Ac-Gly-BoroPro is a selective FAP inhibitor (Ki: 23 nM).

FAP:ki:23 nM

Ac-Gly-BoroPro selectively inhibits FAP relative to other prolyl peptidases. FAP reacts readily with submicromolar concentrations of Ac-Gly-BoroPro, reaching steady-state inhibition levels rapidly (Ki: 23 nM). In contrast, DPP-4 requires higher Ac-Gly-BoroPro concentrations for inhibition and a longer time to reach steady-state inhibition levels (Ki=377±18 nM). Ac-Gly-BoroPro inhibits other prolyl peptidases (DPP-7, DPP-8, DPP-9, prolyl oligopeptidase, and acylpeptide hydrolase) with Ki values ranging from 9- to 5400-fold higher than that for FAP inhibition.

Ki values for inhibition of proteases by Ac-Gly-BoroPro are determined using the method of progress curves for analysis of tight-binding competitive inhibitors. Various concentrations of Ac-Gly-BoroPro are reacted with FAP (1.0 nM) and DPP-4 (0.1 nM) in the presence of Ala-Pro-AFC (500 μM for FAP; 100 μM for DPP-4), and time-dependent inhibition of each protease is monitored. Reactions contained inhibitor concentrations at least 20-fold greater than protease concentrations, such that the protease-inhibitor complex does not significantly deplete the free inhibitor.