Entwicklung, Synthese und Charakterisierung neuartiger Furininhibitoren

In the present work, novel effective peptidomimetic Inhibitors for the proprotein convertase furin were synthesized and characterized. Several compounds were designed based on the structure of the irreversible inhibitor Decanoyl-RVKR-chloromethyl ketone, which contain in place of the P1 Arg-chloromethyl ketone moiety decarboxylated arginin- or lysin derivatives as well as some other basic groups. Initially, the decanoyl residue was replaced with an acetyl or phenylacetyl group and the lysin in P2-position was replaced with and arginin. Following the synthesis, the determination of the inhibitor constants showed that the compound Phenylacetyl-RVR-4-(amidino)benzylamide (17) was a highly effective Inhibitor (0.81 nM). For optimization, different series of inhibitors were synthesized based on this lead structure 17. Therefore, natural and unnatural amino acids and amino acid derivatives were inserted successively in all other positions (P2-P5). It could be shown that the exchange of the P4-arginin as well as the elimination of the basic group in P2 position led to a drastic reduction of the inhibitory efficiency. The systematic incorporation of all proteinogenic amino acids in P3 position led to compounds which could inhibit furin comparable to inhibitor 17. Besides valin, isoleucin, phenylalanin or tyrosin are also well suited in this position. In contrast, the determination of the inhibitory constants of compounds with acidic amino acids in P3-position showed a reduced inhibitory potency, probably due to electrostatic repulsion from the negatively charged active site of furin. Different fatty acids were introduced in P5 position in course of the following synthesis. The determination of the inhibitory constants showed that up to a length of 10 carbon atoms the inhibitory activity was comparable to the lead structure. The further prolongation of the carbon chain led to a gradual loss of the inhibitory efficiency. The introduction of basic residues in P5 position yielded highly potent Inhibitors of which 3-(Guanidinomethyl)phenylacetyl-RVR-4-(amidino)benzylamide (38) inhibits Furin most efficient with a Ki-value of 5.6 pM. In further course of the work, the stability of some synthesized compounds was analyzed via HPLC. Under the applied conditions no degradation or decay of the chosen compounds could be observed. For example, inhibitor 17 was stable in aqueous solution over 10 days at room temperature. In collaboration with the Garten laboratory (Institute of Virology, University of Marburg) some compounds were tested in cell culture for cytotoxicity. The inhibitors with negligible cytotoxic effects were further used in virological assays for the determination of the inhibition of virus spread and proliferation of highly pathogenic avian influenza (H7N1). Among others, inhibitor 17 and 4-(Guanidinomethyl)phenylacetyl-RVR-4-(amidino)benzylamide (40) were able to reduce efficiently the virus proliferation. The quantitative determination of infectious viruses in cell cultures over a period of several days showed a significant reduction in virus spread. After 45 h, the virus titer reached a value of only 1.7 % in comparison to the control without inhibitor. To purify recombinant synthesized furin for utilization in crystallization experiments, some N-terminally biotinylated inhibitors based on the lead structure were synthesized for the application in affinity chromatography. These derivatives were used in collaboration with the workgroup of Mr. Than (Institute for Age Research, Fritz Lipmann-Institut, Jena) for the purification of mouse furin. Therefore, commercially available streptavidin columns were loaded with the biotinylated compounds. In comparison to the classical purification methods, these affinity columns were well suited for an effective purification of furin. The small molecular inhibitors synthesized during this work are the most efficient reversible furin inhibitors worldwide until today. Therefore, they are well suited for further tests in cell cultures or even in animal models.