Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:EXPT01586 (G418)
 2,237 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Inhibitors of human 5alpha-reductase type II are promising drug candidates for the treatment of benign prostatic hyperplasia which is associated with high prostatic DHT levels. In this study we describe the evaluation of potential inhibitors in a new cell assay. First a plasmid (pRcCMV-5alphaII) for the expression of human 5alpha-reductase type II was constructed by the use of the vector pRcCMV and transfected into the African green monkey fibroblast-like cell line COS1. By selection with G418 sulfate, ten COS1 single cell clones were obtained of which three stably exhibited high 5alpha-reductase activity. One single cell clone (COS1-5alphaIIST) was selected for further investigations. By Southern blot analysis, fluorescence in situ hybridization (FISH) and comparative PCR experiments it turned out that the expression plasmid pRcCMV-5alphaII has been integrated into the chromosome, resulting in a long-term stable expression of the foreign 5alpha-reductase gene. The newly established cell line was used for testing novel compounds on their inhibitory effect on human 5alpha-reductase type II. Using this whole cell assay, inhibitors with IC(50) values in the nanomolar range could be identified.
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PMID:Stable expression of human 5alpha-reductase type II in COS1 cells due to chromosomal gene integration: a novel tool for inhibitor identification. 1159 8

Dihydrotestosterone (DHT) is the most potent male hormone that causes androgenetic alopecia. The type II 5alpha-reductase is an enzyme that catalyzes the conversion of testosterone (T) to DHT, therefore it can be expected that specific inhibitors for type II 5alpha-reductase may improve the pathophysiologic status of androgenetic alopecia. In this study, we attempted to establish the reliable and convenient screening model for type II 5alpha-reductase inhibitors. After transfection of human cDNA for type II 5alpha-reductase into HEK293 cells, the type II 5alpha-reductase over-expressing stable cells were selected by G418 treatment. RT-PCR and Western blot analyses confirmed that type II 5alpha-reductase gene was expressed in the stable cells. In in vitro enzymatic assay, 10 microg of stable cell extract completely converted 1 microCi (approximately 0.015 nmol) of T into DHT. The type II 5alpha-reductase activity was inhibited by finasteride in a dose-dependent manner, confirming the reliability of screening system. In cell culture condition, 2 x 10(5) of stable cells completely converted all the input T (approximately 0.03 nmol) into DHT by 4h incubation, demonstrating that the stable cell line can be used as a cell-based assay system. Using this system, we selected the extracts of Curcumae longae rhizoma and Mori ramulus as the potential inhibitors for type II 5alpha-reductase. These results demonstrate that the type II 5alpha-reductase over-expressing stable cell line is a convenient and reliable model for screening and evaluation of inhibitors.
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PMID:Establishment of type II 5alpha-reductase over-expressing cell line as an inhibitor screening model. 1764 96

Human steroid 5alpha-reductase type II (hSRD5A2) and dihydrotestosterone (DHT) play important roles in benign prostatic hyperplasia (BPH). The aim of our study was to establish a novel model to investigate the inhibitory effects of extracts and compounds of Chinese herb medicine on hSRD5A2. The gene, hSRD5A2, was artificially synthesized and cloned into pcDNA3.1(+) vector, which was transfected into CHO cells by liposome. Transfected cells were screened through G418 and MTX. The expressed protein of hSRD5A2 by cells was purified and detected by western blotting. A minimum reactive system comprising hSRD5A2 and testosterone (T) as substrate together with NADPH as hydrogen donor was established for screening inhibitors of hSRD5A2. The reaction system was optimized in the concentrations of T, NADPH, and hSRD5A2 and reaction temperature, time, and activity of hSRD5A2 were determined by the production of DHT. Furthermore, we screened some extracts and compounds of Chinese herb medicine using this model. The concentrations of T, NADPH, and hSRD5A2 were 0.02 microM, 0.8 mM, and 0.05 U/microl, respectively, in the model; maximum activity of hSRD5A2 was achieved at 37 degrees C and 60 min reaction, and mangiferin had significant inhibitory effect on the activity of hSRD5A2. The model in this study is convenient and reliable for screening and evaluation of inhibitors of hSRD5A2; mangiferin may be a potential medicine for the treatment of BPH.
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PMID:Establishment of a novel model for studying the effects of extracts of Chinese herb medicine on human type II 5alpha-reductase in vitro. 2082 78