α ₁ -Antitrypsin Portland, a bioengineered serpin highly selective for furin: Application as an antipathogenic agent

Abstract

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 ␣ 1 -antitrypsin Portland (␣ 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 Hisand FLAG-tagged ␣ 1 -PDX (␣ 1 -PDX͞hf) revealed the selectivity of the ␣ 1 -PDX͞hf 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 ␣ 1 -PDX͞hf 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, ␣ 1 -PDX͞hf partitions with equal probability to undergo proteolysis by furin at the Cterminal side of the reactive center -Arg 355 -Ile-Pro-Arg 358 -2 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 ␣ 1 -PDX͞hf to differentially inhibit members of the proprotein convertase family. Finally, we propose a structural model of the ␣ 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.

Furin regulates the intracellular activation and the uptake rate of cell surface-associated MT1-MMP

“…The expression of other PCs was very low, if any, in glioma cells. These results support the role of PC7 (a PC that is resistant to PDX) (Jean et al, 1998) in the activation of MT1-MMP that was observed in WT/PDX cells.…”

“…In agreement, intracellularly expressed PDX significantly, albeit incompletely, inhibited the activation of MT1-MMP (Figures 4 and 5). These results suggest that PC7, which is less sensitive than furin to PDX (Jean et al, 1998), might be involved in the processing of MT1-MMP in WT/PDX cells.…”

“…Both A 87 MRRPR 92 kC 93 GVPD and A 105 NVRRKR 111 kY 112 AIQ peptides were highly susceptible to furin and both, following the cleavage with furin, generated the AMRRPR 92 and ANVRRKR 111 cleavage fragments, respectively (Figure 2). A furin inhibitor, dec-RVKRcmk (k i ¼ 2 nM) (Jean et al, 1998) Figure 1 Schematic representation of the domain structure of membrane type-1 matrix metalloproteinase (MT1-MMP). S, PRO, CAT, H, PEX, TM and CT: the signal peptide, the prodomain, the catalytic domain, the hinge region, the hemopexin domain, the transmembrane domain and the cytoplasmic tail, respectively.…”

Inhibition of furin‐like enzymes blocks interleukin‐1α/oncostatin M–stimulated cartilage degradation

“…Dec-RVKR-CH 2 Cl is a potent inhibitor of furin and other members of the pro-protein convertase (PC) family, including PACE-4, PC2, PC3, PC6, and PC7 (50). The addition of Dec-RVKR-CH 2 Cl to bovine nasal cartilage explant cultures that had been stimulated to resorb with IL-1␣ and OSM produced a dosedependent reduction in the release of collagen fragments ( Figure 1).…”

“…of Dec-RVKR-CH 2 Cl required to block collagen release in the cartilage assay, compared with that actually required to inhibit furin and other PCs (50), is likely to be due to this compound having a short half-life and having to penetrate the highly charged cartilage matrix.…”