title: [Establishment of a Mycobacterium tuberculosis proteasome inhibitor model in vitro].
abstract: OBJECTIVE
  To establish a model for screening Mycobacterium tuberculosis (M. tuberculosis) proteasome inhibitors in vitro.
  
  
  METHODS
  The proteasome inhibitor in vitro screening model was established according to the principle that the fluorescent substrate Suc-Leu-Leu-Val-AMC (S-LLVY-AMC) was hydrolyzed by the proteasome and released AMC (7-amino-4-methylcoumarin) which was fluorescent, and the fluorescence value was proportional to the proteasome activity. Conditions which could affect proteasome activity including the time of the reaction, concentration of proteasome, or SDS, were evaluated. The model was validated by using the known proteasome inhibitor MG132 and 8 natural products. Linear correlation and regression analysis were performed by the factors affecting fluorescence values.
  
  
  RESULTS
  In the model for screening M. tuberculosis proteasome inhibitors in vitro, the reaction system consisted of 0.34 mg/L SDS, 25 mg/L protein and 64 µmol/L S-LLVT-AMC, and the reaction time was 30 min. By using MG132 and natural compounds A-H to validate the MTB proteasome screening model, the calculated IC50 of MG132 was 49 µmol/L. In a final concentration of 200 µmol/L, the inhibition rates of 8 drugs on MTB proteasome were 0.0%, 3.1%, 7.8%, 45.2%, 52.4%, 69.5%, 69.6%, 88.7% respectively, confirming that the model was repeatable and reliable.
  
  
  CONCLUSIONS
  In this study, we established a model for screening M. tuberculosis proteasome inhibitors in vitro, which could provide a technical platform for fast and large scale screening of mycobacterial proteasome inhibitors.
authors.affiliation: Department of Microbiology and Immunity, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China.
authors.forename: Jun-xiang
authors.initials: JX
authors.lastname: Song
authors.forename: Xin
authors.initials: X
authors.lastname: Jiang
mesh_terms: D001557:Benzenesulfonates / Q000494:pharmacology*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015590:Fluorophotometry; D007976:Leupeptins / Q000494:pharmacology; D008962:Models, Theoretical; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism*; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D061988:Proteasome Inhibitors; D011930:Reaction Time
chemical_list: D001557:Benzenesulfonates; D007976:Leupeptins; D009097:Multienzyme Complexes; D061988:Proteasome Inhibitors; D003546:Cysteine Endopeptidases; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde