Inhibition of KAT3B acetyltransferase activity by ngKATIs was assessed by a separation-based assay70 (link). Reactions consisted of buffer (50 mM HEPES, pH 7.5, 50 mM NaCl, 2 mM EDTA, 2 mM DTT, 0.05% Triton-X-100) with KAT3B (P300, 150 nM) and FITC-Ahx-RGKGGKGLGKGG [Ahx = 6-aminohexanoic acid] substrate (2 μM) were plated in 384-well microplates and equilibrated at RT for 10 min in the presence or absence of inhibitor. Reactions were initiated by addition of acetyl-CoA (1 μM final concentration) with 30 μL final assay volume and quenched during steady-state kinetics (<15% product accumulation) by addition of 5 μL of 0.5 M neutral hydroxylamine. Quenched reaction aliquots were then transferred to a PerkinElmer Lab-Chip EZ-Reader instrument for microfluidic electrophoresis and fluorometric analysis. Optimized separation conditions were downstream voltage −500 V, upstream voltage −2500 V, and pressure −1.5 psi. Percent conversion is calculated by ratiometric measurement of substrate/product peak heights, corrected for nonenzymatic background acetylation. Percent activity KAT3B represents acetylation relative to vehicle control. Concentration responses were analyzed by nonlinear least-squares regression fits to a 4PL equation. Data are mean ± SD from three technical replicates.
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