Study Of Drug Screening

Chinese herbs contain a large number of various structures compounds, which are substance base of Chinese herbs' effectivity. Although some compounds isolated from herbs have definitely bioactivity, most compounds are not clear defined. The aim of the discourse was to clarify the active components of herbs that have hormone-like action and antiviral activity. First, we developed cell-based sensitive fluorescent assay for high throughput screening and identified an active component isolated from Epimedium brevicornum. Second, using CPE/MTS assay, we screening about 3,700 compounds and 200 herb extracts. Several herbs showed potent antiviral activity against SARS-CoV or influenza virus. Bio-guided assay identified some active single compounds and fractions. All above provided new candidate agents for treatment of related diseases, it also provided the examples of herb substance base. Part Ⅰ Screening and identification of small molecular non-peptide agonist of parathyroid hormone receptor and calcitonin receptor. Parathyroid Hormone (PTH) and Calcitonin (CT), the most important regulatory factors in bone remodeling, have actions that promotes bone formation and inhibit bone resopsion. Both PTH and CT were used for treatment of skeletall disorders, such as osteoporosis. In such treatment, PTH and CT preparations are used parenterally and no oral preparation is available at present, due to poor oral absorption and drug resistance result from receptor expression down-regulation. Thus, our aims are develop sensitive assays for screening for PTH receptor and CT receptor from Chinese herb library, to discover orally administered molecules, especially non-peptide small molecules for treatment of osteoporosis. The main results in this part were described as following. One. Developing cell-based green fluorescence protein reporter gene assays for screening leads for PTHR and CTR. Using molecule clone technique, we constructed pcDNA3.1-CTR/hygro expression vector and sequenced and confirmed. The pcDNA3.1-CTR/hygro expression vector, pCRE-d2EGFP/neovector and pcPTHR/neo expression vector were identified by observation of activators induced fluorescencs after transient transfection vectors in HEK293 cells. Then reporter gene and receptor gene were stably transfected in HEK 293 and CHO-K1 cells with antibiotic selection and limited dilution. Five cell lines were abtained and designed as 293-PTHR/CRE-dEGFP cell, CHO-PTHR/CRE-dEGFP cell, 293-CTR/CRE-dEGFP cell, CHO-CTR/CRE-dEGFP cell, and 293-CRE-dEGFP respectively. To optimize the 293-PTHR/CRE-dEGFPcells and 293-CTR/CRE-dEGFP cells assay for high throughput screen (HTS), several parameters including cell density, optium time for agonist stimulation, DMSO sensitivity, etc., were examined. The results showed that EC50 of hPTH and hCT in 293-PTHR/CRE-d2EGFPcells and 293-CTR/CRE-d2EGFP cells were 2.3×10^-8 M 和 7.6×10^-6 M respectively, which were the same as the cAMP functional assay. Time course for agonist stimulation showed that agonist induced fluorescence with high density from 8 h to 18 h. Two. High throughput screening Chinese herb library. More than one hundred and forty-four thousand fractions and single compounds isolated from ninety hundred Chinese herbs were screened using 293-PTHR/CRE-d2EGFP cell assay and 293-CTR/CRE-d2EGFP cell assay. The hit rate of 0.12% and confirm rate of 17.6% were calculated after primary screen using 293-PTHR/CRE-d2EGFP cells assay. The hit rate of 0.09% and confirm rate of 24.9% were calculated after primary screen using 293-CTR/CRE-d2EGFP cells assay. Eb5, isolated from Epimedium brevicornum, showed active PTHR/cAMP/PKA/CRE signal pathway with EC50 value 0.13 μg/ml。Cytotoxity test indicated that cytotoxity of Eb5 is larger than 100 μg/ml. Three. Study of proliferation, gene regulation, and mineralization function of MC3T3-E1 cells after treatment of Eb5 Further functional study indicated that Eb5 promoted the proliferation, increased mRNA expression of type I collagen (COL I), alkaline phosphatase (ALPase), and osteocalci...