Phosphono Bisbenzguanidines as Irreversible Dipeptidomimetic Inhibitors and Activity‐Based Probes of Matriptase‐2

Abstract

Matriptase-2, a type II transmembrane serine protease, plays a key role in human iron homeostasis. Inhibition of matriptase-2 is considered as an attractive strategy for the treatment of iron-overload diseases, such as hemochromatosis and β-thalassemia. In the present study, synthetic routes to nine dipeptidomimetic inactivators were developed. Five active compounds (41-45) were identified and characterized kinetically as irreversible inhibitors of matriptase-2. In addition to a phosphonate warhead, these dipeptides possess two benzguanidine moieties as arginine mimetics to provide affinity for matriptase-2 by binding to the S1 and S3/S4 subpockets, respectively. This binding mode was strongly supported by covalent docking analysis. Compounds 41-45 were obtained as mixtures of two diastereomers and were therefore separated into the single epimers. Compound 45 A, with S configuration at the N-terminal amino acid and R configuration at the phosphonate carbon atom, was the most potent matriptase-2 inactivator with a rate constant of inactivation of 2790 m(-1) s(-1) and abolished the activity of membrane-bound matriptase-2 on the surface of intact cells. Based on the chemotyp of phosphono bisbenzguanidines, the design and synthesis of a fluorescent probe (51 A) by insertion of a coumarin label is described. The in-gel fluorescence detection of matriptase-2 was demonstrated by applying 51 A as the first activity-based probe for this enzyme.

A BODIPY-Tagged Phosphono Peptide as Activity-Based Probe for Human Leukocyte Elastase

“…The protease activities were assayed photometrically or fluorometrically and the reactions were followed over 60 min. 16,23,33 Epimer 1A showed a strong inhibition of HLE with a secondorder rate constant of 399000 M −1 s −1 , a moderate effect against PPE, and inactivity against the other proteases (Table 1). Compound 1B exhibited no inhibitory activity against the investigated proteases except HLE, which was inhibited with a weak second-order rate constant of 1000 M −1 s −1 ( Table 1).…”

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

“…In continuation of a previous study, our design of an ABP for matriptase is based on two linker‐connected benzguanidines, a known substructure of matriptase inhibitors . One benzguanidine moiety is very likely to be oriented towards the deep, negatively charged S1 pocket .…”

“…Scheme outlines the convergent synthetic approach to the ABP 18 , similar to the one that has previously been established for other benzguanidino phosphonates . Details are given in the Supporting Information.…”

Recent Developments in Peptidyl Diaryl Phoshonates as Inhibitors and Activity-Based Probes for Serine Proteases

“…Two guanidinophenyl residues were applied as arginine mimetics in construction of dipeptide diaryl phosphonate analog inhibitors of matriptases [21,22]. Matriptase activates several substrates, which play critical roles in tumorigenesis, while matriptase-2 is a transmembrane serine protease involved in regulation of iron homeostasis though a signaling pathway.…”