ALPHA(1)-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 path ogenic molecules has made this endoprotease a target for the developme nt of potent and selective antiproteolytic agents. Here, we demonstrat e the utility of the protein-based inhibitor alpha(1)-antitrypsin Port land (alpha(1)-PDX) as an antipathogenic agent that can be used prophy lactically to block furin-dependent cell killing by Pseudomonas exotox in A. Biochemical analysis of the specificity of a bacterially express ed His- and FLAG-tagged alpha(1)-PDX (alpha(1)-PDX/hf) revealed the se lectivity of the alpha(1)-PDX/hf reactive site loop for furin (K-i, 60 0 pM) but not for other proprotein convertase family members or other unrelated endoproteases. Kinetic studies show that alpha(1)-PDX/hf inh ibits 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 probabil ity to undergo proteolysis by furin at the C-terminal side of the reac tive center -Arg(355)-Ile-Pro-Arg(358)- --> or to form a kinetically t rapped SDS-stable complex with the enzyme. This partitioning between t he complex-forming and proteolytic pathways contributes to the ability of alpha(1)-PDX/hf to differentially inhibit members of the proprotei n convertase family. Finally, we propose a structural model of the alp ha(1)-PDX-reactive site loop that explains the high degree of enzyme s electivity of this serpin and which can be used to generate small mole cule furin inhibitors.