ABSTRACT
The effect of incorporating alpha,alpha'-diethylglycine and alpha-aminocyclopentane carboxylic acid at the P(2) position of inhibitors on mu-calpain inhibition was studied. Compound 3 with alpha,alpha'-diethylglycine was over 20-fold more potent than 2 with alpha-aminocyclopentane carboxylic acid. Additionally, 3 was over 35-fold selective for mu-calpain compared to cathepsin B, while 2 was 3-fold selective for cathepsin B compared to mu-calpain. Thus, the conformation induced by the P(2) residue influenced the activities of the compounds versus the closely related cysteine proteases, and suggests an approach to the discovery of selective mu-calpain inhibitors.
Subject(s)
Amino Acids/chemistry , Biomimetic Materials/chemical synthesis , Calpain/antagonists & inhibitors , Cysteine Proteinase Inhibitors/chemical synthesis , Cysteine Proteinase Inhibitors/pharmacology , Peptides/chemical synthesis , Animals , Biomimetic Materials/chemistry , Biomimetic Materials/pharmacology , Cathepsin B/antagonists & inhibitors , Drug Design , Humans , Peptides/chemistry , Peptides/pharmacology , SwineABSTRACT
Calpain is a cytosolic cysteine endopeptidase that has been implicated in a number of disorders including cancer. We have synthesized and studied the mu-calpain inhibitory activity and cytotoxicity of peptidyl aldehydes and peptidyl alpha-ketoamides with N-substituted D-proline or L-thiaproline residues at the P2-postion. The most potent and most selective members of the series were (R)-1-(4-nitrophenylsulfonyl)-N-((R,S)-1-oxo-3-phenylpropan-2-yl)pyrrolidine-2-carboxamide (1j) and (R)-1-(4-iodophenylsulfonyl)-N-((R,S)-1-oxo-3-phenylpropan-2-yl)pyrrolidine-2-carboxamide (1n). The compounds inhibited mu-calpain with Ki values of 0.02 microM and 0.03 microM, respectively, and displayed over 180-fold (1j) and 130-fold (1n) greater affinity for mu-calpain compared to cathepsin B. The cytotoxic effect of the compounds was evaluated in two leukemia cell lines (Daudi and Jurkat) and three solid tumor cell lines (DU-145, PC-3, and HeLa). Generally the compounds were modestly cytotoxic and displayed no correlation between the cytotoxic activity and mu-calpain inhibition.
Subject(s)
Aldehydes/pharmacology , Calpain/antagonists & inhibitors , Proline/chemistry , Pyrrolidinones/pharmacology , Sulfones/pharmacology , Thiazoles/chemistry , Aldehydes/chemical synthesis , Aldehydes/chemistry , Cathepsin B/antagonists & inhibitors , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Screening Assays, Antitumor , Enzyme Activation/drug effects , HeLa Cells , Humans , Molecular Structure , Pyrrolidinones/chemistry , Stereoisomerism , Structure-Activity Relationship , Sulfones/chemistry , ThiazolidinesABSTRACT
Calpain is a class of Ca(2+)-dependent cysteine proteases and has been suggested to be involved in several important signaling cascades. A series of novel aldehyde calpain inhibitors identified in our laboratory were more potent and specific than commercially available calpain inhibitors, and were used to assess the involvement of calpain in cancer. Our inhibitors demonstrated potent anti-proliferative activity in four cancer cell lines (PC-3, HeLa, Jurkat and Daudi) with IC(50)'s ranging from 2 to >30 microM. A non-cancer cell line (CV-1) was 4-7-fold less sensitive than the cancer cell lines. Apoptotic activity was determined and appeared to be inversely correlated to calpain expression levels in the different cell types. Leukemia cell lines (i.e., Daudi and Jurkat) with undetectable m-calpain were more susceptible to the apoptotic effects in response to calpain inhibition, while apoptosis was not detected in PC-3 prostate cancer cells, which highly express m-calpain. The extent of apoptosis in HeLa cells was moderate under identical conditions. Apoptosis induced by calpain inhibition was accompanied by caspase-3 activation. Furthermore, cell cycle analysis showed that aldehyde calpain inhibitors arrested cells at the G2/M boundary in a concentration-dependent manner. These results indicate that aldehyde calpain inhibitors exhibit their cytotoxic effects via induction of G2/M arrest and apoptosis. Importantly, the compounds failed to exert any inhibitory effects toward 20S proteasome. Collectively, our results suggest that calpain is a novel target for the treatment of a variety of cancer diseases and provide leads for further discovery and development of calpain inhibitors.
Subject(s)
Aldehydes/pharmacology , Apoptosis/drug effects , Calpain/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , Prostatic Neoplasms/metabolism , Caspase 3 , Caspases/metabolism , Cell Division/drug effects , Cell Line, Tumor , Enzyme Activation/drug effects , G2 Phase/drug effects , HeLa Cells/drug effects , HeLa Cells/metabolism , Humans , Jurkat Cells/drug effects , Jurkat Cells/metabolism , Male , Prostatic Neoplasms/enzymology , Prostatic Neoplasms/genetics , Proteasome Endopeptidase Complex/metabolism , Proteasome InhibitorsABSTRACT
Four new peptidyl aldehydes bearing proline mimetics at the P(2)-position were synthesized and studied as inhibitors of calpain I, cathepsin B, and selected serine proteases. The ring size of the P(2)-constraining residue influenced the inhibitory potency and selectivity of the compounds for calpain I compared to the other proteases.
