Potent and selective inactivation of cysteine proteinases with N-peptidyl-O-acyl hydroxylamines (original) (raw)
Related papers
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.
Potent Inactivation of Cathepsins S and L by Peptidyl (Acyloxy)methyl Ketones
Biological Chemistry Hoppe-Seyler, 1994
Peptidyl (acyloxy)methyl ketones (Z-Aa-Aa-CH 2-O-CO-R), a new class of irreversible inhibitors whose chemical reactivity can be modulated by varying the substitution pattern of the carboxylate leaving group, are shown to be extremely potent inactivators of the lysosomal cysteine proteinases cathepsin L and cathepsin S. The highest /c 2 /Kj values measured were found to exceed 10 6 M" 1 s~1 for both cathepsin L and cathepsin S. The rate of inactivation can be controlled by varying the dipeptidyl moiety or the carboxylate leaving group, with the second-order rate constants for both enzymes found to be strongly dependent on the p K a values of the leaving group. The specificities of the cathepsins S and L reveal a different selectivity towards the nature of substitution of the aryl P leaving group of the inhibitor. This new inhibitor class opens the possibility of the design of selective and specific inhibitors for lysosomal cysteine proteinases.
Synthesis of Peptidyl Ene Diones: Selective Inactivators of the Cysteine Proteinases
Chemical Biology & Drug Design, 2007
A series of synthetic peptides in which the C-terminal carboxyl grouping (-CO 2 H) of each has been chemically converted into a variety of ene dione derivatives (-CO-CH¼CH-CO-X; X ¼ -H, -Me, -OBut, -OEt, -OMe, -CO-OMe), have been prepared and tested as inactivators against typical members of the serine and cysteine protease families. For example, the sequences Cbz-Pro-Phe-CH¼CH-CO-OEt (I) which fulfils the known primary and secondary specificity requirements of the serine protease chymotrypsin, and Cbz-Phe-Ala-CH¼CH-CO-OEt (II) which represents a general recognition sequence for cysteine proteases such as cathepsins B, L and S, have been tested as putative irreversible inactivators of their respective target proteases. It was found that, whereas II, for example, functioned as a time-dependent, irreversible inactivator of each of the cysteine proteases, I behaved only as a modest competitive reversible inhibitor of chymotrypsin. Within the simple ester sequences Cbz-Phe-Ala-CH¼CH-CO-R, the rank order of inhibitor effectiveness decreases in the order R ¼ -OMe > -OEt >> -OBut. It was also found that the presence of both an unsaturated double bond and an ester (or a-keto ester) moiety were indispensable for obtaining irreversible inactivators. Of the irreversible inactivators synthesized, Cbz-Phe-Ala-CH¼CH-CO-CO-OEt (which contains a highly electrophilic a-keto ester grouping) was found to be the most effective exhibiting, for example, second-order rate constants of approximately 1.7 · 10 6 M )1 min )1 and approximately 4.9 · 10 4 M )1 min )1 against recombinant human cathepsin S and human spleenic cathepsin B, respectively. This initial study thus holds out the promise that this class of inactivator may well be specific for the cysteine protease subclass.
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.
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
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).
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.
Archives of Biochemistry and Biophysics, 1992
Proteolytically active complexes of the proteinase cathepsin L, with an endogenous inhibitor of cysteine proteinases, were purified from sheep liver. The complexes were active against the synthetic substrate Z-Phe-Arg-NHMec and also the proteins axocasein and gelatin. The composition of the complexes was demonstrated by Western blotting, after reducing and nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis with monospecific antibodies raised against purified sheep liver cathepsin L and purified sheep liver cysteine proteinase inhibitor (probably stefin B). Similar complexes could be formed in vitro, by coincubation of purified sheep liver cathepsin L with the purified sheep liver cystatin at a pH of 5.5 or higher. 0 ISSZ Academic PMS, IEW.