A new label-free screen for steroid 5α-reductase inhibitors using LC-MS

Steroids. 2016 Dec:116:67-75. doi: 10.1016/j.steroids.2016.10.007. Epub 2016 Oct 24.


Steroid 5α-reductase (S5αR) plays an important role in metabolizing testosterone into active androgen dihydrotestosterone (DHT) which is involved in many androgen dependent disorders, such as androgenic alopecia, benign prostatic hyperplasia and acne. The method for screening for S5αR inhibition is key in finding new antagonists. In this study, the label-free S5αR inhibitory assay using LC-MS was developed. S5αR type 1 enzyme was obtained from LNCaP prostate cancer cells. The enzymatic assay was optimised for enzyme-substrate (testosterone) concentration, NADPH-cofactor concentration, solvent tolerance, enzyme activity stability and incubation time. The developed assay was validated by measuring the signal to background ratio (S/B), the signal to noise ratio (S/N), the signal window (SW) and the zeta factor Z' in accordance with published bioassay guidelines. The enzymatic reaction was performed in 96-well plates and DHT formation was determined by LC-MS. S/B, S/N, SW and Z' factor were well above acceptable criteria and the reproducibility was good using Z' factor other 3days and further validated by dutasteride and finasteride inhibition. The method was successfully applied to quantify S5αR inhibitory activity of some Thai herbal extracts. Two plant extracts, Impatiens balsamina L. and Curcuma longa L. showed IC50 at 5.4±0.2 and 9.0±1.2μgmL-1 and are therefore promising sources of new S5αR inhibitors. The assay has high selectability and reproducibility and suited to medium throughput screening required by phytochemistry.

Keywords: Anti-androgen; LC-MS; Label-free 5-alpha reductase inhibitory assay; Steroid 5-alpha reductase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3-Oxo-5-alpha-Steroid 4-Dehydrogenase / metabolism*
  • 5-alpha Reductase Inhibitors / chemistry
  • 5-alpha Reductase Inhibitors / pharmacology*
  • Cell Line, Tumor
  • Chromatography, Liquid / methods*
  • Curcuma / chemistry
  • Dihydrotestosterone / metabolism
  • Enzyme Assays
  • Enzyme Inhibitors / chemistry
  • Enzyme Inhibitors / pharmacology
  • Humans
  • Impatiens / chemistry
  • Male
  • Mass Spectrometry / methods*
  • Plant Extracts / chemistry
  • Plant Extracts / pharmacology
  • Reproducibility of Results


  • 5-alpha Reductase Inhibitors
  • Enzyme Inhibitors
  • Plant Extracts
  • Dihydrotestosterone
  • 3-Oxo-5-alpha-Steroid 4-Dehydrogenase