Synthese, Charakterisierung und Anwendung neuer Inhibitoren der Proproteinkonvertase Furin

The type-I transmembrane protein furin belongs to the family of proprotein convertases (PCs) and contains a Ca2+-dependent subtilisin-like serine protease domain. Furin is ubiquitously distributed in human tissues and catalyzes the maturation of inactive precursor proteins in physiological and pathogenic processes. It is involved in various viral and bacterial infections, tumorigenesis, neurodegenerative disorders, diabetes, and atherosclerosis. Furin has a strong preference to process its substrates at a multibasic cleavage site, like Arg-Xaa-Arg/Lys-Arg-Xaa. Based on this sequence highly potent substrate analogue inhibitors containing decarboxylated arginine mimetics in P1 position have been previously developed. Among a series of inhibitors containing various proteinogenic P3 amino acids strong differences in their inhibitory potencies have been observed, whereby the strongest inhibition was found for the Val and Ile analogues. Further optimization of the lead structure Phac-Arg-Xaa-Arg-4-amidinobenzylamide by incorporation of unnatural hydrophobic P3-residues provided new furin inhibitors with improved potency. The most potent analogues contain tert.leucine (Ki = 0,17 nM) or penicillamine (Ki = 0,25 nM) as P3 residue. Combination of P3 tert.leucine with basic substituted phenylacetyl residues in P5 position resulted in picomolar furin inhibitors. The most potent derivative MI-1148 (4-GMe-Phac-Arg-Tle-Arg-4-Amba) was characterized as tight-binding inhibitor of furin possessing a Ki value of 5.5 pM. Selected derivatives were used in various cell culture assays to demonstrate their potency as anti-infective drugs. Low micromolar concentrations of inhibitors MI-1148 and of its closely related analogue MI-1150 effectively inhibit the cleavage of the hemagglutinin (HA) from highly pathogenic influenza strains (H5N1, H7N1) and their subsequent virus propagation in MDCK cells. Further decreased inhibitor concentrations reduce the propagation of canine distemper virus in VeroSLAMdogtag cells. Moreover, nanomolar concentrations of MI-1148 and its closely related analogue MI-1150 were sufficient to protected macrophages against anthrax and diphtheria toxin. The protective effect against the bacterial toxins, as wells as their antiviral activity suggest that inhibitors of furin could represent promising lead structures for the development of new broad-spectrum anti-infective drugs. The analogs of this series also inhibit the related PC1/3 with similar potency, whereas PC2 is less affected. It was possible to solve a crystal structure of human furin in complex with MI-1148, which revealed numerous key interactions explaining the excellent low picomolar potency of this analogue. In an additional approach the N-terminal segment of the lead structure was modified. Many furin substrates possess additional basic residues in the P5 – P8 region. For this reason, a constant Arg-Val-Arg-4-Amba segment was maintained in P4-P1 positions and its N-terminus has been elongated with a different number of arginine residues in D- and/or L-configuration. These N-terminally unprotected derivatives inhibit furin in the subnanomolar range between 30 – 90 pM, whereby a perfect fit of the enzyme kinetic data to the equation for tight-binding inhibition was not possible, although determination coefficients >0.97 have been calculated. Compared to the most potent inhibitors these compounds showed a reduced inhibition of influenza virus propagation in MDCK cells at a concentration of 50 µM. Furthermore, the lead structure was extended with L-arginine residues in P5- and P6 position. The best derivative of this series MI-1103 (Phac-Arg-Arg-Arg-Val-Arg-4-Amba) inhibits furin with an inhibition constant of 13,8 pM. To decrease the molecular weight of this inhibitor type, several compounds without P5-moiety have been prepared. These compounds possess reduced inhibitory potency in enzyme kinetic studies. Nevertheless the best compounds of this series (5-guanidino)-valeryl-Val-Arg-4-Amba (MI-1146) and its P3-Tle analogue MI-1152 inhibit furin with Ki-values of 2.5 and 1.3, respectively. Both derivatives also showed a concentration dependent protection of macrophages against anthrax toxin, although their efficacy was relatively low.