A Method Established For Screening Nicotinamide Phosphoribosyltranserase Inhibitors

Objective:To establish a method for screening NAMPT inhibitors.Methods:Using1,4-Bismaleimidobutane(BMB) to cross-link the double mutants of NAMPT, G355C/D393C, thus 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 with280nm excitation and333nm emmision. The enzamatic activity of NAMPT was determined by using nuclear magnetic resonance (NMR) in vitro. The cell viability was determined by using MTT assay.Results:FK866at0.1-1.0μM concentration-dependently decreased the spontaneous fluorescence of NAMPT but no of BMB-NAMPT. Rosmaric, cynarine and1,3-dicaffeoylquinic acid at0.5-50μM concentration-dependently decreased the spontaneous fluorescence of both NAMPT and BMB-NAMPT. The inhibition on two proteins was equivalent. FK866at molecular ratio of1:1(FK866:NAMPT protein) significantly inhibit the catalysis of NAMPT. However, rosmarinic acid, cynarine and1,3-dicaffeoylquinic acid at molecular ratio of100:1(compounds:NAMPT protein) failed to inhibit the catalysis of NAMPT. FK866at10-7M time-dependently (24,48,72h) inhibited the cell viability of A549cell. Rosmarinic acid, cynarine and 1,3-dicaffeoylquinic acid at10-10M increased cell viability of A549cell at high concentration.Conclusion: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 and1,3-dicoffeoylquinic acid can bind to NAMPT out its enzymatic active site. Objective:To Establish a cell model for screening nonenzymic nicotinamide phosphoribosyltranserase (NAMPT) blockers.Methods:NAMPT proteins whose catalysis completely missed were obtained through the mutation; Catalysis of NAMPT without ATP was detected by using fluorescence spectrometry; expression of IL-6mRNA was determined by Real time-polymerase chain reaction (PCR), and we observed all kinds of NAMPT proteins inducing function of IL-6mRNA expression in BV2cells and THP-1cells. We got BV2cell lines which stability transfected pIL-6-promoter-luc plasmid, and used it for high throughput screening of IL-6mRNA expression.Results:H247A, R311D. R392E， K423E mutations equally could weaken the catalysis of NAMPT protein significantly, the catalysis of R392E among NAMPT mutants was disappear completely. NAMPT proteins could induce THP-1cells and BV2cells expression of IL-6mRNA, and its role negatively correlated with the strength of the catalyst. The cell model based on IL-6report gene could be used for rapidly detecting nonenzymic action of NAMPT.Conclusion:R392E mutant attacking BV2cells or THP-1cells, then detecting expression level of IL-6mRNA, could be used for screening for nonenzymic blocker of NAMPT, and BV2cells which stably expressed IL-6report gene could be used as cell model of high throughput screening.