ABSTRACT
Human immunodeficiency virus type 1 (HIV-1) infection is highly dependent on its conical fullerene capsid, which is composed of approximately 1 500 capsid proteins. In recent years, capsid protein has been recognized as an ideal target for design and screening drugs for AIDS therapy. Screening methods are the key to develop HIV-1 capsid inhibitors. A variety of screening approaches for HIV-1 capsid protein inhibitor, which have been reported in the literature are reviewed in the article.
ABSTRACT
Aim To establish a cell model targeting on GLP-1 R,and evaluate its function by the cAMP assay,for screening the new class of GLP-1 analogues as anti-diabetic candidates. Methods An eukaryotic expression vector pEGFP-GLP-1 R was constructed and transfected into HEK293A cells.After selecting with G41 8,a cell line stably expressing GLP-1 R-GFP was estab-lished.The expression and the cellular distribution of GLP-1 R-GFP fusion protein were investigated by Western blot and fluo-rescence microscopy.Then,the activity of GLP-1 analogue Lira-glutide was evaluated by monitoring the content of cAMP via HTRF using this cell model.Results GLP-1 R-GFP-293A cell line was successfully established.GLP-1 R-GFP fusion proteins were mainly distributed in the cell membrane.The dose-respon-sive relationship experiments revealed that cAMP could be effec-tively stimulated by Liraglutide using this cell model.Conclu-sion This cell model could be used to detect the bioactivity of GLP-1 analogues in vitro,which lays a foundation for the screen-ing of GLP-1 analogues and small GLP-1 R agonists.
ABSTRACT
Prostaglandin (PG) E2 is an active substance in pathological and physiological mechanisms, such as inflammation and pain. The in vitro high-throughput assay for screening the inhibitors of reducing PEG2 production is a useful method for finding out antiphlogistic and analgesic candidates. The assay was based on LPS-induced PGE2 production model using a homogeneous time-resolved fluorescence(HTRF) PGE2 testing kit combined with liquid handling automation and detection instruments. The critical steps, including the cell density optimization and IC50 values determination of a positive compound, were taken to verify the stability and sensibility of the assay. Low intra-plate, inter-plate and day-to-day variability were observed in this 384-well, high-throughput format assay. Totally 5 121 samples were selected from the company's traditional Chinese medicine(TCM) material base library and used to screen PGE2 inhibitors. In this model, the cell plating density was 2 000 cells for each well; the average IC₅₀ value for positive compounds was (7.3±0.1) μmol; the Z' factor for test plates was more than 0.5 and averaged at 0.7. Among the 5 121 samples, 228 components exhibited a PGE2 production prohibition rate of more than 50%, and 23 components exhibited more than 80%. This model reached the expected standards in data stability and accuracy, indicating the reliability and authenticity of the screening results. The automated screening system was introduced to make the model fast and efficient, with a average daily screening amount exceeding 14 000 data points and provide a new model for discovering new anti-inflammatory and analgesic drug and quickly screening effective constituents of TCM in the early stage.