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.

Limiting the Number of Potential Binding Modes by Introducing Symmetry into Ligands: Structure-Based Design of Inhibitors for Trypsin-Like Serine Proteases.

In the absence of X-ray data, the exploration of compound binding modes continues to be a challenging task. For structure-based design, specific features of active sites in different targets play a major role in rationalizing ligand binding characteristics. For example, dibasic compounds have been reported as potent inhibitors of various trypsin-like serine proteases, the active sites of which contain several binding pockets that can be targeted by cationic moieties. This results in several possible orientations within the active site, complicating the binding mode prediction of such compounds by docking tools. Therefore, we introduced symmetry in bi- and tribasic compounds to reduce conformational space in docking calculations and to simplify binding mode selection by limiting the number of possible pocket occupations. Asymmetric bisbenzamidines were used as starting points for a multistage and structure-guided optimization. A series of 24 final compounds with either two or three benzamidine substructures was ultimately synthesized and evaluated as inhibitors of five serine proteases, leading to potent symmetric inhibitors for the pharmaceutical drug targets matriptase, matriptase-2, thrombin and factor Xa. This study underlines the relevance of ligand symmetry for chemical biology.

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

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 ß-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.

Active site mapping of trypsin, thrombin and matriptase-2 by sulfamoyl benzamidines.

The benzamidine moiety, a well-known arginine mimetic, has been introduced in a variety of ligands, including peptidomimetic inhibitors of trypsin-like serine proteases. According to their primary substrate specificity, the benzamidine residue interacts with the negatively charged aspartate at the bottom of the S1 pocket of such enzymes. Six series of benzamidine derivatives (1-73) were synthesized and evaluated as inhibitors of two prototype serine proteases, that is, bovine trypsin and human thrombin. As a further target, human matriptase-2, a recently discovered type II transmembrane serine protease, was investigated. Matriptase-2 represents an important regulatory protease in iron homeostasis by down-regulation of the hepcidin expression. Compounds 1-73 were designed to contain a fixed sulfamoyl benzamidine moiety as arginine mimetic and a linker-connected additional substructure, such as a tert-butyl ester, carboxylate or second benzamidine functionality. A systematic mapping approach was performed with these inhibitors to scan the active site of the three target proteases. In particular, bisbenzamidines, able to interact with both the S1 and S3/S4 binding sites, showed notable affinity. In branched bisbenzamidines 66-73 containing a third hydrophobic residue, opposite effects of the stereochemistry on trypsin and thrombin inhibition were observed.

A Bisbenzamidine Phosphonate as a Janus-faced Inhibitor for Trypsin-like Serine Proteases.

A hybrid approach was applied for the design of an inhibitor of trypsin-like serine proteases. Compound 16 [(R,R)- and (R,S)-diphenyl (4-(1-(4-amidinobenzylamino)-1-oxo-3-phenylpropan-2-ylcarbamoyl)phenylamino)(4-amidinophenyl)methylphosphonate hydrochloride], prepared in a convergent synthetic procedure, possesses a phosphonate warhead prone to react with the active site serine residue in a covalent, irreversible manner. Each of the two benzamidine moieties of 16 can potentially be accommodated in the S1 pocket of the target enzyme, but only the benzamidine close to the phosphonate group would then promote an irreversible interaction. The Janus-faced inhibitor 16 was evaluated against several serine proteases and caused a pronounced inactivation of human thrombin with a second-order rate constant (kinac /Ki) of 59 500 M(-1) s(-1). With human matriptase, 16 showed preference for a reversible mode of inhibition (IC50 =2.6 µM) as indicated by linear progress curves and enzyme reactivation.

A cell-permeable inhibitor to trap Gαq proteins in the empty pocket conformation.

In spite of the crucial role of heterotrimeric G proteins as molecular switches transmitting signals from G protein-coupled receptors, their selective manipulation with small molecule, cell-permeable inhibitors still remains an unmet challenge. Here, we report that the small molecule BIM-46187, previously classified as pan-G protein inhibitor, preferentially silences Gαq signaling in a cellular context-dependent manner. Investigations into its mode of action reveal that BIM traps Gαq in the empty pocket conformation by permitting GDP exit but interdicting GTP entry, a molecular mechanism not yet assigned to any other small molecule Gα inhibitor to date. Our data show that Gα proteins may be "frozen" pharmacologically in an intermediate conformation along their activation pathway and propose a pharmacological strategy to specifically silence Gα subclasses with cell-permeable inhibitors.