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.

Evaluation of bisbenzamidines as inhibitors for matriptase-2.

The serine protease matriptase-2 has attracted much attention as a potential target for the treatment of iron overload diseases. In this study, a series of 27 symmetric, achiral bisbenzamidines was evaluated for inhibitory activity against human matriptase-2, against the closely related enzyme human matriptase, as well as against human thrombin, bovine factor Xa and human trypsin. The conformationally restricted piperazine derivative 19 and the oxamide-derived bisbenzamidine 1 were identified as the most potent inhibitors of this series for matriptase-2 and matriptase, respectively.

The intact Kunitz domain protects the amyloid precursor protein from being processed by matriptase-2.

Proteolytic processing of the amyloid precursor protein (APP) leads to amyloid-ß (Aß) peptides. So far, the mechanism of APP processing is insufficiently characterized at the molecular level. Whereas the knowledge of Aß generation by several proteases has been expanded, the contribution of the Kunitz-type protease inhibitor domain (KPI) present in two major APP isoforms to the complex proteolytic processing of APP is poorly understood. In this study, we have identified KPI-containing APP as a very potent, slow-binding inhibitor for the membrane-bound proteolytic regulator of iron homeostasis matriptase-2 by forming stable complexes with its target protease in HEK cells. Inhibition and complex formation depend on the intact KPI domain. By inhibiting matriptase-2, KPI-containing APP is protected from matriptase-2-mediated proteolysis within the Aß region, thus preventing the generation of N-terminally truncated Aß.

En Route to New Therapeutic Options for Iron Overload Diseases: Matriptase-2 as a Target for Kunitz-Type Inhibitors.

The cell-surface serine protease matriptase-2 is a critical stimulator of iron absorption by negatively regulating hepcidin, the key hormone of iron homeostasis. Thus, it has attracted much attention as a target in primary and secondary iron overload diseases. Here, we have characterised Kunitz-type inhibitors hepatocyte growth factor activator inhibitor 1 (HAI-1) and HAI-2 as powerful, slow-binding matriptase-2 inhibitors. The binding modes of the matriptase-2-HAI complexes were suggested by molecular modelling. Different assays, including cell-free and cell-based measurements of matriptase-2 activity, determination of inhibition constants and evaluation of matriptase-2 inhibition by analysis of downstream effects in human liver cells, demonstrated that matriptase-2 is an excellent target for Kunitz inhibitors. In particular, HAI-2 is considered a promising scaffold for the design of potent and selective matriptase-2 inhibitors.

3,1-Benzothiazines, 1,4-Benzodioxines and 1,4-Benzoxazines as Inhibitors of Matriptase-2: Outcome of a Focused Screening Approach.

The liver enzyme matriptase-2 is a multi-domain, transmembrane serine protease with an extracellular, C-terminal catalytic domain. Synthetic low-molecular weight inhibitors of matriptase-2 have potential as therapeutics to treat iron overload syndromes, in particular in patients with ß-thalassemia. A sub-library of 64 compounds was screened for matriptase-2 inhibition and several active compounds were identified. (S)-Ethyl 2-(benzyl(3-((4-carbamidoylphenoxy)methyl)-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)amino)-2-oxoacetate ((S)-12) showed an IC50 value of less than 10 µM. Structure-activity relationships were discussed and proposals to design new matriptase-2 inhibitors were made.

3-Cyano-3-aza-ß-amino Acid Derivatives as Inhibitors of Human Cysteine Cathepsins.

Nitrile-type inhibitors are known to interact with cysteine proteases in a covalent-reversible manner. The chemotype of 3-cyano-3-aza-ß-amino acid derivatives was designed in which the N-cyano group is centrally arranged in the molecule to allow for interactions with the nonprimed and primed binding regions of the target enzymes. These compounds were evaluated as inhibitors of the human cysteine cathepsins K, S, B, and L. They exhibited slow-binding behavior and were found to be exceptionally potent, in particular toward cathepsin K, with second-order rate constants up to 52 900 × 10(3) M(-1) s(-1).