ABSTRACT
Dipeptidyl peptidase I (DPPI, cathepsin C) is a lysosomal cysteine protease that can activate zymogens of several different serine proteases by one step or sequential removal of dipeptides from the N-termini of the pro-protease protein substrates. To find DPPI inhibitors more suitable for cellular applications than diazomethyl ketones, we synthesized three types of inhibitors: dipeptide acyloxymethyl ketones, fluoromethyl ketones, and vinyl sulfones (VS). The acyloxymethyl ketones inhibited DPPI slowly and are moderate inhibitors of cellular DPPI. The fluoromethyl ketones were potent, but the inhibited DPPI regained activity quickly. The dipeptide vinyl sulfones were effective inhibitors for DPPI, but they also inhibited cathepsins B, H, and L weakly. The best inhibitor, Ala-Hph-VS-Ph, had a k2/K(I) of 2,000,000M(-1)s(-1). The vinyl sulfones also inhibited intracellular DPPI, and for this application the more stable inhibitors exhibit better potency. We conclude that vinyl sulfones are promising inhibitors to study the intracellular functions of DPPI.
Subject(s)
Cathepsin C/antagonists & inhibitors , Ketones/chemistry , Sulfones/chemistry , Animals , Cathepsin C/chemistry , Cell Line , Drug Design , Enzyme Activation/drug effects , Enzyme Inhibitors/chemical synthesis , Humans , Ketones/chemical synthesis , Mast Cells/enzymology , Rats , Spleen/enzymology , Sulfones/chemical synthesisABSTRACT
Granzyme M is a member of a family of granule serine proteases that participate in target cell death initiated by cytotoxic lymphocytes. The enzyme is almost exclusively expressed in NK cell types. Granzyme M cleaves at the carboxy side of amino acids with long, hydrophobic side chains like Met, Leu, and Nle. To further study the substrate specificity of the enzyme, a series of peptide thiobenzyl esters was synthesized. The hydrolysis of the substrates with murine and human recombinant forms of granzyme M was observed. The results show that the enzyme has a strong preference for Pro at the P2 position and Ala, Ser, or Asp at the P3 position. These results suggest that the protein residues of the S2 and S3 subsites form important binding interactions that aid in the selection of specific natural substrates for granzyme M. A series of inhibitors was also tested with granzyme M. None of the inhibitors were effective inactivators of granzyme M, including the general serine protease inhibitor, 3,4-dichloroisocoumarin, which is usually a potent inactivator of serine proteases. This suggests that inhibition of granzyme M may be difficult. Also reported for the first time is the method utilized to isolate granzyme M used in this and previous publications. The observations in this paper will be valuable in development of new potent inhibitors for granzyme M as well as assist in determining the biological function of the enzyme.
Subject(s)
Peptides/chemistry , Peptides/metabolism , Serine Endopeptidases/metabolism , Serine Proteinase Inhibitors/pharmacology , Sulfhydryl Compounds/chemistry , Sulfhydryl Compounds/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Binding Sites , Cattle , Cell Line , Chymotrypsin/metabolism , Granzymes , Humans , Hydrolysis , Kinetics , Mice , Peptides/pharmacology , Rats , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Deletion , Serine Endopeptidases/genetics , Serine Endopeptidases/isolation & purification , Serine Proteinase Inhibitors/chemistry , Spodoptera , Substrate Specificity , Sulfhydryl Compounds/pharmacologyABSTRACT
The broadly reactive cysteine protease dipeptidyl peptidase I (DPPI, cathepsin C) is thought to activate all progranzymes (zymogens of lymphocyte serine proteases) to form mature granzymes. We synthesized dipeptide 7-amino-4-methylcoumarin (AMC) substrates containing progranzyme activation sequences and showed that they were efficiently hydrolyzed by DPPI. However, DPPI will not hydrolyze Ile-Ile-AMC, the N-terminal dipeptide sequence found in mature granzymes. Introduction of the nonphysiological homophenylalanine (Hph) residue at P1 resulted in the best substrate Ala-Hph-AMC for DPPI (k(cat)/K(m)=9,000,000M(-1)s(-1)). The charged N-terminal amino group of the substrate was essential and replacement of the NH(2) group with OH or NH(CH(3)) in Gly-Phe-AMC reduced the k(cat)/K(m) value by two to three orders of magnitude. A hydrazide azaglycine analog, NH(2)NHCO-Phe-AMC, was not hydrolyzed at pH 5.5, but underwent slow hydrolysis at lower pHs where the amino group is partially protonated. DPPI also failed to hydrolyze NH(2)COCH(2)-Phe-AMC, where the NH(2) group is unprotonated. The results reported in this paper should be useful in the design of better DPPI inhibitors to block granzyme maturation and granzyme-dependent apoptosis.
