Fig. 3.
SBI-797812 impacts PP consumption and pHisNAMPT reactivity. a SBI-797812 stimulates NAMPT-mediated PP consumption. NAMPT (100 nM) was incubated with ATP (2 mM), PP (20 μM) or ATP + PP for 2 h at 37 °C in TMD buffer. Where indicated, SBI-797812 (5 μM) was also included. Samples assayed for PP using the colorimetric assay. Data expressed as means ± s.d.; n = 3. *p < 0.001 vs. No NAMPT (ATP + PP); #p < 0.001 vs. NAMPT (ATP + PP). One-way ANOVA with Tukey’s multiple comparisons test. b Transition structure of the predicted reaction between pHisNAMPT and PP. The computational model used an original X-ray data set (PDB: 3DHF and 3DKL): pHis247, residues Asp313 and Asp279 are mimicked by acetate groups, two magnesium atoms (grey spheres) were incorporated to coordinate PP along with water molecules. The catalyzed reaction proceeds via late SN2 attack of the pHis247 with dN–P = 2.38 Å and dP–O = 1.96 Å (blue dashed lines). c Production of P3 from ATP and PP by NAMPT. NAMPT incubated for 2 h with ATP (2 mM), PP (100 μM) in absence or presence of SBI-797812 (5 μM). Samples also run with 20 μM NMN where indicated. Samples analyzed by LC-MS/MS for P3. Data expressed as means ± s.d.; n = 5. *p < 0.0001. One-way ANOVA with Tukey’s multiple comparisons test. d SBI-797812 stabilized pHisNAMPT in presence of NMN or PP. NAMPT (20 μg/ml) incubated with ATP (2 mM) for 10 min at 37 °C (all lanes). Also present were: 5 μM SBI-797812 (lanes 3–4), 10 μM NMN (lanes 5–6), NMN + SBI-797812 (lanes 7–8), 10 μM PP (lanes 9–10), PP + SBI-797812 (lanes 11–12). Samples were analyzed by western blotting using anti-1-pHisAb. pHisNAMPT was detected with a LICOR infrared imager. For Fig. 3a, c, d, source data are provided as a Source Data file