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. 2019 Jul 19;10:3241. doi: 10.1038/s41467-019-11078-z

Fig. 2.

Fig. 2

SBI-797812 mechanism of action. a NAMPT activation by SBI-797812 requires ATP. NAMPT (30 nM) incubated with NAM (10 μM) and PRPP (50 μM) and, where indicated, ATP (2 mM) and/or SBI-797812 (2 μM). Reactions performed at 37 °C for 1 or 4 h without or with ATP, respectively. NMN detected with the fluorescence assay. Data normalized to NMN produced in 1 h. Data expressed as means ± s.d.; n = 4. *p < 0.001 compared to NAMPT or (NAMPT + ATP). One-way ANOVA with Tukey’s multiple comparisons test. b SBI-797812 increased NAMPT affinity for ATP. Without SBI-797812 (open circle), NAMPT (30 nM) incubated with NAM (10 μM), PRPP (50 μM), and varying ATP (0–2 mM). With 2 μM SBI-797812 (filled circle), reactions were identical except 15 nM NAMPT was used. Reactions performed for 1 h at 37 °C. NMN measured with the fluorescence assay. Data normalized for NMN production in the absence of ATP and expressed as U nmol−1 NAMPT; means ± s.d. are shown; n = 3. Curve fitting using GraphPad Prism. c Impact of SBI-797812 on NAMPT equilibrium reaction. NAMPT (200 nM) mixed with 5 μM NAM, 100 μM PRPP, 5 μM NMN, 100 μM PP with (closed) and without (open) SBI-797812 (1 μM). Samples incubated at 37 °C and aliquots removed sequentially. At 3 h, 2 mM ATP was spiked. Incubation continued at 37 °C with aliquots sampled as indicated. NAM (blue) and NMN (red) assayed by LC-MS/MS. Data expressed as means ± s.d.; n = 4. d Comprehensive profiling of NAMPT reaction substrates/products. NAM (25 μM), PRPP (50 μM), and ATP (2 mM) incubated for 1 h at 37 °C in buffer without (BG) or with NAMPT (100 nM). Where indicated, SBI-797812 (5 μM) was included. Samples assayed for NAM, NMN and ADP by LC-MS/MS. Pi and PP determined by colorimetric assay. Data expressed as means ± s.d.; n = 6 except for BG (NAM, NMN, and ADP assays), n = 5. *P < 0.0001, vs. BG in each group; #p < 0.0001, NAMPT vs. (NAMPT + SBI-797812) in each group. 1-way ANOVA with Tukey’s multiple comparisons test. e SBI-797812 blunted NAD+-mediated feedback inhibition of NAMPT activity. NAMPT (30 nM) incubated with NAM (10 μM), PRPP (50 μM), ATP (2 mM) for 1 h at 37 °C. SBI-797812 (2 μM) or NAD+ (250 μM) included as indicated. NMN measured by LC-MS/MS. Data expressed as means ± s.d.; n = 4. *p < 0.0001 vs. Control; #p < 0.0001 vs. NAD+. 1-way ANOVA with Tukey’s multiple comparisons test. For Fig. 2a–e, source data are provided as a Source Data file