Elongated and Shortened Peptidomimetic Inhibitors of the Proprotein Convertase Furin

Abstract

Novel elongated and shortened derivatives of the peptidomimetic furin inhibitor phenylacetyl-Arg-Val-Arg-4-amidinobenzylamide were synthesized. The most potent compounds, such as N^α (carbamidoyl)Arg-Arg-Val-Arg-4-amidinobenzylamide (K_i =6.2 pm), contain additional basic residues at the N terminus and inhibit furin in the low-picomolar range. Furthermore, to decrease the molecular weight of this inhibitor type, compounds that lack the P5 moiety were prepared. The best inhibitors of this series, 5-(guanidino)valeroyl-Val-Arg-4-amidinobenzylamide and its P3 tert-leucine analogue displayed K_i values of 2.50 and 1.26 nm, respectively. Selected inhibitors, together with our previously described 4-amidinobenzylamide derivatives as references, were tested in cell culture for their activity against furin-dependent infectious pathogens. The propagation of the alphaviruses Semliki Forest virus and chikungunya virus was strongly inhibited in the presence of selected derivatives. Moreover, a significant protective effect of the inhibitors against diphtheria toxin was observed. These results confirm that the inhibition of furin should be a promising approach for the short-term treatment of acute infectious diseases.

Keywords: Semliki Forest virus; alphaviruses; chikungunya virus; diphtheria toxin; furin; inhibitors.

Figure 1

Known 4-Amba derived furin inhibitors 1–3 used as reference compounds.^[13,16]

Figure 2

Inhibition of furin (0.95 nM in assay) by compounds 3, 4 and 12 in the presence of the substrate Phac-Arg-Val-Arg-Arg-AMC (12.5 μM in assay). The data were fitted to the equation for reversible tight-binding inhibition (equation 1). The calculated curve of derivative 12 (●, dotted line) deviates significantly from the measured data in the “elbow region” (marked by the box, R² = 0.974), whereas the calculated curve of inhibitor 4 (○, solid line) and reference compound 3 (■, dashed line; R² = 0.999) fit well with the experimental data (R² = 0.999).

Figure 3

Infection of BHK-21 cells with SFV at a multiplicity of infection (MOI) of 0.01 in presence of the indicated furin inhibitors (25 μM in assay). Viral titers were determined by plaque assay 24 h post-infection. The dashed line illustrates the control titer in the absence of a furin inhibitor. Data represent the mean ± SE for three independent experiments.

Figure 4

Infection of BHK-21 cells with SFV at an MOI of 0.01 in the presence of the selected furin inhibitors 2 ( ), 3 ( ), 9 ( ) and 16 (●). Titers were determined by plaque assay at 24 h post-infection. Data represent the mean ± SE for three independent experiments.

Figure 5

Inhibition of E3-E2 (p62) cleavage by compounds 3, 9 and 16 (25 μM in assay) compared to a control in the absence of inhibitor. BHK-21 cells were infected with SFV at an MOI of 10. Proteins E3-E2 and mature E2 (49 kDa) were immunochemically detected with an E2 antibody after gel electrophoresis followed by Western blot analysis. Relative molecular mass of marker proteins displayed on the left-hand side.

Figure 6

Infection of BHK-21 cells with CHIKV in the presence of the indicated furin inhibitors (25 μM) at an MOI of 0.1. The titers at 24 h post-infection were determined by plaque assay. Data represent the mean ± SE obtained of at least three independent experiments.

Figure 7

Protection of Vero cells against diphtheria toxin intoxication in the presence of 200 (gray), 50 (white) and 10 nM (black) inhibitor (n = 2).