Figure 1.

Discovery of NOTUM inhibitors from a set of activated ureas and carbamates by competitive ABPP. (A) Gel-based ABPP and Western blot analysis of conditioned media (CM) from HEK293T cells transiently transfected with cDNAs encoding WT-NOTUM, a catalytically inactive S232A-NOTUM, or control protein (METAP2). Gel-based ABPP was performed by incubating CM proteome with the FP-Rh probe (1 μM, 30 min, room temperature). Western blotting was performed with an anti-NOTUM antibody (Sigma-Aldrich HPA023041). (B) General structures of the triazole urea¹⁸ and NHH carbamate¹⁷ classes of SH-directed inhibitors. (C) Gel-based ABPP of a collection of triazole urea and NHH carbamate compounds against the CM of WT-NOTUM-transfected HEK293T cells. CM samples were pretreated with 10 μM compound (30 min, 37 °C) followed by FP-Rh (1 μM, 30 min, room temperature), prior to SDS-PAGE analysis and in-gel fluorescence scanning. (D,E) Structures (D) and concentration-dependent inhibition profiles (E) for two NHH carbamate inhibitors of NOTUM: ABC28 and ABC63.