, 2020.
Article in Chinese | WPRIM | ID: wpr-849621

ABSTRACT

Objective To construct a high-throughput screening model for transient receptor potential vanilloid 4 (TRPV4) channel modulators based on calcium-activated chloride channels (CaCC). Methods RT-PCR was used to detect the endogenous expression of TRPV4 in Fischer rat thyroid (FRT) cells. The PCR products obtained were subjected to nucleic acid sequencing using gel-recovery technology. Western blotting was employed to detect the expression of TRPV4 protein in FRT cells. The liposome transfection method was applied to construct the FRT cell model that co-expressed anoctamin 1 (ANO1) and YFP-H148Q/ I152L. The expressions of ANO1 and YFP-H148Q/I152L in cells were identified by the inverted fluorescence microscope and the fluorescence quenching kinetics test. After adding TRPV4 activators and inhibitors, the fluorescence quenching kinetics experiment was used to test whether the model could screen TRPV4 modulators. The Fura-2 fluorescent probe method was applied to detect the calcium concentration in cells after adding TRPV4 activators; The Z' factor was calculated to evaluate the sensitivity and specificity of the cell model. Results RT-PCR and Western blotting confirmed the endogenous expression of TRPV4 in FRT cells; ANO1 was clearly expressed on the FRT cell membrane and YFP-H148Q/I152L was clearly expressed in the cytoplasm of FRT cells under the inverted fluorescence microscope. The FRT cell model co-expressing ANO1 and YFP-H148Q/I152L was successfully constructed. Fluorescence quenching kinetics experiments confirmed that the model could screen TRPV4 regulators, and the slope value of fluorescence change and the concentration of TRPV4 regulator concentration were in a dose-dependent manner. The model could sensitively detect changes in intracellular calcium concentration, and the slope value could reflect intracellular calcium concentration. The Z' factor was 0.728, which demonstrates its capacity for high-throughput screening. Conclusions We successfully constructed a high-throughput model that could screen TRPV4 modulators sensitively and efficiently.

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