Klára Németh

Peptidyl β-homo-aspartals: Specific inhibitors of interleukin-1β converting enzyme and its homologues (caspases)

Abstract Inhibition of interleukin-1β converting enzyme (ICE), apopain, papain, thrombin and trypsin with substrate like peptidyl L- and D-α-aldehydes and their L-β-homo-aldehyde analogues was investigated. The L-β-homo-aspartals appear to be specific inhibitors for ICE and its homologues; the other enzymes were not inhibited with such L-β-homo aldehydes. Papain shows tolerance for D-residues at P1 depending on their chiral stability.

Peptidyl β‐homo‐aspartals (3‐amino‐4‐carboxybutyraldehydes): New specific inhibitors of caspases

Interleukin-1 beta (IL-1 beta)-converting enzyme (ICE, caspase-1) processes the IL-1 beta precursor to mature inflammatory cytokine IL-1 beta. ICE has been identified as a unique cysteine protease, which cleaves Asp-X bonds, shows resistance to E-64 (an inhibitor of most cysteine proteases) and has a primary structure that is homologous to CED-3, a protein required for apoptosis (programmed cell death) in the nematode Caenorhabditis elegans, and to mammalian cysteine proteases that initiate and execute apoptosis, e.g., apopain/CPP32/caspase-3. The inhibitors of the ICE/CED-3 family or caspases, as they are called recently, may constitute therapeutic agents for amelioration of inflammatory and apoptosis-associated diseases. The most efficient ICE inhibitors 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 P1 of such inhibitors, and the peptidyl (acyloxy)methane analogues having the beta-homo-aspartyl residue [-NH-CH(CH2COOH)-CH2CO-] are inactive. These findings we reexamined in terms of two issues. (a) ICEs resistance to E-64. Since it was thought to be caused by the enzymes unique substrate specificity, we prepared substrate-based analogues, which were not inhibitory suggesting significant structural difference between the active centers of ICE and papain-like enzymes. (b) Tolerance for D-stereochemistry at the P1 of these inhibitors. In view of the mechanism of cysteine protease inhibition by peptidyl X-methanes, we thought that this phenomenon should be a general characteristic of cysteine proteases and the hAsp-containing analogues should behave as reversible inhibitors. Here, we analyzed the inhibition of ICE and apopain in comparison with that of papain, thrombin, and trypsin by peptide L/D-alpha-aldehydes and their L-beta-homo-aldehyde [-NH-CH(R)-CH2-CHO] analogues. The following results were found. (1) The peptidyl L-beta-homo-aspartals are potent inhibitors for caspases. (2) The L-beta-homo analogues of peptide aldehyde inhibitors designed for other proteases are not inhibitory. (3) Unlike trypsin and thrombin (serine proteases), papain (cysteine protease) shows tolerance for D-stereochemistry at the P1 site of peptide aldehydes in proportion to the lability of the alpha-hydrogen of the P1-D-residue. The complete tolerance of ICE for P1-D-Asp may arise from this residues high tendency to epimerization. (4) Reaction of cysteine proteases with peptide aldehyde or peptidyl X-methane inhibitors containing P1-D-residues may include alpha-proton abstraction followed by asymmetric induction leading to P1-L-residue-containing products.

Effect of peptide aldehydes with IL-1β converting enzyme inhibitory properties on IL-1α and IL-1β production in vitro

Tripeptide and pentapeptide aldehydes as substrate-base inhibitors of cysteine proteases were designed in our laboratory for the inhibition of interleukin-1 beta converting enzyme (ICE), a recently described cysteine protease responsible for the processing of IL-1 beta. The biological effectivity of the peptide aldehydes was studied in THP-1 cells and human whole blood. The released and cell-associated IL-1 alpha and IL-1 beta levels were determined by ELISA from the supernatants and cell lysates, respectively. The total IL-1 like bioactivity was assayed by the D10 G4.1 cell proliferation method. The tripeptide aldehyde (Z-Val-His-Asp-H) and pentapeptide aldehyde (Eoc-Ala-Tyr-Val-Ala-Asp-H) significantly reduced IL-1 beta levels in the supernatants in relatively high concentrations (10-100 microM), but the IL-1 alpha release was unaffected by these peptides. However, a considerable decrease in the cell-associated IL-1 beta and IL-1 alpha levels was observed. N-terminal extension of the tripeptide aldehyde yielded even more potent inhibitors. Amino acid substitution at the P2 position did not cause considerable changes in the inhibitory activity. The peptide aldehydes suppressed the IL-1 beta production in a reversible manner, whereas dexamethasone, a glucocorticoid, had a prolonged inhibitory effect. The inhibitory effect of these peptides and that of dexamethasone appeared to be additive. These findings indicate that these peptide aldehydes might be used as IL-beta inhibitory agents in experimental models in which IL-1 beta is a key mediator or ICE is implicated.

Antiapoptotic effect of benzyloxycarbonyl-aspartyl-(β-tertier-butyl ester)-bromomethylketone (z-d(otbu)-bmk), an intermediate of interleukin-lβ converting enzyme inhibitors

Abstract The effect of several interleukin-1β converting enzyme (ICE) inhibitors on apoptosis was examined. The ICE inhibitors tested were peptide aldehydes such as ethyloxycarbonyl-Ala-Tyr-Val-Ala-Asp-aldehyde (Etoco-AYVAD-CHO), acetyl-Tyr-Val-Ala-Asp-aldehyde (Ac-YVAD-CHO), benzyloxycarbonyl-Val-His-Asp-aldehyde (Z-VHD-CHO), a tetrapeptide chloromethylketone, acetyl-Tyr-Val-Ala-Asp-chloromethyl-ketone (Ac-YVAD-Cmk) and their common intermediate benzyloxycarbonyl-Asp-(β-tertier-butyl ester)-bromomethylketone (Z-D(OtBu)-Bmk). Apoptosis was induced with several chemical agents conventionally used for this purpose in THP-1, L929, NB-41A3 cell lines and mouse thymocytes. DNA fragmentation during apoptosis was measured by conventional gel electrophoresis and ELISA. The cell morphology was examined by hematoxylin/eosin staining method. Cell viability was also monitored by MTT assay. Contrary to expectations, the peptide aldehydes listed above and Ac-YVAD-Cmk, known as highly specific ICE inhibitors, did not inhibit the apoptosis of these cell types. However, Z-D(OtBu)-Bmk, which had no relevant inhibitory activity on ICE, potently blocked the DNA fragmentation in THP-1 cells and thymocytes whichever of the inducing agents was used. In the other two cell lines Z-D(OtBu)-Bmk was inactive. The apoptotic cell morphology was also inhibited by Z-D(OtBu)-Bmk. Nevertheless, Z-D(OtBu)-Bmk failed to prevent the loss of mitochondrial activity and the cell destruction in the late phase of apoptosis. These data suggest that ICE is not involved in the apoptotic cell death induced by chemical agents. Thus, Z-D(OtBu)-Bmk, a common intermediate of some ICE inhibitors, may be a useful antiapoptotic agent for studying the early events of apoptosis in some cell types.

Novel peptide inhibitors for interleukin-1β processing in monocytes

Interleukinan inflammatory mediator released by human monocytes, is synthesized as a 269-mer precursor. It is cleaved by a cytoplasmic cysteine protease termed ILconverting enzyme (ICE) at to produce the active cytokine. ICE and its homologues, which are responsible for apoptosis, appear to belong to a new cysteine protease family. Effective ICE inhibitors are peptidyl aspartals (Asp-H) and (acyloxy)methanes [1]. A recent study reported [2] that (i) the analog of the latter having the homo-aspartyl (hAsp) residue, is inactive which indicatesthe key role of Asp CO in recognition; (ii) ICE accepts both Dand LAsp at the of both kinds of inhibitors. It is noted that epimerization occurs slowly at the -Asp and it is not known whether or not ICE catalyzes this process. The mechanism of the inhibition of cysteine proteases by peptidyl halomethanes and (acyloxy)methanes assumes the thiolate can react directly with the (a) as well as the adjacent halomethyl or (acyloxy)methyl group (b) [1]. In path b, one may expect that the thiolate (i) can contact the -proton of the temporarily bound and produce the LAsp-thiomethane product via proton abstraction and asymmetric induction, and (ii) can also attack the CO of hAsp that results in reversible inhibition; lack of such interaction with the -hAsp analog studied may be due to steric hindrance. Starting from these findings and speculations we compared the inhibiting activity of some peptide i-homoaldehydes to their parent -aldehydes against ICE, papain, trypsin and thrombin.

Papain has a tolerance for D-stereochemistry at P1 like caspases

Diastereomeric pairs of peptide aldehydes (1-5) and amides (6-7) as well as the all-L substrates (S1-S4) were prepared by conventional synthesis in solution. The -isomers are known from the literature (e.g. 1 and S1 [2], 2 [3], and 5 [4]) or derived from known structures (3 with 6 from S2 and 4 with S3 and 7 from Ac-Gly-Phe-Nle-H [3]). Inhibiting activities were assessed by substrate assays; ICE inhibition was also tested in a bioassay. Inhibition with peptide aldehydes of papain appeared to be substratedependent, like that of serine proteases [4], i.e. values for measured with S2 and S3 were 2.1 and respectively. and were assayed with S3 (Table 1). Substrate activity was assessed by the amount of amine released. In such terms S3 is about a 1.4 times better substrate for papain than S2. Data from Table 1 show that the -containing isomers are more inhibitory but the D:L potency ratios range from 1.7 (ICE) to 597 (trypsin). For papain inhibitors, medium (2 and 3) and quite low (4) values are obtained suggesting that this classic cysteine protease accepts D-aldehydes at in the order of Cys(Et) > Arg > Phe, i.e. in proportion to the lability of -C-H bond of these residues. By contrast, the high ratio found with 5 indicates that serine protease trypsin has no tolerance for D-residues at