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

The cathepsins comprise a family of lysosomal proteolytic enzymes. Eleven human cathepsins are cysteine proteases of the papain-like subfamily C1A and represent the best characterized group of cathepsins. Cysteine cathepsins are processed to active enzymes during maturation. The final activation of the proenzymes proceeds in the late endosomes either auto- catalytically or catalyzed by other lysosomal proteases. The majority of cysteine cathepsins act as endopeptidases, mainly to degrade proteins that have entered the lysosomal system. Nontraditional roles for cathepsins in the extracellular space as well as in the cytosol and nucleus have also been uncovered. The cleavage of the peptide bond, catalyzed by cysteine cathepsins, occurs as a two-step acyl transfer. It is initiated by the nucleophilic attack of the active site cysteine leading to the release of the first product. In the subsequent step, the thiolester bond of the acyl enzyme is hydrolyzed and the second product is formed.

Cysteine cathepsin-catalyzed proteolytic cleavage plays a critical role in normal cellular functions as well as pathophy-siological events such as osteoporosis, rheumatoid arthritis and osteoarthritis, inflammation, cancer, neurological disorders, multiple sclerosis, pancreatitis, diabetes, and lysosomal storage diseases. For example, cathepsin K, the primary enzyme involved in osteoclastic bone resorption, is generally accepted as an important target for the treatment of osteoporosis. This cathepsin, when secreted by osteoclasts, is capable to degrade several components of the bone matrix, including type I collagen, the main constituent of the organic bone matrix, as well as osteopontin and osteonectin. However, degradation of collagen mediated by cathepsin K also takes place in the lysosomal vesicles within the osteoclasts, and lysosomal collagen degradation was shown to be inhibited by E64d, a membrane-permeable cysteine protease inhibitor. Cathepsins L and B have been demonstrated to play an essential role in the development and progression of cancer. They are involved in the degradation of extracellular matrix proteins, a process promoting tumor angiogenesis, invasion and metastasis of tumor cells. Cathepsin S is crucial in the MHC class II antigen presentation pathway. It represents the major degrading enzyme of the MHC class II invariant chain necessary for loading of antigenic peptides and subsequent antigen presentation by specialized antigen-presenting cell types, such as B cells, macrophages and dendritic cells. Due to the involvement in pathophysiological processes, cysteine cathepsins are well established as targets for drug development. For cathepsins K and S in particular, inhibition of the intracellular activity is desired. Thus, low-molecular weight inhibitors should be capable of entering cells to address their target cathepsins. In this study, two fluorescently labeled prototypic dipeptide nitrile inhibitors for cysteine cathepsins have been designed, their inhibitory profile characterized and their cellular uptake investigated.

Peptidomimetic inhibitors of cysteine cathepsins as well as activity-based probes can be designed by replacing the scissile peptide bond of a peptidic substrate by an electrophilic warhead structure, such as a halomethyl, diazomethyl and acyloxymethyl ketone, an epoxide or aziridine group, a vinyl sulfone or another Michael acceptor, as well as a β-lactam or a cyclopropenone structure. In particular, epoxide derivatives and acyloxymethyl ketones have been successfully devolved to activity-based probes for cysteine cathepsins. The peptidic part of such covalent modifiers accounts for sufficient affinity to be accommodated in the active site of the target protease due to specific non-covalent interactions mainly with the S1–S4 binding pockets. The practically irreversible mode of inactivation results from the attack of the active site cysteine nucleophile at an electrophilic carbon of the warhead. Other types of inhibitors without adjacent leaving groups, such as peptide aldehydes, ketones or nitriles inhibit cysteine proteases as they can sequester the active site cysteine via the reversible formation of thiohemiacetal, thiohemiketal or thio- imidate intermediates, respectively. The peptide nitriles have attracted particular attention in the course of inhibitor development for cysteine proteases. Peptide nitriles have been developed against several human cathepsins and, by utilizing the interactions of the cathepsins’ specificity pockets with the corresponding residues of the inhibitors, strong selectivity has been achieved for the target cathepsin.

Peptide nitriles represent promising drug candidates. For example, the cathepsin K inhibitor odanacatib is currently being developed for the treatment of osteoporosis. The basic compound balicatib, a further potent inhibitor of cathepsin K, possesses lysosomotropic properties leading to a reduced selectivity in cell-based assays. Both odanacatib and balicatib exhibit the characteristic structural features of low-molecular-weight inhibitors of cysteine cathepsins. In cathepsin K, the S2 pocket is hydrophobic and relatively small which is consistent with the preference for a leucine side chain or a cyclohexane moiety. The S3 pocket of cathepsin K is large and shallow. An extended P3 aryl substituent directly attached to the amide carbonyl (as in 2 and 5) was shown to improve potency, e.g. when replacing the simple Cbz group. Strong affinity was obtained with functionalized biaryl groups or with a piperazine directly attached to the phenyl group. During the development of odanacatib, metabolic liabilities have been minimized. Thus, one amide bond was replaced by the isosteric, non-basic F3C-CH-NH motif providing an increase in potency and selectivity, and the 1,1-cyclopropane ring was introduced. Recently, the activated cathepsin K pool in resorbing osteoclasts was localized using an analogous inhibitor of odanacatib in which the SO2Me group was replaced by a linker-connected BODIPY fluorophore. The accessibility of cathepsin K in intracellular vesicles and in resorption lacunae was demonstrated with this inhibitor.



Franziska Kohl, Janina Schmitz, Michael Gütschow*. Org. Biomol. Chem., 2015, 13,10310–10323


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