Peptide .alpha.-keto ester, .alpha.-keto amide, and .alpha.-keto acid inhibitors of calpains and other cysteine proteases

Abstract

A series of dipeptidyl and tripeptidyl alpha-keto esters, alpha-keto amides, and alpha-keto acids having leucine in the P2 position were synthesized and evaluated as inhibitors for the cysteine proteases calpain I, calpain II, cathepsin B, and papain. In general, peptidyl alpha-keto acids were more inhibitory toward calpain I and II than alpha-keto amides, which in turn were more effective than alpha-keto esters. In the series Z-Leu-AA-COOEt, the inhibitory potency decreased in the order: Met (lowest KI) > Nva > Phe > 4-Cl-Phe > Abu > Nle (highest KI) with calpain I, while almost the reverse order was observed for calpain II. Extending the dipeptide alpha-keto ester to a tripeptide alpha-keto ester yielded significant enhancement in the inhibitory potency toward cathepsin B, but smaller changes toward the calpains. Changing the ester group in the alpha-keto esters did not substantially decrease KI values for calpain I and calpain II. N-Monosubstituted alpha-keto amides were better inhibitors than the corresponding alpha-keto esters. alpha-Keto amides with hydrophobic alkyl groups or alkyl groups with an attached phenyl group had the lower KI values. N,N-Disubstituted alpha-keto amides were much less potent inhibitors than the corresponding N-monosubstituted peptide alpha-keto amides. The peptide alpha-keto acid Z-Leu-Phe-COOH was the best inhibitor for calpain I (KI = 0.0085 microM) and calpain II (KI = 0.0057 microM) discovered in this study. It is likely that the inhibitors are transition-state analogs and form tetrahedral adducts with the active site cysteine of cysteine proteases and form hydrogen bonds with the active site histidine and possibly another hydrogen bond donor in the case of monosubstituted amides. Several inhibitors prevented spectrin degradation in a platelet membrane permeability assay and may be useful for the treatment of diseases which involve neurodegeneration.

Cytoprotective Properties of Novel Nonpeptide Calpain Inhibitors in Renal Cells

“…Using purified calpains, the IC 50 values of these two SJA compounds are approximately 0.1 M, in the IC 50 range (0.0057-1.8 M) for many peptidyl catalytic site-directed calpain inhibitors (Li et al, 1993(Li et al, , 1996Harriman et al, 2001). The presence of a methoxy group on the phenyl ring of SJA7019 increases its potency approximately 2-fold compared with SJA7029 using purified calpains.…”

“…For example, calpain inhibitor 1 (N-acetyl-Leu-Leu-norleucinal) is a tripeptidyl aldehyde. A series of peptide ␣-keto amide inhibitors of calpains have been reported (Li et al, 1993(Li et al, , 1996, and the properties of these inhibitors were tested using intact RPTs and a synthetic calpain substrate SLLVY-7-amino-4-methylcoumarin (Harriman et al, 2000). No clear correlation was obtained between the in vitro inhibitory constants of -or m-calpain and cytoprotection, therefore leaving the role of each calpain isozyme in acute renal cell injury undetermined (Harriman et al, 2000).…”

“…No clear correlation was obtained between the in vitro inhibitory constants of -or m-calpain and cytoprotection, therefore leaving the role of each calpain isozyme in acute renal cell injury undetermined (Harriman et al, 2000). In addition, many of the catalytic site-directed peptidyl calpain inhibitors lack intracellular specificity, plasma membrane permeability, and/or potency (Li et al, 1993(Li et al, , 1996Wang et al, 1996;Mellgren, 1997;Sorimachi et al, 1997;Harriman et al, 2000). In contrast, the compound 3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid (PD150606) represents a nonpeptide Ca 2ϩ -binding site-directed calpain inhibitor and has been shown to block RPT cell death following exposure to diverse insults (Edelstein et al, 1996;Waters et al, 1997;Liu et al, 2001).…”

Optimization of peptidyl allyl sulfones as clan CA cysteine protease inhibitors

“…To date, a number of cysteine protease inhibitors have been developed using a variety of reactive functional groups 31 , also referred to as "warheads, " including aldehydes 20 , fluoromethyl ketones 21 , nitriles 32 , α-ketoesters, amides, and acids 33 , tetrafluorophenoxymethyl ketones 34 , epoxysuccinates 35 , thiosemicarbazones 36 , hydrazone derivatives 37,38 , vinyl sulfones 39 and allyl sulfones 40 . These warheads are attached to either a peptidyl or peptidomimetic scaffold 16,41 .…”

QSAR studies on peptide α-ketoamides and α-ketohydroxamate derivatives as calpain I inhibitors

“…These inhibitors permanently inactivate calpain by displacing the active site cysteine thiol to form a sulfide. Reversible inhibitors include peptidyl aldehydes [13,14] and activated ketones [15,16] like a-ketoesters, a-keto-acids, and a-keto-amides. These inhibitors inactivate calpain in a transient manner by forming a reversible covalent bond (hemithioacetal or hemithioketal) with the cysteine thiol [17].…”