title: Probing of primed and unprimed sites of calpains: Design, synthesis and evaluation of epoxysuccinyl-peptide derivatives as selective inhibitors.
abstract: Calpains are intracellular cysteine proteases with important physiological functions. Up- or downregulation of their expression can be responsible for several diseases, therefore specific calpain inhibitors may be considered as promising candidates for drug discovery. In this paper we describe the synthesis and characterization of a new class of inhibitors derived from the analysis of amino acid preferences in primed and unprimed sites of calpains by incorporation of l- or d-epoxysuccinyl group (Eps). Amino acids for replacement were chosen by considering the substrate preference of calpain 1 and 2 enzymes. The compounds were characterized by RP-HPLC, amino acid analysis and ESI-MS. Selectivity of the compounds was studied by using calpain 1 and 2; and cathepsin B. We have identified five calpain specific inhibitors with different extent of selectivity. Two of these also exhibited isoform selectivity. Compound NH2-Thr-Pro-Leu-(d-Eps)-Thr-Pro-Pro-Pro-Ser-NH2 proved to be a calpain 2 enzyme inhibitor with at least 11.8-fold selectivity, while compound NH2-Thr-Pro-Leu-(l-Eps)-Ser-Pro-Pro-Pro-Ser-NH2 possesses calpain 1 enzyme inhibition with at least 4-fold selectivity. The results of molecular modeling calculations suggest that the orientation of the bound inhibitor in the substrate binding cleft is markedly dependent on the stereochemistry of the epoxysuccinyl group.
authors.affiliation: MTA-ELTE Research Group of Peptide Chemistry, Hungarian Academy of Sciences (HAS), Eötvös Loránd University (ELTE), Budapest, Hungary.
authors.forename: Levente E
authors.initials: LE
authors.lastname: Dókus
authors.affiliation: Laboratory of Structural Chemistry and Biology and MTA-ELTE Protein Modeling Group, Eötvös Loránd University, Budapest, Hungary.
authors.forename: Dóra K
authors.initials: DK
authors.lastname: Menyhárd
authors.affiliation: Institute of Enzymology, Research Centre for Natural Sciences of the Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Ágnes
authors.initials: Á
authors.lastname: Tantos
authors.affiliation: MTA-ELTE Research Group of Peptide Chemistry, Hungarian Academy of Sciences (HAS), Eötvös Loránd University (ELTE), Budapest, Hungary; Department of Organic Chemistry, Eötvös L. University, Budapest, Hungary.
authors.forename: Ferenc
authors.initials: F
authors.lastname: Hudecz
authors.affiliation: MTA-ELTE Research Group of Peptide Chemistry, Hungarian Academy of Sciences (HAS), Eötvös Loránd University (ELTE), Budapest, Hungary. Electronic address: banoczi@elte.hu.
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Bánóczi
mesh_terms: D002154:Calpain / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002401:Cathepsin B / Q000037:antagonists & inhibitors / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D004852:Epoxy Compounds / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D008958:Models, Molecular; D008968:Molecular Conformation; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D013329:Structure-Activity Relationship
chemical_list: D004852:Epoxy Compounds; D009842:Oligopeptides; D011480:Protease Inhibitors; D002154:Calpain; C533912:CTSB protein, human; D002401:Cathepsin B
keywords: Calpain; Enzyme inhibitor; Epoxysuccinic acid; Epoxysuccinyl-peptide