Peptidyl beta-homo-aspartals (3-amino-4-carboxybutyraldehydes) new specific inhibitors of caspases

Interleukin-IP (IL-1 beta)-converting enzyme (ICE, caspase-1) processes the IL-IP precursor to mature inflammatory cytokine IL-1 beta. ICE has been id entified as a unique cysteine protease, which cleaves Asp-X bonds, shows re sistance to E-64 (an inhibitor of most cysteine proteases) and has a primar y structure that is homologous to CED-3, a protein required for apoptosis ( programmed cell death) in the nematode Caenorhabditis elegans, and to mamma lian cysteine proteases that initiate and execute apoptosis, e.g., apopain/ CPP32/caspase-3. The inhibitors of the ICE/CED-3 family or caspases, as the y are called recently, may constitute therapeutic agents for amelioration o f inflammatory and apoptosis-associated diseases. The mast efficient ICE in hibitors are peptide aldehydes and peptidyl chloro or (acyloxy)methanes. A recent study revealed that both D- and L-Asp are accepted by ICE at the P-1 of such inhibitors, and the peptidyl (acyloxy)methane analogues having the beta-homo-aspartyl residue [-NH-CH(CH2COOH)-CH2CO-] are inactive. These fi ndings we reexamined in terms of two issues. (a) ICE's resistance to E-64. Since it was thought to be caused by the enzyme's unique substrate specific ity, we prepared substrate-based analogues, which were not inhibitory sugge sting significant structural difference between the active centers of ICE a nd papain-like enzymes. (b) Tolerance for D-stereochemistry at the P-1 of t hese inhibitors. In view of the mechanism of cysteine protease inhibition b y peptidyl X-methanes, we thought that this phenomenon should be a general characteristic of cysteine proteases and the hAsp-containing analogues shou ld behave as reversible inhibitors. Here, we analyzed the inhibition of ICE and apopain in comparison with that of papain, thrombin, and trypsin by pe ptide L/D-alpha-aldehydes and their L-beta-homo-aldehyde [-NH-CH(R)-CH2-CHO ] analogues. The following results were found. (I) The peptidyl L-beta-homo -aspartals are potent inhibitors for caspases. (2) The L-beta-homo analogue s of peptide aldehyde inhibitors designed for other proteases are not inhib itory. (3) Unlike trypsin and thrombin (serine proteases), papain (cysteine protease) shows tolerance for D-stereochemistry at the P-1 site of peptide aldehydes in proportion to the lability of the alpha-hydrogen of the the P -1-D-residue. The complete tolerance of ICE for P-1-D-Asp may arise from th is residue's high tendency to epimerization. (4) Reaction of cysteine prote ases with peptide aldehyde or peptidyl X-methane inhibitors containing P-1- D-residues may include alpha-proton abstraction followed by asymmetric indi ction leading to P-1-L-residue-containing products. (C) 1999 John Wiley & S ons, Inc.