title: [A method based on endogenous fluorescence determination for screening NAMPT inhibitors].
abstract: To establish a method for screening nicotinamide phosphoribosyl transferase (NAMPT) inhibitors based on endogenous fluorescence determination.
  
  
  
  The double mutants of NAMPT, G355C/D393C, was cross-linked by using 1, 4-Bismaleimidobutane (BMB) to block the entrance of enzymatic active site of NAMPT. The binding of compounds to NAMPT was evaluated according to the change of spontaneous fluorescence of NAMPT and BMB-NAMPT with 280 nm excitation and 333 nm emmision. The in vitro enzamatic activity of NAMPT was determined by nuclear magnetic resonance. The cell viability was determined by MTT assay.
  
  
  
  FK866 significantly decreased the spontaneous fluorescence of NAMPT but not of BMB-NAMPT. Rosmaric, cynarine and 1, 3-dicaffeoylquinic acid also decreased the spontaneous fluorescence of both NAMPT and BMB-NAMPT. However, the inhibition on two proteins was equivalent. FK866 significantly inhibit the catalysis of NAMPT. Rosmarinic acid, cynarine and 1, 3-dicaffeoylquinic acid failed to inhibit the catalysis of NAMPT. FK866 inhibited the viability of A549 cells, but rosmarinic acid, cynarine and 1, 3-dicaffeoylquinic acid did not.
  
  
  
  Endogenous fluorescence spectrometry based on NAMPT and BMB-NAMPT protein can be used for screening compounds that bind with NAMPT, and distinguishing the binding site - either within the enzymatic active site or not. Rosmarinic acid, cynarine and 1, 3-dicoffeoylquinic acid can bind to NAMPT out its enzymatic active site.
authors.affiliation: 1. Department of Pharmacology， Zhejiang University School of Medicine， Hangzhou 310058， China; 2. Zhejiang Xiaoshan Hospital， Hangzhou 311200，China.
authors.forename: Xue
authors.initials: X
authors.lastname: Han
authors.affiliation: Wuhan Institute of Physics and Mathematics Chinese Academy of Sciences， Wuhan 430071， China.
authors.forename: Xue
authors.initials: X
authors.lastname: Dong
authors.affiliation: Wuhan Institute of Physics and Mathematics Chinese Academy of Sciences， Wuhan 430071， China.
authors.forename: Wen-xue
authors.initials: WX
authors.lastname: Jiang
authors.affiliation: Department of Pharmacology， Zhejiang University School of Medicine， Hangzhou 310058， China.
authors.forename: Peng
authors.initials: P
authors.lastname: Huang
authors.affiliation: Department of Pharmacology， Zhejiang University School of Medicine， Hangzhou 310058， China.
authors.forename: Wei-ping
authors.initials: WP
authors.lastname: Zhang
authors.affiliation: Wuhan Institute of Physics and Mathematics Chinese Academy of Sciences， Wuhan 430071， China.
authors.forename: Chun
authors.initials: C
authors.lastname: Tang
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005453:Fluorescence; D006801:Humans; D054409:Nicotinamide Phosphoribosyltransferase / Q000037:antagonists & inhibitors*
chemical_list: D054409:Nicotinamide Phosphoribosyltransferase