Journal: Nature Communications
Article Title: Boosting NAD + with a small molecule that activates NAMPT
doi: 10.1038/s41467-019-11078-z
Figure Lengend Snippet: Discovery of SBI-797812, a small molecule NAMPT activator. a Compiled melting temperature (Tm) data for human NAMPT treated with chemical compounds ( N = 57,004) and tested with the PTS assay protocol (green). NAMPT ligands produced an upward Tm shift. DMSO was the Neg Control (blue). CHS-828 (20 μM) was the Pos Control (red). b Structures of NAMPT activators and inhibitors. c Pivotal role of the 4-pyridyl nitrogen in SBI-797812 for NAMPT activation. NAMPT (30 nM), NAM (10 μM), PRPP (50 μM), ATP (2 mM) were incubated 1 h at 37 °C with vehicle or 2 μM SBI-797812, GNI-50, or SBI-796950. NMN was detected using the fluorescence assay. Data are expressed as means ± s.d.; n = 4. *, p < 0.0001 compared to Vehicle. One-way ANOVA with Dunnett’s multiple comparisons test was used. d Dose-dependent activation of human NAMPT by SBI-797812. NMN production assay was performed as above but with 25 μM NAM and varying SBI-797812. NMN levels were normalized for basal NMN production without SBI-797812. Three replicates were run for each SBI-797812 concentration. Michaelis-Menten curve fit was produced with GraphPad Prism software. e NAMPT(G217R) mutant was resistant to both SBI-797812 and FK-866. NAMPT(G217R) (50 nM) was incubated with NAM (10 μM), PRPP (50 μM), ATP (2 mM) and (where indicated) 1 μM SBI-797812 and/or 1 μM FK-866. Reactions were performed for 1 h at 37 °C. NMN was detected with the fluorescence assay. Data are expressed as means ± s.d.; n = 4. * p < 0.0001 compared to NAMPT(G217R). One-way ANOVA with Dunnett’s multiple comparisons test was used. f FK-866 and CHS-828 blocked binding of SBI-797812 to NAMPT. SBI-797812 was added to T8MD-Tween buffer with ATP or the same buffer containing NAMPT, NAMPT + FK-866, or NAMPT + CHS-828. Samples were incubated at 37 °C for 10 min and applied to a spin column to separate NAMPT-bound SBI-797812 from unbound SBI-797812. SBI-797812 (in column eluent) was measured by LC-MS-TOF. Data are expressed as means ± s.d.; n = 4. * p < 0.0001 compared to “No NAMPT”. One-way ANOVA with Dunnett’s multiple comparisons test was used. For Fig. 1c–f, source data are provided as a Source Data file
Article Snippet: The values for V max and K m (ATP hydrolysis) were deduced using the on-line Michaelis-Menten kinetics tool at http://www.graphpad.com/quickcalcs/ttest1/?Format=SEM .
Techniques: Produced, Activation Assay, Incubation, Fluorescence, Concentration Assay, Software, Mutagenesis, Binding Assay, Liquid Chromatography with Mass Spectroscopy
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