Potent inhibitors of furin and furin-like proprotein convertases containing decarboxylated P1 arginine mimetics

Abstract

Furin belongs to the family of proprotein convertases (PCs) and is involved in numerous normal physiological and pathogenic processes, such as viral propagation, bacterial toxin activation, cancer, and metastasis. Furin and related furin-like PCs cleave their substrates at characteristic multibasic consensus sequences, preferentially after an arginine residue. By incorporating decarboxylated arginine mimetics in the P1 position of substrate analogue peptidic inhibitors, we could identify highly potent furin inhibitors. The most potent compound, phenylacetyl-Arg-Val-Arg-4-amidinobenzylamide (15), inhibits furin with a K(i) value of 0.81 nM and has also comparable affinity to other PCs like PC1/3, PACE4, and PC5/6, whereas PC2 and PC7 or trypsin-like serine proteases were poorly affected. In fowl plague virus (influenza A, H7N1)-infected MDCK cells, inhibitor 15 inhibited proteolytic hemagglutinin cleavage and was able to reduce virus propagation in a long-term infection test. Molecular modeling revealed several key interactions of the 4-amidinobenzylamide residue in the S1 pocket of furin contributing to the excellent affinity of these inhibitors.

Figure 1

Dixon plot of inhibitor 15. Kinetic measurements were performed with four different substrate concentrations (pyroGlu-Arg-Thr-Lys-Arg-AMC: ● 5, ○ 12.5, ▲ 20, and △ 50 μM) in presence of 0.95 nM furin using various inhibitor concentrations ≥ 10 nM. The dashed line represents 1/V_max, which was obtained from a Michaelis-Menten curve measured at the same time in parallel on the same 96-well plate (K_m = 4.9 μM, V_max = 28.4 RFU/s).

Figure 2

Inhibition of fowl plague virus hemagglutinin cleavage by compound 15 and Dec-Arg-Val-Lys-Arg-CMK as control. (A) Confluent MDCK cell cultures were infected with egg-grown influenza virus A/FPV/Rostock/34 (H7N1) at a MOI of 10 per cell. Inhibition of HA cleavage (PSKKRKKR↓GLFG) at different inhibitor concentrations was analyzed after cell lysis 16 h post infection. Proteins from virus-infected cells were subjected to SDS-PAGE and Western blotting. Viral proteins were immunochemically detected by using rabbit anti-FPV serum and ECL reaction kit (Pierce): HA0 (82 kDa), nucleoprotein NP (56 kDa) and HA1 (∼50 kDa), whereas HA2 (32 kDa) was not detectable by the antiserum used. (B) Quantification of HA cleavage inhibition. Three independent experiments for both inhibitors were performed using a modified Western blot analysis technique allowing quantification by a near-IR dye-labelled second monoclonal antibody applicable for the LI-COR Odyssey Image-System. The maximum amount of HA0 obtained by inhibition with 50 μM Dec-Arg-Val-Lys-Arg-CMK was normalized to 100 % cleavage inhibition. Other HA0 band intensities at different concentrations were measured and equalized by standardization of each HA0 value correlating with the corresponding nucleoprotein NP band (56 kDa).

Figure 3

Inhibition of multiple cycle replication of FPV in cell culture in the absence (filled bars) and in the presence of inhibitor 15 (white bars). Cultures of MDCK cells were inoculated with the Rostock strain of FPV at an MOI of 10^-5 PFU per cell and incubated in DMEM without FCS at 37°C. The inhibitor was added to the medium reaching a final concentration of 25 μM. At different times (10, 18, 32, 48, 60, and 72 h p.i.) the virus amount released into the medium was measured by hemagglutination titration (HAU), at 10 h no released viruses was detected in both groups. Mean values of four independent experiments are shown.

Figure 4

Stereoview of the modeled complex between inhibitor 18 (Dec-Arg-Val-Lys-Amba) and mouse furin. A) The inhibitor (ball-and-stick model) is shown in front of the solid surface of the catalytic domain, colored according to its calculated negative (-15 e/kT, red) and positive (15 e/kT, blue) electrostatic potential. Most of the amino acids responsible for the strong negative surface potential in the vicinity of the active site are labelled. Asp306 (denoted by an asterisk) is actually located below the enzyme surface at the bottom of the S1-pocket, making a direct contact with the amidino-moiety of the inhibitor (see also panel B). B) Stick model of the surrounding residues together with the inhibitor (ball-and-stick model, carbon in light green). Furin carbons are colored grey, oxygens red and nitrogens blue. The catalytic residues are shown as thicker dark-grey sticks, and the calcium 2 ion at the bottom of the S1-pocket as a purple sphere. For clarity water molecules were omitted and only important enzyme residues involved in interactions to the inhibitor and/or being responsible for the strong negative surface potential are labelled. Strong H-bond and salt bridge contacts discussed in the text are indicated by dotted green lines. Panel B was slightly rotated with respect to panel A to improve the visibility. A pdb file of the modelled complex is available as supporting information.

Scheme 1. Synthesis of inhibitors 3-8

Reagents and conditions: (a) Tritylchloride resin, 2 equiv diamine, dry THF, 2 h; (b) Fmoc SPPS, double couplings with 4 equiv amino acid (or phenylacetic acid), HOBt and HBTU, respectively and 8 equiv DIPEA; (c) TFA/TIS/H₂O (95/2.5/2.5, v/v/v), 2 h; (d) 3 equiv 1H-pyrazole-1-carboxamidine · HCl, 4 equiv DIPEA in DMF/1 M Na₂CO₃ solution (1/1, v/v), 16 h.

Scheme 2. Synthesis of inhibitors 13–15

Reagents and conditions: (a) Loading of 2-chlorotrityl chloride resin, Fmoc-Arg(Pbf)-OH, 4 equiv DIPEA, dry DCM, 2h; (b) Fmoc SPPS, conditions see scheme 1; (c) 1 % TFA in DCM, 2 × 30 min; (d) 1, 1.1 equiv PyBOP, 3 equiv 6-Cl-HOBt, 3 equiv DIPEA in DMF, 2 h; (e) TFA/TIS/H₂O (95/2.5/2.5, v/v/v), 2 h; (f) 1-Boc-4-(aminomethyl)piperidine, 1.1 equiv PyBOP, 3 equiv 6-Cl-HOBt, 3 equiv DIPEA in DMF, 2 h; (g) 3 equiv 1H-pyrazole-1-carboxamidine · HCl, 4 equiv DIPEA in DMF, 16 h.