Highly potent inhibitors of proprotein convertase furin as potential drugs for treatment of infectious diseases

Abstract

Optimization of our previously described peptidomimetic furin inhibitors was performed and yielded several analogs with a significantly improved activity. The most potent compounds containing an N-terminal 4- or 3-(guanidinomethyl)phenylacetyl residue inhibit furin with K(i) values of 16 and 8 pM, respectively. These analogs inhibit other proprotein convertases, such as PC1/3, PC4, PACE4, and PC5/6, with similar potency, whereas PC2, PC7, and trypsin-like serine proteases are poorly affected. Incubation of selected compounds with Madin-Darby canine kidney cells over a period of 96 h revealed that they exhibit great stability, making them suitable candidates for further studies in cell culture. Two of the most potent derivatives were used to inhibit the hemagglutinin cleavage and viral propagation of a highly pathogenic avian H7N1 influenza virus strain. The treatment with inhibitor 24 (4-(guanidinomethyl)phenylacetyl-Arg-Val-Arg-4-amidinobenzylamide) resulted in significantly delayed virus propagation compared with an inhibitor-free control. The same analog was also effective in inhibiting Shiga toxin activation in HEp-2 cells. This antiviral effect, as well as the protective effect against a bacterial toxin, suggests that inhibitors of furin or furin-like proprotein convertases could represent promising lead structures for future drug development, in particular for the treatment of infectious diseases.

SCHEME 1.

Synthesis of inhibitors 25 and 26. The N-terminal Boc-protected P5-P2 segment was prepared on a 2-chlorotrityl chloride resin using 3-(Boc-aminomethyl)phenylacetic acid for the coupling of the P5 group and otherwise using a standard Fmoc protocol. a, cleavage from resin with 1% TFA in DCM, two times for 30 min, drying in vacuo; b, 1.5 eq 4-aminomethylbenzamidine·2 HCl, 1.7 eq PyBOP, 4.5 eq 6-Cl-1-hydroxybenzotriazole, 10 eq DIPEA in DMF, 2 h; c, TFA/TIS/H₂O (95:2.5:2.5, v/v/v), 3 h at 35 °C, precipitation in cold diethyl ether, preparative reversed phase HPLC; d, 5 eq 1H-pyrazole-1-carboxamidine·HCl in 1 m Na₂CO₃, 24 h, purification by preparative HPLC. DCM, dichloromethane; PyBOP, benzotriazol-1-yl-N-oxy-tris(pyrrolidino)phosphonium hexafluorophosphate; DIPEA, diisopropylethylamine; DMF, N,N-dimethylformamide; TIS, triisopropylsilane.

FIGURE 1.

Dixon plot for the stearoyl inhibitor 11, the shown data are the means ± S.E. of three independent measurements. The nonlinear curves (substrate concentrations 32.5 (●) and 13 (○) μm) do not permit the calculation of a true K_i value according to a classical competitive mechanism. Only for the purpose of better visualization, the data points have here been connected by fitting to an exponential growth curve. The dashed line parallel to the x axis represents 1/V_max.

FIGURE 2.

IC₅₀ determination for inhibitors 6 (●), 10 (▴), 11 (♢), 13 (♦), and 14 (○). The shown data are the means ± S.E. of three independent measurements and were performed at a fixed substrate concentration of 8.13 μm. The data were fitted to Equation 1.

FIGURE 3.

Concentration dependence of light scattering (in %) determined for inhibitors 3 (☐), 7 (●), 11 (○), 13 (▴), and 14 (△) in the buffer used for enzyme kinetics. Dotted lines indicated no micelle formation in the concentration range used.

FIGURE 4.

Active site titration attempts of furin using Dec-Arg-Val-Lys-Arg-CMK. A, determination of the remaining enzyme activity after preincubation of furin with different concentrations of the irreversible inhibitor Dec-Arg-Val-Lys-Arg-CMK (times for preincubation indicated in the graphs). B, concentration of active furin obtained was dependent on the preincubation time, providing a relatively constant value of ∼1.2 nm only after 2 h of preincubation.

FIGURE 5.

Enzyme kinetic analysis of inhibitor 26 in presence of 8.13 μm substrate. The measured data were fitted to Equation 2, providing a furin concentration of 0.81 nm as a parameter (solid line). In contrast, fits performed using different constant enzyme concentrations were unsatisfactory (e.g. furin fixed to a concentration of 1 nm (dotted line) and fixed to 0.6 nm (dashed line).

FIGURE 6.

Inhibition of HA cleavage by different concentrations of compounds 22 and 24. A, MDCK cell cultures were infected with FPV at a multiplicity of infection of 100. Inhibition of HA cleavage at different inhibitor concentrations was analyzed 24 h post-infection. Proteins from virus particles (HA0, 82 kDa; nucleoprotein (NP), 56 kDa; HA1, 50 kDa; and neuraminidase (NA), 50 kDa) were immunochemically detected after SDS-PAGE followed by Western blot analysis. B, quantification of HA cleavage inhibition by Western blot analysis (n = 2, including S.E.). The maximum amount of HA0, obtained by inhibition with 50 μm of inhibitor 24, was normalized to 100% cleavage inhibition. Other HA0 band intensities at different concentrations were measured and normalized by standardization of each HA0 value correlating with the corresponding nucleoprotein band. Experimental details are provided in the supplemental material.

FIGURE 7.

Inhibition of multiple cycle replication of FPV in cell culture in the absence (○) and in the presence of 50 μm of inhibitor 22 (▾) and 24 (△). Cultures of MDCK cells were inoculated with FPV at a multiplicity of infection of 0.0001. The inhibitor was added to the medium at a final concentration of 50 μm. At different times (0, 18, 24, and 48 h post-infection) the amount of infectious virus (as plaque-forming units (PFU) per ml) released into the medium was determined by a microplaque assay (44).

FIGURE 8.

Stability measurements with inhibitor 24 at two different concentrations in pure cell culture medium (30 μm (▾) and 10 μm (●)) and in medium containing MDCK cells (30 μm (▿) and 10 μm (○)). In all cases, 100 μl of the inhibitor containing medium or supernatant from the cell culture was analyzed by analytical HPLC, which corresponds to ∼3.7 μg in the case of the 30 μm solution and to 1.2 μg in the case of the 10 μm solution. The shown data are the mean of two measurements, including S.E.

FIGURE 9.

Intoxication of HEp-2 cells by Shiga-toxin (n ≥3 for all experiments). A, intoxication was performed in the presence of selected inhibitors (all used at 25 μm; control lacking inhibitor in black, ID₅₀ 4.4 ng/ml; inhibitor 24 in red, ID₅₀ 26.1 ng/ml; inhibitor 1 in blue, ID₅₀ 7.83 ng/ml; Phac-Val-d-Arg-Arg-4-Amba in magenta, ID₅₀ 6.1 ng/ml; benzylsulfonyl-d-Ser-Lys(Cbz)-4-Amba in green, ID₅₀ 3.69 ng/ml). B, intoxication was performed in presence of various concentrations of inhibitor 24. No protection was observed at an inhibitor concentration of 0.1 μm.