Peptidic Inhibitors and a Fluorescent Probe for the Selective Inhibition and Labelling of Factor XIIIa Transglutaminase

Factor XIIIa (FXIIIa) is a transglutaminase of major therapeutic interest for the development of anticoagulants due to its essential role in the blood coagulation cascade. While numerous FXIIIa inhibitors have been reported, they failed to reach clinical evaluation due to their lack of metabolic stability and low selectivity over transglutaminase 2 (TG2). Furthermore, the chemical tools available for the study of FXIIIa activity and localization are extremely limited. To combat these shortcomings, we designed, synthesised, and evaluated a library of 21 novel FXIIIa inhibitors. Electrophilic warheads, linker lengths, and hydrophobic units were varied on small molecule and peptidic scaffolds to optimize isozyme selectivity and potency. A previously reported FXIIIa inhibitor was then adapted for the design of a probe bearing a rhodamine B moiety, producing the innovative KM93 as the first known fluorescent probe designed to selectively label active FXIIIa with high efficiency (k(inact)/K-I = 127,300 M-1 min(-1)) and 6.5-fold selectivity over TG2. The probe KM93 facilitated fluorescent microscopy studies within bone marrow macrophages, labelling FXIIIa with high efficiency and selectivity in cell culture. The structure-activity trends with these novel inhibitors and probes will help in the future study of the activity, inhibition, and localization of FXIIIa.

Automated summary

Factor XIIIa (FXIIIa) is an important transglutaminase enzyme in the blood coagulation cascade and is a target for the development of anticoagulant drugs. However, current FXIIIa inhibitors lack metabolic stability and have low selectivity over transglutaminase 2 (TG2), preventing clinical evaluation. In this study, we designed and evaluated 21 novel inhibitors targeting FXIIIa, with varying electrophilic warheads, linker lengths, and hydrophobic units to optimize selectivity and potency. We also developed a fluorescent probe, KM93, which selectively labels active FXIIIa with high efficiency and 6.5-fold selectivity over TG2. The structure-activity trends observed with these inhibitors and probe will contribute to future studies on the activity, inhibition, and localization of FXIIIa.