A. Matsui

Structure based development of novel specific inhibitors for cathepsin L and cathepsin S in vitro and in vivo

Specific inhibitors for cathepsin L and cathepsin S have been developed with the help of computer‐graphic modeling based on the stereo‐structure. The common fragment, N‐(L‐trans‐carbamoyloxyrane‐2‐carbonyl)‐phenylalanine‐dimethylamide, is required for specific inhibition of cathepsin L. Seven novel inhibitors of the cathepsin L inhibitor Katunuma (CLIK) specifically inhibited cathepsin L at a concentration of 10−7 M in vitro, while almost no inhibition of cathepsins B, C, S and K was observed. Four of the CLIKs are stable, and showed highly selective inhibition for hepatic cathepsin L in vivo. One of the CLIK inhibitors contains an aldehyde group, and specifically inhibits cathepsin S at 10−7 M in vitro.

Study of the functional share of lysosomal cathepsins by the development of specific inhibitors.

To analyze the functional share of individual cathepsins, we developed powerful and specific inhibitors for individual cathepsins using computer graphics of substrate binding pockets based on X-ray crystallography. These new inhibitors were named CLIK group. Epoxy succinate peptide derivatives, CLIK-066, 088, 112, 121, 148, 181, 185 and 187, are typical specific inhibitors for cathepsin L. Aldehyde derivatives CLIK-060 and CLIK-164 showed specific inhibition against cathepsin S and cathepsin K, respectively. We found that pyridoxal phosphate (PLP), a coenzyme form of vitamin B6, inhibits all cathepsins and also new artificially synthesized pyridoxal derivatives, CLIK-071 and -072, in which the phosphate esters of PLP were replaced by propionic acid, exhibited strong inhibition for cathepsins. Furthermore, CLIK-071 was easy to incorporate into cells and showed powerful inhibition for intracellular cathepsins. Using these selective inhibitors, the allotment of individual cathepsin functions in cells has been studied as follows. Cathepsin L and/or K participate in bone resorption based on bone type-1 collagen degradation and the L-type protease inhibitors suppressed the bone resorption. Cathepsins B and S participate in antigen presentations based on antigen processing and invariant chain degradation, respectively. Also cathepsin L participates in cell apoptosis mediated by caspase III activation.

Novel physiological functions of cathepsins B and L on antigen processing and osteoclastic bone resorption

Lysosomal cathepsin B plays an essential role in the processing of ovalbumin as an exogenous antigen to produce the complex between antigenic-peptide and major histocompatibility-complex class II. Administration of cathepsin B inhibitors, E-64, CA-074 and vitamin B6, caused the strong suppression of the Th-2 type immune responses. We found that pyridoxal phosphate (PAP), a coenzyme form of vitamin B6, inhibits the activities of cathepsin B and L in vitro and vitamin B6 administration induces the inhibition of the lysosomal cathepsin activities in vivo. The production of an antigenic epitope (I323-R339) of ovalbumin by antigen presenting cells was suppressed by cathepsin B specific inhibitors. The ovalbumin dependent production of immunoglobulins (IgE and IgG1) and of the corresponding interleukin (IL-4) was suppressed by cathepsin B inhibitors, while the production of IgG2a and interferon (INF-gamma) was increased. The switch of helper T lymphocyte functions from the type-2 to the type-1 may be induced by the cathepsin B inhibition. The experimental bone pit formation, i.e., osteoclastic bone collagen degradation test, induced by parathyroid hormone was markedly suppressed by the administration of pyridoxal, because of the inhibition of cathepsin L type cysteine proteases in bone.

A novel apoptosis cascade mediated by lysosomal lactoferrin and its participation in hepatocyte apoptosis induced by d‐galactosamine

A new apoptosis cascade mediated by lysosomal lactoferrin was found in apoptotic liver induced by d‐galactosamine. Caspase‐3 and lactoferrin were increased in the apoptotic liver cytoplasm and serum transaminases were elevated. Recombinant lactoferrin stimulated procaspase‐3 processing at 10−6–10−7 M to an extent similar to that by granzyme B in vitro. Lactoferrin changed procaspase‐3 structure susceptible to the processing. Synthetic peptide Y679‐K695 in lactoferrin molecule inhibited the processing of procaspase‐3 by lactoferrin. Lactoferrin in lysosomes was decreased and lactoferrin released into cytoplasm was increased quantitatively in d‐galactosamine induced apoptotic liver, and procaspase‐3 in cytoplasm was processed to caspase‐3.