Fig. 4: Identification of NAMPT as a target of OT-82 and the mechanism of OT-82-induced cell death.

a Identification of NAMPT as the protein target of OT-82 by affinity chromatography followed by functional confirmation. MV4–11 cell extract (3 mg protein/reaction) was incubated for 1 h at 40 °C with Affi-Gel 10 coupled with “active” compound (OT-82), “inactive” compound (a non-cytotoxic OT-82 structural analog), or a combination of the two (“competition”). Eluted proteins were subjected to gel electrophoresis and silver staining (top) or transferred to nitrocellulose membrane for western blotting with NAMPT antisera (bottom). Comparison of inhibitory effects of OT-82 and FK866 on the in vitro activity of recombinant NAMPT indicates close similarity of dose dependence of OT-82 with a bona fide NAMPT inhibitor. Presence of 10 μM of nicotinic acid (NA) protects human leukemia-derived cell lines from OT-82-induced cell death: MV4–11 and U937 cells were incubated with the indicated concentrations of OT-82 in the absence or presence of 10 μM NA for 72 h. Viability was measured by resazurin assay. b Characterization of events preceding cell death from OT-82 treatment. Effect of different doses of OT-82 on NAD and ATP concentrations was measured after the indicated time points in MV4–11 cells following OT-82 treatment using Enzyfluo and ATPlite 1 luminescence kits, respectively. Data are shown as relative effect (%) compared with untreated cells (mean ± SE; n = 2–3). The right panel indicates the impact of OT-82 dose on viability and apoptosis markers (activation of caspase-3, depolarization of the mitochondrial membrane, and the proportion of cells with sub-G1 DNA content) in MV4–11 cells following 48 h of incubation with different concentrations of OT-82. See Supplementary Methods for details.