title: A luminescence assay for natural product inhibitors of the Mycobacterium tuberculosis proteasome.
abstract: BACKGROUND
  Mycobacterium tuberculosis (Mtb) causes a large global burden of disease, with a high mortality rate in healthy and immuno-compromised patients. A number of molecular targets have been identified for treatment of this disease, including the Mtb proteasome. The Mtb proteasome enhances Mtb survival during nitrosative and oxidative stress in the latent, non-replicative phase. Therefore, Mtb proteasome inhibition could help to combat Mtb infections that do not respond to current therapies.
  
  
  OBJECTIVE
  To develop and validate a novel biochemical assay to assess Mtb proteasome activity in the presence of organic and aqueous plant test extracts.
  
  
  METHODS
  Fluorescence (photoluminescence) and luminescence (chemiluminescence) assays were investigated as potential methods to determine the robustness and repeatability for use in screening natural product extracts for Mtb proteasome inhibitors.
  
  
  RESULTS
  The fluorescence assay, used widely for Mtb proteasome activity assays, was subject to interference due to the natural fluorescence of compounds in many of the extracts; there is little interference with the luminescence approach. As proof of principle, we used the luminescence assay to screen a small set of plant test extracts.
  
  
  CONCLUSIONS
  Luminescence is the more suitable assay for assay of plant natural product extracts. The sensitivities of the luminescence and fluorescence assays are comparable. A Z'-factor of 0.58 for the luminescence assay makes it suitable for medium-to-high throughput screening efforts.
authors.affiliation: Department of Chemistry and Biochemistry, University of the Sciences, Philadelphia, PA, USA.
authors.forename: Amber
authors.initials: A
authors.lastname: Gunderwala
authors.affiliation: Department of Biological Sciences, University of the Sciences, Philadelphia, PA, USA.
authors.forename: John
authors.initials: J
authors.lastname: Porter
mesh_terms: D001688:Biological Products / Q000494:pharmacology*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D005453:Fluorescence; D049449:Luminescence; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects*; D015203:Reproducibility of Results
chemical_list: D001688:Biological Products; D015853:Cysteine Proteinase Inhibitors; D046988:Proteasome Endopeptidase Complex
keywords: Mycobacterium tuberculosis; luminescence; natural products; proteasome