Potent Inactivation of Cathepsins S and L by Peptidyl (Acyloxy)methyl Ketones (original) (raw)

Potent and selective inactivation of cysteine proteinases with N-peptidyl-O-acyl hydroxylamines

The Biochemical journal, 1989

A series of N-peptidyl-O-acyl hydroxylamines was synthesized and tested as inactivators of cysteine proteinases. Depending on the structure of the peptidyl residue of the inhibitors, rapid and complete irreversible inactivation of the lysosomal cathepsins, B, L and S, may be achieved. The most effective inhibitors display second-order rate constants of the inactivation in the range 10(5)-10(6) M-1.s-1. By contrast, the activity of the aminoendopeptidase cathepsin H is only negligibly affected by the N-terminal-protected peptidyl inhibitors.

N-Peptidyl-O-carbamoyl amino acid hydroxamates: Irreversible inhibitors for the study of the S2′ specificity of cysteine proteinases

FEBS Letters, 1993

A series of new inhibitors for cysteine proteinases with the general structure Z-Phe-Gly-NHO-CO-Aa (Aa = amino acids) was synthesized and tested as inhibitors of papain-like enzymes (cathepsins S, L, B and papain). Like N-peptidyl-O-acyl hydroxamates the inhibitors inactivate cysteine proteinases by a sulfenamidation of the active site cysteine residue. The most effective inhibitors display second order-rate constants of inactivation in the range of lo'-lo4 M-'Y'. Since the structure of the N-peptidyl-O-carbamoyl amino acid hydroxamates allows the variation of the leaving group this class of inhibitors was used as a new tool for the evaluation of the S,' specificity of cysteine proteinases.

Design and evaluation of inhibitors for dipeptidyl peptidase I (Cathepsin C)

Archives of Biochemistry and Biophysics, 2004

Dipeptidyl peptidase I (DPPI, cathepsin C) is a lysosomal cysteine protease that can activate zymogens of several different serine proteases by one step or sequential removal of dipeptides from the N-termini of the pro-protease protein substrates. To find DPPI inhibitors more suitable for cellular applications than diazomethyl ketones, we synthesized three types of inhibitors: dipeptide acyloxymethyl ketones, fluoromethyl ketones, and vinyl sulfones (VS). The acyloxymethyl ketones inhibited DPPI slowly and are moderate inhibitors of cellular DPPI. The fluoromethyl ketones were potent, but the inhibited DPPI regained activity quickly. The dipeptide vinyl sulfones were effective inhibitors for DPPI, but they also inhibited cathepsins B, H, and L weakly. The best inhibitor, Ala-Hph-VS-Ph, had a k 2 =K I of 2,000,000 M À1 s À1 . The vinyl sulfones also inhibited intracellular DPPI, and for this application the more stable inhibitors exhibit better potency. We conclude that vinyl sulfones are promising inhibitors to study the intracellular functions of DPPI.

In Search of Selective Inhibitors of Cysteine Protease, Cathepsin K

International Journal of Peptide Research and Therapeutics, 2005

Two potential azapeptide inhibitors of cathepsin K were designed and synthesized. To analyze in detail interactions between these azainhibitors and the investigated cysteine protease, molecular dynamics simulations were performed. For the obtained compounds the equilibrium constants for dissociation of inhibitor -enzyme complex, K i , were determined. The examined azapeptides proved to be not as potent inhibitors of cathepsin K as they were expected to be according to the results of simulations. However, these calculations provide valuable information about probable structures of this type of peptidomimetics in the catalytic pocket of cathepsin K, which could be useful in designing of more selective inhibitors of this cysteine protease.

Peptidyl vinyl sulphones: a new class of potent and selective cysteine protease inhibitors: S2P2 specificity of human cathepsin O2 in comparison with cathepsins S and L

Biochemical Journal, 1996

Peptidyl vinyl sulphones are a novel class of extremely potent and specific cysteine protease inhibitors. They are highly active against the therapeutically important cathepsins O2, S and L. The highest kinact/Ki values exceed 107 M-1·s-1 for cathepsin S and 105 M-1·s-1 for cathepsins O2 and L. To study the primary specificity site of the novel human cathepsin O2 and the effectiveness of this novel class of inhibitors, a series of peptidyl vinyl sulphones with variations in the P2 residue was synthesized. Leucine in the P2 position was proven to be the most effective residue for cathepsin O2 and also for cathepsins S and L. Cathepsins O2 and S share a decreased accessibility towards P2 hydrophobic non-branched residues such as aminohexanoic acid (norleucine), methionine and oxidized methionine, but are distinguished by their different affinity towards phenylalanine in the P2 position. In contrast, cathepsin S accepts a broader range of hydrophobic residues in its S2 subsite than cat...

Peptidyl O-acyl hydroxamates: Potent new inactivators of cathepsin B

Biochemical and Biophysical Research Communications, 1988

Peptidyl O-acyl hydroxamates having appropriate active-site recognition features are very potent time-dependent inhibitors of the cysteine proteinase cathepsin B. The inhibition is irreversible, and the inactivation rate is strongly dependent on peptide structure and correct positioning of the P1 amino acid carbonyl group. Lipophilic O-acyl groups provide the most rapid inactivators, as exemplified by the inhibitor O-mesitoyl N-benzyloxycarbonyl-L-phenylalanyI-L-alanine hydroxamate (kmax/K i = 640,000 M-ls-1). © 1988 Aoad~mi c P ..... ~nc. The lysosomal cysteine proteinase cathepsin B (EC 3.4.22.1) has been implicated in a number of conditions associated with abnormal protein degradation, such as muscular dystrophy (1), bone resorption (2), myocardial infarction (3), malignancy (4), and inflammation (5). In establishing the role of cathepsin B in these and other biological phenomena, studies of the effects of selective cathepsin B inhibitors have been and will continue to be of critical importance. Cathepsin B inhibitors utilized in these investigations include the peptidyl aldehyde leupeptin (6), chloromethyl ketones (7), diazomethyl ketones (8,9), and peptidyl epoxides such as E-64 (10). More recently developed inhibitors include peptidyl monofluoromethyl ketones (11,12) and peptidyl methyl sulfonium salts (13). Of particular note is the progress in the development of the peptidyl epoxide "EST", now in clinical trials for the treatment of Duchenne muscular dystrophy (1,14).

Potency and Selectivity of the Cathepsin L Propeptide as an Inhibitor of Cysteine Proteases †

Biochemistry, 1996

The cathepsin L propeptide (phcl-2) was expressed in Saccharomyces cereVisiae using a human procathepsin L/R-factor fusion construct containing a stop codon at position -1 (the C-terminal amino acid of the proregion). Since the yield after purification was very low, the cathepsin L propeptide was also obtained by an alternate procedure through controlled processing of an inactive mutant of procathepsin L (Cys25Ser/Thr110Ala) expressed in Pichia pastoris, by small amounts of cathepsin L. The peptide resulting from the cleavage of the proenzyme (phcl-1) was then purified by HPLC. The purified propeptides were characterized by N-terminal sequencing and mass spectrometry and correspond to incomplete forms of the proregion (87 and 81 aa for phcl-1 and phcl-2 respectively, compared to 96 aa for the complete cathepsin L propeptide). The two peptides were found to be potent and selective inhibitors of cathepsin L at pH 5.5, with K i values of 0.088 nM for phcl-1 and 0.66 nM for phcl-2. The K i for inhibition of cathepsin S was much higher (44.6 nM with phcl-1), and no inhibition of cathepsin B or papain could be detected at up to 1 µM of the propeptide. The inhibitory activity was also found to be strongly pHdependent. Two synthetic peptides of 75 and 44 aa corresponding to N-terminal truncated versions of the propeptide were also prepared by solid phase synthesis and displayed K i values of 11 nM and 2900 nM, respectively, against cathepsin L. The data obtained for the 4 propeptide derivatives of various lengths indicate that the first 20 residues in the N-terminal region of the propeptide are more important for inhibition than the C-terminal region which contributes little to the overall inhibitory activity. † NRCC Publication No. 39920. Supported by postdoctoral fellowships from the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, Brazil (E.C.), as well as NATO and the Ministère