Fig 1: BDW568 as prodrug was metabolized into an active form by CES1. (a) Immunoblotting of single-clone CES1 knockout cells (THP-1 IFNAR2–/–) using two sgRNA sequences (loading control, γ-tubulin). Data are representative of two independent experiments. (b) BDW568 activity in single-clone CES1 knockout THP-1 reporter cells using two sgRNA sequences (NTC, nontargeting control using scrambled sgRNA sequence). (c) Ultra-Performance Liquid Chromatography (UPLC) profiles of representative injections of authentic BDW568 and BDW-OH samples (lanes 1 and 4), extracts of BDW568-treated cells at 15 min and 120 min (lanes 2 and 3), and coinjection of authentic BDW-OH and BDW568 metabolite at 15 min (lane 5). (d) BDW-OH-versus-time curve in CES1+/+ or CES1–/– THP-1 reporter cells. The error bars represent ± s.d. for biological triplicates of one experiment. (e) Michaelis–Menten curve for recombinant CES1 compared to the enzyme-free reaction in PBS. The hydrolysis of BDW568 was measured in the presence or absence of 100 μg/mL human recombinant CES1 in PBS by LC-MS. Data are presented as mean ± s.d. for biological triplicates from one experiment. (f) Chemical structures and dose–response curves of BDW568, BDW-tBu, and BDW-NH-Me in THP-1 reporter cells. All dose–response curves are representative of three experiments; error bars represent ± s.d. for four biological replicates. (g) STD NMR assay of BDW-OH (300 μM) and STING-AQ (30 μM) in 10% D2O, 5% DMSO-d6 and 10 mM DTT. The assay was performed using a train of low power (50 Hz) Gaussian pulses (total saturation time = 7.5 s) at 30 and 0.85 ppm for off- and on-resonance saturation, respectively. The vertical scale of the STD spectra is increased by a factor of 8 relative to the off-resonance spectrum.