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

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — 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