In vitro competitive ABPP of β-lactone (±)-5. (A)
Gel-based ABPP of (±)-5 in various
human cancer cell and mouse tissue proteomes showing inhibition of
multiple serine hydrolases (highlighted in red boxes). Proteomes were
treated with DMSO or (±)-5 (1.0 or 25 μM)
for 30 min followed by FP-Rh (30 min). (B) Concentration-dependent
inhibition of serine hydrolase activities in PC3 cell proteomes treated
with DMSO or (±)-5 (0.001–10 μM), showing
significant inhibition of serine hydrolase activities migrating at
30 and 45 kDa (red asterisks). (C) ABPP-SILAC analysis of (±)-5 at 1.0 μM (heavy amino acid-labeled proteome) versus
DMSO (light amino acid-labeled proteome) in PC3 cell proteomes, revealing
inhibition of ABHD10, CTSA, SCPEP1, and ACOT1/2. (D) ABPP-SILAC analysis
of (±)-5 (1.0, 0.1, and 0.01 μM), 2 (10 μM),
or DMSO (heavy) versus DMSO (light) in PC3 cell proteomes showing
dose-dependent inhibition of the four primary targets of (±)-5 observed at 1.0 μM. For C and D, data are presented
as the mean ± standard deviations of heavy/light ratios for multiple
unique peptides from each serine hydrolase.