Establishment of an in vitro screening model for steroid 5 alpha-reductase inhibitors with the microplate reader / 中华男科学杂志
National Journal of Andrology
;
(12): 483-486, 2013.
Article
in Chinese
| WPRIM
| ID: wpr-350875
ABSTRACT
<p><b>OBJECTIVE</b>To establish an in vitro screening model for steroid 5 alpha-reductase inhibitors using the microplate reader.</p><p><b>METHODS</b>Steroid 5 alpha-reductase was obtained from the liver of female rats, an in vitro screening model for steroid 5 alpha-reductase inhibitors established using the 96-well plate and microplate reader after determination of the enzymatic activity, and the reliability of the model verified with the known 5 alpha-reductase inhibitors epristeride and finasteride. Added to the 96-well plate were the final concentrations of testosterone (0-40 micromol/L), NADPH (22 micromol/L), epristeride (0-60 nmol/L) or finasteride (0-60 nmol/ L) and steroid 5 alpha-reductase (20 microl), the total volume of each well adjusted to 200 microl with Tris-Hcl buffer. The 96-well plate was placed in the microplate reader, mixed and incubated at 37 degrees C, followed by detection of the A340nm value at 0 and 10 min and analysis of the data.</p><p><b>RESULTS</b>The Km value of steroid 5 alpha-reductase was 3.794 micromol/L, with a Vmax of 0.271 micromol/(L. min). The Ki of epristeride was 148.2 nmol/L, with an IC50 of 31.5 nmol/L, and the enzymatic reaction kinetic curve suggested that epristeride was an uncompetitive enzyme inhibitor. The Ki of finasteride was 158. 8 nmol/L, with an IC50 of 13.6 nmol/L. The enzymatic reaction kinetic curve showed that both epristeride and finasteride were competitive enzyme inhibitors, similar to those reported in the published literature.</p><p><b>CONCLUSION</b>A screening model was successfully established, which could rapidly and effectively screen steroid 5 alpha-reductase inhibitors in vitro.</p>
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Immunoenzyme Techniques
/
Rats, Sprague-Dawley
/
Drug Evaluation, Preclinical
/
High-Throughput Screening Assays
/
5-alpha Reductase Inhibitors
/
Methods
Type of study:
Diagnostic study
/
Screening study
Limits:
Animals
Language:
Chinese
Journal:
National Journal of Andrology
Year:
2013
Type:
Article
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