α1-antitrypsin Portland, a bioengineered serpin highly selective for furin:: Application as an antipathogenic agent

The important role of furin in the proteolytic activation of many pathogenic molecules has made this endoprotease a target for the development of potent and selective antiproteolytic agents. Here, we demonstrate the utility of the protein-based inhibitor alpha(1)-antitrypsin Portland (alpha(1)-PDX) as an antipathogenic agent that can be used prophylactically to block furin-dependent cell killing by Pseudomonas exotoxin A. Biochemical analysis of the specificity of a bacterially expressed His- and FLAG-tagged alpha(1)-PDX (alpha(1)-PDX/hf) revealed the selectivity of the alpha(1)-PDX/hf reactive site loop for furin (K-i, 600 pM) but not for other proprotein convertase family members or other unrelated endoproteases. Kinetic studies show that alpha(1)-PDX/hf inhibits furin by a slow tight-binding mechanism characteristic of serpin molecules and functions as a suicide substrate inhibitor. Once bound to furin's active site, alpha(1)-PDX/hf partitions with equal probability to undergo proteolysis by furin at the C-terminal side of the reactive center -Arg(355)-Ile-Pro-Arg(358)- --> or to form a kinetically trapped SDS-stable complex with the enzyme. This partitioning between the complex-forming and proteolytic pathways contributes to the ability of alpha(1)-PDX/hf to differentially inhibit members of the proprotein convertase family. Finally, we propose a structural model of the alpha(1)-PDX-reactive site loop that explains the high degree of enzyme selectivity of this serpin and which can be used to generate small molecule furin inhibitors.