Improving the selectivity of 3-amidinophenylalanine-derived matriptase inhibitors.

A rational structure-based approach was employed to develop novel 3-amidinophenylalanine-derived matriptase inhibitors with improved selectivity against thrombin and factor Xa. Of all 23 new derivatives, several monobasic inhibitors exhibit high matriptase affinities and strong selectivity against thrombin. Some inhibitors also possess selectivity against factor Xa, although less pronounced as found for thrombin. A crystal structure of a selective monobasic matriptase inhibitor in complex with matriptase and three crystal structures of related compounds in trypsin and thrombin have been determined. The structures offer an explanation for the different selectivity profiles of these inhibitors and contribute to a more detailed understanding of the observed structure-activity relationship. Selected compounds were tested in vitro against a matriptase-dependent H9N2 influenza virus strain and demonstrated a concentration-dependent inhibition of virus replication in MDCK(II) cells.

Mutational and structural studies uncover crucial amino acids determining activity and stability of 17ß-HSD14.

17ß-Hydroxysteroid dehydrogenase type 14 (17ß-HSD14) catalyzes the conversion of highly active estrogens and androgens into their less active oxidized forms in presence of NAD+ as cofactor. The crystal structure of 17ß-HSD14 has been determined, however, the role of individual amino acids likely involved in the enzymatic function remains poorly understood. Objective of this study was to further characterize the enzyme by site-directed mutagenesis considering five amino acids next to the catalytic center. The tools used for the characterization of the enzyme variants are X-ray crystallography and enzyme kinetics. Lys158 was confirmed to belong to the catalytic triad. Tyr253', located on the C-terminal loop of the adjacent monomer, enters into the active site of the neighboring monomer and interacts with the catalytic Tyr154. Therefore, Tyr253' helps to tie the two monomers together. Cys255, located at the interface between both monomers, can form a disulfide bridge with the Cys255' from the adjacent monomer. In contrast to the contact provided by Tyr253, the latter interaction is not crucial for dimer formation. His93 and Gln148 are located at the rim of the substrate binding pocket. His93 does not interact directly with the ligand in the active site. However, it influences the turnover of the enzyme. The Gln148 restricts in size the access tunnel of the substrate to the binding pocket.

A Fluorescent-Labeled Phosphono Bisbenzguanidine As an Activity-Based Probe for Matriptase.

Activity-based probes are compounds that exclusively form covalent bonds with active enzymes. They can be utilized to profile enzyme activities in vivo, to identify target enzymes and to characterize their function. The design of a new activity-based probe for matriptase, a member of the type II transmembrane serine proteases, is based on linker-connected bis-benzguanidines. An amino acid, introduced as linker, bears the coumarin fluorophore. Moreover, an incorporated phosphonate allows for a covalent interaction with the active-site serine. The resulting irreversible mode of action was demonstrated, leading to enzyme inactivation and, simultaneously, to a fluorescence labeling of matriptase. The ten-step synthetic approach to a coumarin-labeled bis-benzguanidine and its evaluation as activity-based probe for matriptase based on in-gel fluorescence and fluorescence HPLC is reported. HPLC fluorescence detection as a new application for activity-based probes for proteases is demonstrated herein for the first time.