Epoxysuccinyl peptide-derived cathepsin B inhibitors: Modulating membrane permeability by conjugation with the C-terminal heptapeptide segment of penetratin

Besides its physiological role in lysosomal protein breakdown, extralysosomal cathepsin B has recently been implicated in apoptotic cell death. Highly specific irreversible cathepsin B inhibitors that are readily cellpermeant should be useful tools to elucidate the effects of cathepsin B in the cytosol. We have covalently functionalised the poorly cellpermeant epoxysuccinyl based cathepsin B inhibitor [RGlyGlyLeu(2S, 3S)tEpsLeuProOH; R=OMe] with the C-terminal heptapeptide segment of penetratin (R=εAhxArg ArgNleLysTrpLysLysNH(2)). The high inhibitory potency and selectivity for cathepsin B versus cathepsin L of the parent compound was not affected by the conjugation with the penetratin heptapeptide. The conjugate was shown to efficiently penetrate into MCF-7 cells as an active inhibitor, thereby circumventing an intracellular activation step that is required by other inhibitors, such as the prodruglike epoxysuccinyl peptides E64d and CA074Me

Surface Cathepsin B Protects Cytotoxic Lymphocytes from Self-destruction after Degranulation

The granule exocytosis cytotoxicity pathway is the major molecular mechanism for cytotoxic T lymphocyte (CTL) and natural killer (NK) cytotoxicity, but the question of how these cytotoxic lymphocytes avoid self-destruction after secreting perforin has remained unresolved. We show that CTL and NK cells die within a few hours if they are triggered to degranulate in the presence of nontoxic thiol cathepsin protease inhibitors. The potent activity of the impermeant, highly cathepsin B–specific membrane inhibitors CA074 and NS-196 strongly implicates extracellular cathepsin B. CTL suicide in the presence of cathepsin inhibitors requires the granule exocytosis cytotoxicity pathway, as it is normal with CTLs from gld mice, but does not occur in CTLs from perforin knockout mice. Flow cytometry shows that CTLs express low to undetectable levels of cathepsin B on their surface before degranulation, with a substantial rapid increase after T cell receptor triggering. Surface cathepsin B eluted from live CTL after degranulation by calcium chelation is the single chain processed form of active cathepsin B. Degranulated CTLs are surface biotinylated by the cathepsin B–specific affinity reagent NS-196, which exclusively labels immunoreactive cathepsin B. These experiments support a model in which granule-derived surface cathepsin B provides self-protection for degranulating cytotoxic lymphocytes

(2S,3S)-Oxirane-2,3-dicarboxylic acid: A privileged platform for probing human cysteine cathepsins

Schaschke N. (2S,3S)-Oxirane-2,3-dicarboxylic acid: A privileged platform for probing human cysteine cathepsins. JOURNAL OF BIOTECHNOLOGY. 2007;129(2):308-315

Mast cell tryptase beta as a target in allergic inflammation: An evolving story

Sommerhoff CP, Schaschke N. Mast cell tryptase beta as a target in allergic inflammation: An evolving story. CURRENT PHARMACEUTICAL DESIGN. 2007;13(3):313-332

The arginine mimicking beta-amino acid beta(3)hPhe(3-H(2)N-CH(2)) as S1 ligand in cyclotheonamide-based beta-tryptase inhibitors

Janke D, Sommerhoff CP, Schaschke N. The arginine mimicking beta-amino acid beta(3)hPhe(3-H(2)N-CH(2)) as S1 ligand in cyclotheonamide-based beta-tryptase inhibitors. Bioorganic & Medicinal Chemistry. 2011;19(23):7236-7243.beta-Tryptase, a mast-cell specific serine protease with trypsin-like activity, has emerged in the last years as a promising novel therapeutic target in the field of allergic inflammation. Recently, we have developed a potent and selective beta-tryptase inhibitor based on the natural product cyclotheonamide E4 by implementing a basic P3 residue that addresses the determinants of the extended substrate specificity of beta-tryptase. To further improve the affinity/selectivity profile of this lead structure, we have now investigated beta-homo-3-aminomethylphenylalanine as S1 ligand. In contrast to the corresponding beta-homo amino acids derived from lysine or arginine, we demonstrate that this particular basic beta-homo amino acid is a privileged S1 ligand for the development of beta-tryptase inhibitors. Besides affinity, selectivity and reduced basicity, these novel cyclotheonamide E4 analogs show excellent stability in human plasma and serum. (C) 2011 Elsevier Ltd. All rights reserved

Bivalent inhibition of beta-tryptase: Distance scan of neighboring subunits by dibasic inhibitors

Schaschke N, Dominik A, Matschiner G, Sommerhoff CP. Bivalent inhibition of beta-tryptase: Distance scan of neighboring subunits by dibasic inhibitors. BIOORGANIC & MEDICINAL CHEMISTRY LETTERS. 2002;12(6):985-988

Conformational and molecular modeling studies of beta-cyclodextrin-heptagastrin and the third extracellular loop of the cholecystokinin 2 receptor

Giragossian C, Schaschke N, Moroder L, Mierke DF. Conformational and molecular modeling studies of beta-cyclodextrin-heptagastrin and the third extracellular loop of the cholecystokinin 2 receptor. BIOCHEMISTRY. 2004;43(10):2724-2731

E-64 analogues as inhibitors of cathepsin B. On the role of the absolute configuration of the epoxysuccinyl group

Schaschke N, AssfalgMachleidt I, Machleidt W, Turk D, Moroder L. E-64 analogues as inhibitors of cathepsin B. On the role of the absolute configuration of the epoxysuccinyl group. BIOORGANIC & MEDICINAL CHEMISTRY. 1997;5(9):1789-1797

Inhibition of human mu-calpain by conformationally constrained calpastatin peptides

Pfizer J, Assfalg-Machleidt I, Machleidt W, Schaschke N. Inhibition of human mu-calpain by conformationally constrained calpastatin peptides. BIOLOGICAL CHEMISTRY. 2008;389(1):83-90.The 27-mer peptide CP1 B-[1-27] derived from exon 1B of calpastatin stands out among the known inhibitors for mu- and m-calpain due to its high potency and selectivity. By systematical truncation, a 20-mer peptide, CP1B-[4-23], was identified as the core sequence required to maintain the affinity/selectivity profile of CP1B-[1-27]. Starting with this peptide, the turn-like region Glu(10)(i)-Leu(11)(i+1)-Gly(12)(i+2)-Lys(13)(i +3) was investigated. Sequence alignment of subdomains 1B, 2B, 3B and 4B from different mammalians revealed that the amino acid residues in position i+1 and i+2 are almost invariably flanked by oppositely charged residues, pointing towards a turn-like conformation stabilized by salt bridge/H-bond interaction. Accordingly, using different combinations of acidic and basic residues in position i and i+3, a series of conformationally constrained variants of CP1 B-[4-23] were synthesized by macrolactamization utilizing the side chain functionalities of these residues. With the combination of Glu(i)/Dab(i+3), the maximum of conformational rigidity without substantial loss in affinity/selectivity was reached. These results clearly demonstrate that the linear peptide chain corresponding to subdomain 1B reverses its direction in the region Glu(10)-Lys(13) upon binding to mu-calpain, and thereby adopts a loop-like rather than a tight turn conformation at this site