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. 2020 Mar 11;11:1308. doi: 10.1038/s41467-020-15111-4

Fig. 2. Identification of 6-(4-aminophenyl)-N-(3,4,5-trimethoxyphenyl)pyrazin-2-amine (APA), a compound selectively toxic to slow NAT2 cells.

Fig. 2

a, b Validation of RKO (a) and DLD-1 (b) CRC cell models of rapid and slow NAT2. Cell lysates from clones and parental cells were subjected to PAGE and immunoblotting with an α-MYC-Tag antibody to detect recombinant NAT2 and α-tubulin detection as loading control. c, d Quantification of NAT2 catalytic activity in RKO and DLD-1 cell clones. The velocity at which the NAT2-specific substrates amonafide (yellow) and procainamide (blue) become acetylated by different enzymatic variants at 10 µM was measured by LC-MS/MS in RKO (c) and DLD-1 (d) clones. Mean of two independent experiments. e Workflow of the selection of the chemical compounds for screening. f, g The dose-response for APA is NAT2 dependent in both RKO (f) and DLD-1 (g) CRC cells. Rapid or slow acetylator NAT2 clones, parental cells and vector controls were treated with different concentrations of APA and cell viability was measured by MTT assay after 72 h. The mean and S.D. of three independent experiments is shown. Data were analyzed using two-way ANOVA. **P = 0.0035 in (f) and 0.0074 in (g), ****P < 0.0001.