Cathepsin B: Active site mapping with peptidic substrates and inhibitors.

The potential of papain-like cysteine proteases, such as cathepsin B, as drug discovery targets for systemic human diseases has prevailed over the past years. The development of potent and selective low-molecular cathepsin B inhibitors relies on the detailed expertise on preferred amino acid and inhibitor residues interacting with the corresponding specificity pockets of cathepsin B. Such knowledge might be obtained by mapping the active site of the protease with combinatorial libraries of peptidic substrates and peptidomimetic inhibitors. This review, for the first time, summarizes a wide spectrum of active site mapping approaches. It considers relevant X-ray crystallographic data and discloses propensities towards favorable protein-ligand interactions in case of the therapeutically relevant protease cathepsin B.

Synthesis and kinetic evaluation of ethyl acrylate and vinyl sulfone derived inhibitors for human cysteine cathepsins.

A series of inhibitors targeting human cathepsins have been designed and synthesized following a combinatorial approach. The compounds bear an α,ß-unsaturated phenyl vinyl sulfone or ethyl acrylate warhead and a peptidomimetic portion aligned to the non-primed binding region. Biochemical evaluation toward four human cathepsins was carried out and the kinetic characterization confirmed an irreversible mode of inhibition. Compound 6c combining the most advantageous building blocks for cathepsin S inhibition was identified as a potent cathepsin S inactivator exhibiting a second-order rate constant of 30600 M-1 s-1.

Evaluation of dipeptide nitriles as inhibitors of rhodesain, a major cysteine protease of Trypanosoma brucei.

A series of dipeptide nitriles known as inhibitors of mammalian cathepsins were evaluated for inhibition of rhodesain, the cathepsin L-like protease of Trypanosoma brucei. Compound 35 consisting of a Leu residue fitting into the S2 pocket and a triarylic moiety consisting of thiophene, a 1,2,4-oxadiazole and a phenyl ring fitting into the S3 pocket, and compound 33 with a 3-bromo-Phe residue (S2) and a biphenyl fragment (S3) were found to inhibit rhodesain in the single-digit nanomolar range. The observed steep structure-activity relationship could be explained by covalent docking simulations. With their high selectivity indices (ca. 200) and the good antitrypanosomal activity (8µM) the compounds represent promising starting points for new rhodesain inhibitors.

Cathepsin B Inhibitors: Combining Dipeptide Nitriles with an Occluding Loop Recognition Element by Click Chemistry.

An active site mapping of human cathepsin B with dipeptide nitrile inhibitors was performed for a combinatorial approach by introducing several points of diversity and stepwise optimizing the inhibitor structure. To address the occluding loop of cathepsin B by a carboxylate moiety, click chemistry to generate linker-connected molecules was applied. Inhibitor 17 exhibited K i values of 41.3 nM, 27.3 nM, or 19.2 nM, depending on the substrate and pH of the assay. Kinetic data were discussed with respect to the conformational selection and induced fit models.

Design, characterization and cellular uptake studies of fluorescence-labeled prototypic cathepsin inhibitors.

Besides their extracellular activity crucial for several pathophysiological conditions, human cysteine cathepsins, in particular cathepsins K and S, represent important intracellular targets for drug development. In the present study, a prototypic dipeptide nitrile inhibitor structure was equipped with a coumarin moiety to function as a fluorescent reporter group. In a second inhibitor, a PEG linker was introduced between the dipeptide nitrile and the fluorophore. These tool compounds 6 and 7 were characterized by kinetic investigations as covalent reversible inhibitors of human cathepsins L, S, K and B. Probe 6 showed a pronounced inhibitory activity against cathepsins K and S, which was corroborated by modeling of inhibition modes. Probe 7 was highly potent (Ki = 93 nM) and selective for cathepsin S. To examine the ability of both probes to enter living cells, human embryonic kidney 293 cells were targeted. At a concentration of 10 µM, cellular uptake of probe 6 was demonstrated by fluorescence measurement after an incubation time of 30 min and 3 h, respectively. The probe's concentration in cell lysates was ascertained on the basis of the emission at 492 nm upon excitation at 450 nm, and the results were expressed as concentrations of probe 6 relative to the protein concentration originating from the lysate. After incubation of 10 µM of probe 6 for 3 h, the cellular uptake was confirmed by fluorescence microscopy. HPLC was used to assess the probes' lipophilicity, and the obtained

Active Site Mapping of Human Cathepsin†F with Dipeptide Nitrile Inhibitors.

Cleavage of the invariant chain is the key event in the trafficking pathway of major histocompatibility complex class II. Cathepsin S is the major processing enzyme of the invariant chain, but cathepsin F acts in macrophages as its functional synergist which is as potent as cathepsin S in invariant chain cleavage. Dedicated low-molecular-weight inhibitors for cathepsin F have not yet been developed. An active site mapping with 52 dipeptide nitriles, reacting as covalent-reversible inhibitors, was performed to draw structure-activity relationships for the non-primed binding region of human cathepsin F. In a stepwise process, new compounds with optimized fragment combinations were designed and synthesized. These dipeptide nitriles were evaluated on human cysteine cathepsins F, B, L, K and S. Compounds 10 (N-(4-phenylbenzoyl)-leucylglycine nitrile) and 12 (N-(4-phenylbenzoyl)leucylmethionine nitrile) were found to be potent inhibitors of human cathepsin F, with Ki values <10 nM. With all dipeptide nitriles from our study, a 3D activity landscape was generated to visualize structure-activity relationships for this series of cathepsin F inhibitors.

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).

A coumarin-labeled vinyl sulfone as tripeptidomimetic activity-based probe for cysteine cathepsins.

A coumarin-tetrahydroquinoline hydride 8 was synthesized as a chemical tool for fluorescent labeling. The rigidified tricyclic coumarin structure was chosen for its suitable fluorescence properties. The connection of 8 with a vinyl sulfone building block was accomplished by convergent synthesis thereby leading to the coumarin-based, tripeptidomimetic activity-based probe 10, containing a Gly-Phe-Gly motif. Probe 10 was evaluated as inactivator of the therapeutically relevant human cysteine cathepsins S, L, K, and B: it showed particularly strong inactivation of cathepsin S. The detection of recombinant and native cathepsin S was demonstrated by applying 10 to in-gel fluorescence imaging.

An access to aza-Freidinger lactams and E-locked analogs.

Freidinger lactams, possessing a peptide bond configuration locked to Z, are important key elements of conformationally restricted peptidomimetics. In the present work, the C(α)H(i+1) unit has been replaced by N, leading to novel aza-Freidinger lactams. A synthesis to corresponding building blocks and their E-locked analogs is introduced. The versatile buildings blocks reported here are expected to serve as useful elements in peptide synthesis.

Selective nitrile inhibitors to modulate the proteolytic synergism of cathepsins S and F.

A series of dipeptide nitriles with different P3 substituents was designed to explore the S3 binding pocket of cathepsin S. Racemic 7-16 and the enantiopure derivative (R)-22 proved to be potent inhibitors of human cathepsin S and exhibited notable selectivity over human cathepsins L, K, and B. Inhibition of cathepsin F, the functional synergist of cathepsin S, was not observed. The azadipeptide analogue of 22, compound 26, was highly potent but nonselective.