STF-31 has a cell growth inhibition profile similar to that of known NAMPT inhibitors and inhibits recombinant NAMPT. (A) Chemical structures of STF-31 and compound 146. (B) Heat-map visualization of pairwise correlations from unsupervised clustering of 496 compounds using AUC values. (C) AUC-AUC comparison between STF-31, APO-866, and CAY-10618 across 560 cell lines. Each vertical line represents a cell line, and these are aligned according to their sensitivity to STF-31. The Pearson correlation coefficient for STF-31 and each known (biochemically validated) NAMPT inhibitor is given. (D) The Spearman (rank) correlation between basal gene-expression levels and AUC values across up to 688 adherent cell lines was calculated for 18,988 transcripts, and correlation coefficients were plotted as box-and-whisker plots, with outliers (black dots) representing the 1st and 99th percentiles and NAMPT highlighted in green. (E) Recombinant NAMPT activity was measured using a coupled-enzyme system at 30 °C. Concentration–response curves were fit using non-linear regression. Each data point is mean ± SD (n = 3).

Nicotinic acid blocks the effect of STF-31 and other NAMPT inhibitors in NAPRT1-expressing cells. (A) HCT116 and HT1080 cells were tested for viability after 72 h treatment with compound 146 and known NAMPT inhibitors in the presence or absence of 10 μM nicotinic acid (NA). Each data point is mean ± SD (n = 3). (B) Sensitivity of HCT116 cells tranfected with control or NAPRT1 siRNA to compound 146 in the presence or absence of NA. Each data point is mean ± SD (n = 3). (C) Sensitivity of HCT116 against compound 146 in different media conditions. Each data point is mean (n = 2). (D) NAPRT1-dependent effects of nicotinic acid (NA) on STF-31 sensitivity across 772 CCLs. Sensitive cell lines were defined as having an AUC < 3.5, and insensitive cell lines as an AUC > 5.5. Red bars = mean ± SD.

Identification of recurrent genomic alterations in compound 146-resistant cell lines. (A) Workflow of the generation of compound 146-resistant clones in HCT116 cells. (B) Sensitivity of compound 146-resistant clones to compound 146 after 72 h incubation. Each data point is mean (n = 2). (C) Work flow to identify single nucleotide variants (SNVs) present in all resistant clones and predicted to have a high functional impact by MutationAssessor. (D) Sensitivity of compound 146-resistant cells to two known NAMPT inhibitors. Each data point is mean (n = 2).

NAMPT H191R confers resistance to the growth inhibition effect of STF-31, compound 146, and a known NAMPT inhibitor. (A) NAMPT and GLUT1 protein expression in parental HCT116 cells, HCT116 cells engineered with either WT or H191R NAMPT, and a HCT116 cell line selected for resistance to compound 146 (α-tubulin; loading control). (B) Effects of STF-31, compound 146, and APO-866 after 72 h treatment in HCT116 cells expressing wild-type or H191R NAMPT. Each data point is mean ± SD (n = 3).