Selective and potent furin inhibitors protect cells from anthrax without significant toxicity

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Abstract

Furin and related proprotein convertases cleave the multibasic motifs R-X-R/K/X-R in the precursor proteins and, as a result, transform the latent proproteins into biologically active proteins and peptides. Furin is present both in the intracellular secretory pathway and at the cell surface. Intracellular furin processes its multiple normal cellular targets in the Golgi and secretory vesicle compartments while cell-surface furin appears to be essential only for the processing of certain pathogenic proteins and, importantly, anthrax. To design potent, safe and selective inhibitors of furin, we evaluated the potency and selectivity of the derivatized peptidic inhibitors modeled from the extended furin cleavage sequence of avian influenza A H5N1. We determined that the N- and C-terminal modifications of the original RARRRKKRT inhibitory scaffold produced selective and potent, nanomolar range, inhibitors of furin. These inhibitors did not interfere with the normal cellular function of furin because of the likely functional redundancy existing between furin and other proprotein convertases. These furin inhibitors, however, were highly potent in blocking the furin-dependent cell-surface processing of anthrax protective antigen-83 both in vitro and cell-based assays and in vivo. We conclude that the inhibitors we have designed have a promising potential as selective anthrax inhibitors, without affecting major cell functions.

Introduction

A variety of proteins, including metalloproteinases, growth factors, and adhesion molecules as well as bacterial and viral pathogens, are initially synthesized as precursors. Specific processing is required to transform these latent proproteins into biologically active molecules (Seidah et al., 2008). Furin and related proprotein convertases (PCs) cleave the multibasic motifs R-X-R/K/X-R and transform proproteins into biologically active proteins (Thomas, 2002). Seven structurally related furin-family proteases (furin, PACE4, PC1/3, PC2, PC4, PC5/6 and PC7) have been identified in humans (Fugere and Day, 2005). Two additional, more distantly related PCSK8 and PCSK9 are implicated in cholesterol and lipid metabolism and they exhibit less stringent cleavage preferences (Horton et al., 2007, Pasquato et al., 2006).

Furin is the most studied enzyme among all of the PCs (Bassi et al., 2005, Khatib et al., 2002, Scamuffa et al., 2006). Furin is synthesized as a pre-proprotein which contains a signal peptide, a prodomain, a subtilisin-like catalytic domain, a middle P domain, a cysteine-rich region, a transmembrane anchor and a cytoplasmic tail. The N-terminal prodomain functions as a potent auto-inhibitor (Bhattacharjya et al., 2007, Fugere et al., 2002). To become proteolytically active, furin requires proteolytic removal of its inhibitory prodomain (Lazure, 2002, Thomas, 2002). Some proportion of the furin molecules cycles between the trans-Golgi network and the cell surface (Thomas, 2002). Because of the overlapping substrate preferences and cell/tissue expression, there is some redundancy in the PCs’ functionality. Thus, furin knockout is lethal in mice (Scamuffa et al., 2006). In contrast, conditional furin knockout mice targeting the liver showed no obvious adverse effects thus suggesting that other PCs can compensate the molecular ablation of furin in cells/tissues (Roebroek et al., 2004).

Furin is also implicated in the processing of membrane fusion proteins and pro-toxins of bacteria and viruses, including anthrax and botulinum toxins, influenza A H5N1 (bird flu), flaviviruses, and Marburg and Ebola viruses (Chiron et al., 1997, Collier and Young, 2003, Decha et al., 2008, Feldmann et al., 1999, Garten et al., 1994, Gordon and Leppla, 1994, Moulard and Decroly, 2000, Rockwell et al., 2002, Rott et al., 1995, Stadler et al., 1997, Zambon, 2001). Inhibition of furin represses aggressive viral and bacterial diseases (Basak et al., 2001, Chen et al., 1998, Jiao et al., 2006, Sarac et al., 2002, Shiryaev et al., 2007) suggesting that furin and related PCs are promising drug targets in infectious diseases. Because furin is required for the processing and activation of multiple normal human proteins, it has been assumed that wide-range inhibitors of furin and other PCs would interfere with normal cell functions and possibly elevate the level of side-effects.

No natural protein inhibitors of furin are known. The peptidic inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone (dec-RVKR-cmk) and α1-antitrypsin variant Portland are used to inhibit furin in vitro and in cell-based tests. The original α1-antitrypsin serpin is a natural inhibitor of neutrophil elastase (Travis and Salvesen, 1983). After a natural mutation of the active site Met358 to Arg the mutant serpin becomes a potent inhibitor of thrombin (Lewis et al., 1978). The additional, genetically engineered mutation generates α1-antitrypsin Portland that is a 0.5 nM inhibitor of furin (Anderson et al., 1993, Jean et al., 1998). Dec-RVKR-cmk and α1-antitrypsin Portland are poorly selective and, in addition to furin, they target other PCs (Benjannet et al., 1997). It is not clear if the toxicity of these compounds is the result of the inhibition of multiple cellular PCs or furin alone.

We have designed furin inhibitors modeled from the furin cleavage sequence (TPQRERRRKKR↓GL) of avian influenza A H5N1. Our results suggest that furin inhibitors can provide host protection against multiple furin-dependent, but otherwise unrelated pathogens, including anthrax (Remacle et al., 2008, Shiryaev et al., 2007). The peptides we have designed included β-Ala-TPRARRRKKRT-amide (Ki = 23 nM against furin). We have had, however, a concern that a broad-range inhibition of PCs would interfere with the intracellular processing of physiological targets and, especially, TGFβ1 (Pesu et al., 2008).

Here, we characterized the efficacy and selectivity of the modified derivatives of the original inhibitory peptide. As a result, we designed the potent and selective inhibitors of furin. These inhibitors do not significantly interfere with the intracellular processing of MT1-MMP and TGFβ1 but they perform as potent, selective and safe anthrax antagonists.

Section snippets

Reagents

Reagents were purchased from Sigma unless indicated otherwise. A murine 3G4 monoclonal antibody against the MT1-MMP's catalytic domain, a TMB/M substrate and a hydroxamate inhibitor of MMPs (GM6001) were from Chemicon. A goat polyclonal TGFβ1 antibody (C-16) was from Santa Cruz Biotechnology. Decanoyl-Arg-Val-Lys-Arg-chloromethylketone (dec-RVKR-cmk, an inhibitor of PCs) was from Bachem. The protease inhibitor mixture set III, the Protein G-agarose beads and the fluorescence pyroglutamic

Rationale

Multiple growth factors, hormones and cell receptors are synthesized as inactive precursors (Egeblad and Werb, 2002, Lopez-Otin and Bond, 2008). These precursors are transformed into active proteins by the autolytically activated furin-like PCs (Fugere and Day, 2005, Seidah et al., 2008). Because of its narrow cleavage preferences, furin predominantly cleaves only the RXR/KR↓ motif in the peptides and proteins (Remacle et al., 2008). Furin cleaves de novo synthesized precursor proteins in the

Acknowledgements

The work was supported by NIH Grants CA83017, CA77470, AI078048, AI059572 and RR020843 (to AYS) and AI055789 (to RCL) and by Canadian Institutes of Health Research and the Ministère du Développement économique, de l’innovation et de l’Exportation du Québec research grants (to RD). Authors would like to thank Drs. Mojgan Sabet and Donald G. Guiney (UCSD, La Jolla, CA, USA) for their help in performing animal experiments with anthrax spores.

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