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

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.

Fig. 1. Synthetic inhibitors of furin (SSM-1, SSM-2 and SSM-3)

The EC50 values of the inhibitors (277 nM, 101 nM and 54 nM, respectively) were determined in the reactions using furin and Pyr-RTKR-AMC (Jiao et al., 2006).

Fig. 2. Cleavage of anthrax PA83 by furin

PA83 was incubated for 1 h at 37°C with furin (enzyme-substrate molar ratio 1:1000) in the presence of the inhibitory peptides (top) and synthetic inhibitors (bottom). The reactions were analyzed by SDS-gel electrophoresis followed by Coomassie staining. DEC, dec-RVKR-cmk.

Fig. 3. Cleavage of HIV gp160, HA and MT1-MMP by furin

A, Cleavage of gp160 by furin at the indicated enzyme-substrate molar ratio. Where indicated, dec-RVKR-cmk (5 μM) was added to the reactions. B, Inhibition of furin proteolysis of gp160 by the inhibitors. C, Inhibition of furin proteolysis of HA by the inhibitors. D, Inhibition of furin proteolysis of MT1-MMP by the inhibitors. The reactions were analyzed by SDS-electrophoresis followed by Coomassie staining (HA), immunoblotting (MT1-MMP), and silver staining (HIV-1 gp160). DEC, dec-RVKR-cmk.

Fig. 4. Processing of anthrax PA83 in cell-based assays

Glioma U251 cells were co-incubated for 3 h at 37°C with biotin-labeled PA83 (1 μg/ml) and the indicated concentrations of the inhibitory peptides (top) and synthetic inhibitors (bottom). The cell lysates were examined by Western blotting. DEC, dec-RVKR-cmk.

Fig. 5. Processing of cellular MT1-MMP and TGFβ1

A, Breast carcinoma MCF-MT1-E240A-FLAG cells were co-incubated for 18 h with the inhibitors. The cell lysates were immunocaptured using anti-FLAG beads. The samples were analyzed by Western blotting with the MT1-MMP 3G4 antibody. B, Fibrosarcoma HT1080 cells were co-incubated for 24 h with the inhibitors. Medium aliquots were precipitated using 10% TCA. Precipitates were analyzed by Western blotting with the TGFβ1 antibody. DEC, dec-RVKR-cmk.

Fig. 6. Uptake of the FITC-modified peptides by cells

HT 1080 cells were incubated for 1 h in the presence of fluorescein-tagged AO-RARRRKKRT and β-Ala-RARRRKKRT peptides (10 μM). The cell samples were treated with trypsin before FACS analysis to remove the cell membrane-adsorbed peptides (the bottom panel). Values represent geometric means which corresponds to the mean of the fluorescence.

Fig. 7. Cell toxicity assays

Murine RAW264.7 macrophages were co-incubated for 24 h at 37°C with the peptide (top) and synthetic inhibitors (bottom). The cell viability was measured using an ATP-Lite kit.

Fig. 8. The AO-RARRRKKRT peptide and Cipro protect A/J mice from post-exposure inhalation anthrax

Mice (8 animals/group) were infected intranasally with B. anthracis spores. Treatment with the peptide started 24 h post-exposure and continued for the next 6 days. On the fourth day following infection, mice were given Cipro daily. Control received PBS alone.

Fig. 9. The structure of furin modeled in the complex with the RARRRKKRT peptide

The partial sequence alignment of furin, PC5/6 and PACE4 is shown in the upper part of the left panel. Only the sequence regions which form the substrate binding site are shown. The functionally important residues are color coded. RARRRKKRT is shown as sticks. Right panel, close-up of the S5 sub-site. Furin and PACE4 residues are orange and pink, respectively. The distance between the ε-nitrogen of the P5 Arg of the inhibitor and the oxygen of the Glu²⁵⁷ carboxyl group of furin is 2.5Å. Because of the presence of Asp²⁵⁷ at the S5, this distance is 3.5Å in PC5/6 and PACE4.