ABSTRACT
Successive multiple ionic-polymer layers (SMILs) have long since proved their worth in capillary electrophoresis as they ensure stable electroosmotic flow (EOF) and relatively high separation efficiency. Recently, we demonstrated that plotting the plate height (H) against the solute migration velocity (u) enabled a reliable quantitative evaluation of the coating performances in terms of separation efficiency. In this work, various physicochemical and chemical parameters of the SMIL coating were studied and optimized in order to decrease the slope of the ascending part of the H vs u curve, which is known to be controlled by the homogeneity in charge of the coating surface and by the possible residual solute adsorption onto the coating surface. SMILs based on poly(diallyldimethylammonium chloride) (PDADMAC) and poly(sodium styrene sulfonate) (PSS) were formed and the effect of each polyelectrolyte molar mass and of the number of polyelectrolyte layers (up to 21 layers) was studied. The use of polyethylene imine as an anchoring first layer was considered. More polyelectrolyte couples based on PDADMAC, polybrene, PSS, poly(vinyl sulfate), and poly(acrylic acid) were tested. Finally, zwitterionic polymers based on the poly(α-l-lysine) scaffold were synthesized and used as the last layer of SMILs, illustrating their ability to finetune the EOF, while maintaining good separation efficiency.
Subject(s)
Electrophoresis, Capillary , Polyethylenes , Polymers , Quaternary Ammonium Compounds , Polyelectrolytes , Cations , Electrophoresis, Capillary/methods , Proteins/analysis , PolyethyleneimineABSTRACT
The zymogens of the neutrophil serine proteases elastase, proteinase 3, and cathepsin G are converted proteolytically into their pro-inflammatory active forms by the action of cathepsin C. The inhibition of this cysteine protease therefore is an interesting therapeutic approach for the treatment of inflammatory disorders with a high neutrophil burden such as COPD. Based on E-64c-hydrazide as lead structure, we have recently developed a covalently acting cathepsin C inhibitor using a n-butyl residue attached at the amine nitrogen of the hydrazide moiety to efficiently address the deep hydrophobic S2 pocket. To further optimize the affinity and selectivity profile of this inhibitor, the S1'-S2' area was now investigated by a combinatorial approach, showing that Nle-tryptamide is a ligand superior to the initially used Leu-isoamylamide. Using the neutrophil precursor line U937 as a cell culture model, this optimized inhibitor blocks the intracellular cathepsin C activity and thereby suppresses the activation of neutrophil elastase.
Subject(s)
Cathepsin C , Hydrazines , Cathepsin C/metabolism , Hydrazines/pharmacology , Leukocyte Elastase/metabolism , Serine Proteases , LeucineABSTRACT
Taar1 is a G protein-coupled receptor (GPCR) confined to primary cilia of rodent thyroid epithelial cells. Taar1-deficient mouse thyroid follicles feature luminal accumulation of thyroglobulin suggesting that Taar1 acts as a regulator of extra- and pericellular thyroglobulin processing, which is mediated by cysteine cathepsin proteases present at the apical plasma membrane of rodent thyrocytes. Here, by immunostaining and confocal laser scanning microscopy, we demonstrated co-localization of cathepsin L, but only little cathepsin B, with Taar1 at primary cilia of rat thyrocytes, the FRT cells. Because proteases were shown to affect half-lives of certain receptors, we determined the effect of cathepsin activity inhibition on sub-cellular localization of Taar1 in FRT cells, whereupon Taar1 localization altered such that it was retained in compartments of the secretory pathway. Since the same effect on Taar1 localization was observed in both cathepsin B and L inhibitor-treated cells, the interaction of cathepsin activities and sub-cellular localization of Taar1 was thought to be indirect. Indeed, we observed that cathepsin inhibition resulted in a lack of primary cilia from FRT cells. Next, we proved that primary cilia are a necessity for Taar1 trafficking to reach the plasma membrane of FRT cells, since the disruption of primary cilia by treatment with ß-cyclodextrin resulted in Taar1 retention in compartments of the secretory pathway. Furthermore, in less well-polarized rat thyrocytes, namely in FRTL-5â¯cells lacking primary cilia, Taar1 was mainly confined to the compartments of the secretory pathway. We conclude that Taar1 localization in polarized thyroid epithelial cells requires the presence of primary cilia, which is dependent on the proteolytic activity of cysteine cathepsins B and L.
Subject(s)
Cathepsin B/metabolism , Cathepsin L/metabolism , Cilia/drug effects , Endoplasmic Reticulum/metabolism , Receptors, G-Protein-Coupled/metabolism , Thyroid Epithelial Cells/metabolism , Animals , Cathepsin B/antagonists & inhibitors , Cathepsin L/antagonists & inhibitors , Cell Line , Protein Transport/drug effects , Thyroid Epithelial Cells/cytologyABSTRACT
Pancreatitis is associated with premature activation of digestive proteases in the pancreas. The lysosomal hydrolase cathepsin B (CTSB) is a known activator of trypsinogen, and its deletion reduces disease severity in experimental pancreatitis. Here we studied the activation mechanism and subcellular compartment in which CTSB regulates protease activation and cellular injury. Cholecystokinin (CCK) increased the activity of CTSB, cathepsin L, trypsin, chymotrypsin, and caspase 3 in vivo and in vitro and induced redistribution of CTSB to a secretory vesicle-enriched fraction. Neither CTSB protein nor activity redistributed to the cytosol, where the CTSB inhibitors cystatin-B/C were abundantly present. Deletion of CTSB reduced and deletion of cathepsin L increased intracellular trypsin activation. CTSB deletion also abolished CCK-induced caspase 3 activation, apoptosis-inducing factor, as well as X-linked inhibitor of apoptosis protein degradation, but these depended on trypsinogen activation via CTSB. Raising the vesicular pH, but not trypsin inhibition, reduced CTSB activity. Trypsin inhibition did not affect apoptosis in hepatocytes. Deletion of CTSB affected apoptotic but not necrotic acinar cell death. In summary, CTSB in pancreatitis undergoes activation in a secretory, vesicular, and acidic compartment where it activates trypsinogen. Its deletion or inhibition regulates acinar cell apoptosis but not necrosis in two models of pancreatitis. Caspase 3-mediated apoptosis depends on intravesicular trypsinogen activation induced by CTSB, not CTSB activity directly, and this mechanism is pancreas-specific.
Subject(s)
Apoptosis , Cathepsin B/metabolism , Pancreas/enzymology , Pancreatitis/pathology , Peptide Hydrolases/metabolism , Animals , Cathepsin B/antagonists & inhibitors , Enzyme Activation , Mice , Mice, Inbred C57BL , Mice, Knockout , Pancreatitis/enzymology , Subcellular Fractions/enzymologyABSTRACT
Cysteine cathepsins are expressed in most tissues, including the gastrointestinal tract. We demonstrated an involvement of mouse intestinal cathepsin B in extracellular matrix remodeling for regeneration from trauma. The present study aimed at elucidating roles of cysteine cathepsins in the non-traumatized gastrointestinal tract of mice. Thus we investigated expression and localization patterns of cathepsin B and its closest relative, cathepsin X, along the length of the gastrointestinal tract, and determined the effects of their absence. Cathepsin B showed the highest protein levels in the anterior segments of the gastrointestinal tract, whereas the highest activity was observed in the jejunum, as revealed by cathepsin B-specific activity-based probe labeling. Cathepsin X was most abundant in the jejunum and protein levels were elevated in duodenum and colon of Ctsb-/- mice. The segmental pattern of cathepsin expression was reflected by a compartmentalized distribution of junction proteins and basal lamina constituents, changes in tissue architecture and altered activities of the brush border enzyme aminopeptidase N. In conclusion, we observed different compensatory effects and activity levels of cysteine peptidases along the length of the small and large intestines in a segment-specific manner suggesting specific in situ functions of these enzymes in particular parts of the gastrointestinal tract.
Subject(s)
Cathepsin B/metabolism , Cathepsins/genetics , Cathepsins/metabolism , Gastrointestinal Tract/cytology , Gastrointestinal Tract/metabolism , Animals , Cadherins/metabolism , Cathepsin B/genetics , Ileum/cytology , Ileum/metabolism , Jejunum/cytology , Jejunum/metabolism , Mice , Mice, KnockoutABSTRACT
Cathepsinâ C is a papain-like cysteine protease with dipeptidyl aminopeptidase activity that is thought to activate various granule-associated serine proteases. Its exopeptidase activity is structurally explained by the so-called exclusion domain, which blocks the active-site cleft beyond the S2 site and, with its Asp 1 residue, provides an anchoring point for the Nâ terminus of peptide and protein substrates. Here, the hydrazide of (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane (E-64c) (k2/Ki =140±5 M(-1) s(-1)) is demonstrated to be a lead structure for the development of irreversible cathepsinâ C inhibitors. The distal amino group of the hydrazide moiety addresses the acidic Asp 1 residue at the entrance of the S2 pocket by hydrogen bonding while also occupying the flat hydrophobic S1'-S2' area with its leucine-isoamylamide moiety. Furthermore, structure-activity relationship studies revealed that functionalization of this distal amino group with alkyl residues can be used to occupy the conserved hydrophobic S2 pocket. In particular, the n-butyl derivative was identified as the most potent inhibitor of the series (k2/Ki =56 000±1700 M(-1) s(-1)).
Subject(s)
Cathepsin C/antagonists & inhibitors , Leucine/analogs & derivatives , Protease Inhibitors/chemistry , Binding Sites , Catalytic Domain , Cathepsin C/metabolism , Crystallography, X-Ray , Hydrazines/chemistry , Hydrophobic and Hydrophilic Interactions , Kinetics , Leucine/chemical synthesis , Leucine/chemistry , Leucine/metabolism , Protease Inhibitors/chemical synthesis , Protease Inhibitors/metabolism , Structure-Activity RelationshipABSTRACT
BACKGROUND: Miraziridine A, a natural peptide isolated from a marine sponge, is a potent cathepsin B inhibitor with a second-order rate constant of 1.5 × 10(4) M(-1) s(-1). In the present study, miraziridine A was isolated from the Red Sea sponge Theonella swinhoei on the basis of chromatographic and spectrometric techniques. We conclude that T. swinhoei from the Red Sea represents an alternative source of the aziridinylpeptide miraziridine A to the previously identified Theonella mirabilis from Japan. We confirmed that the metabolite is produced by marine sponges from different geographical locations. CONTEXT: Marine sponges have been proven to be a rich source of secondary metabolites exhibiting a huge diversity of biological activities, including antimicrobial, antitumor and immunomodulatory activities. Theonella species (order Lithistida, Demospongiae) have been shown to be a source of anti-protease and anti-HIV secondary metabolites. AIMS: To identify the protease inhibitor mirazirine A in the marine sponge Theonella swinhoei. MATERIAL AND METHODS: The marine sponge Theonella swinhoei was collected by SCUBA diving in the Red Sea in Eilat (Israel). Sponge material was lyophilized and further extracted successively with cyclohexane, dichloromethane and methanol to obtain three crude extracts. LC-MS analysis was performed to confirm the presence of Miraziridine A in the dichloromethane fraction. RESULTS: In the present study, miraziridine A was isolated from the Red Sea sponge T. swinhoei on the basis of chromatographic and spectrophotometric techniques. CONCLUSIONS: We conclude that T. swinhoei from the Red Sea represents an alternative source of the aziridinylpeptide miraziridine A to the previously identified Theonella mirabilis from Japan.
ABSTRACT
Proteolytic activation of the fusion protein of the highly pathogenic Nipah virus (NiV F) is a prerequisite for the production of infectious particles and for virus spread via cell-to-cell fusion. Unlike other paramyxoviral fusion proteins, functional NiV F activation requires endocytosis and pH-dependent cleavage at a monobasic cleavage site by endosomal proteases. Using prototype Vero cells, cathepsin L was previously identified to be a cleavage enzyme. Compared to Vero cells, MDCK cells showed substantially higher F cleavage rates in both NiV-infected and NiV F-transfected cells. Surprisingly, this could not be explained either by an increased F endocytosis rate or by elevated cathepsin L activities. On the contrary, MDCK cells did not display any detectable cathepsin L activity. Though we could confirm cathepsin L to be responsible for F activation in Vero cells, inhibitor studies revealed that in MDCK cells, cathepsin B was required for F-protein cleavage and productive replication of pathogenic NiV. Supporting the idea of an efficient F cleavage in early and recycling endosomes of MDCK cells, endocytosed F proteins and cathepsin B colocalized markedly with the endosomal marker proteins early endosomal antigen 1 (EEA-1), Rab4, and Rab11, while NiV F trafficking through late endosomal compartments was not needed for F activation. In summary, this study shows for the first time that endosomal cathepsin B can play a functional role in the activation of highly pathogenic NiV.
Subject(s)
Cathepsin B/metabolism , Endosomes/enzymology , Henipavirus Infections/enzymology , Henipavirus Infections/virology , Nipah Virus/metabolism , Viral Fusion Proteins/metabolism , Animals , Cathepsin B/genetics , Cathepsin L/genetics , Cathepsin L/metabolism , Cell Line , Dogs , Endocytosis , Endosomes/virology , Henipavirus Infections/genetics , Henipavirus Infections/physiopathology , Humans , Mice , Mice, Knockout , Nipah Virus/genetics , Viral Fusion Proteins/geneticsABSTRACT
Human strains of Staphylococcus aureus secrete two papain-like proteases, staphopain A and B. Avian strains produce another homologous enzyme, staphopain C. Animal studies suggest that staphopains B and C contribute to bacterial virulence, in contrast to staphopain A, which seems to have a virulence unrelated function. Here we present a detailed study of substrate preferences of all three proteases. The specificity of staphopain A, B and C substrate-binding subsites was mapped using different synthetic substrate libraries, inhibitor libraries and a protein substrate combinatorial library. The analysis demonstrated that the most efficiently hydrolyzed sites, using Schechter and Berger nomenclature, comprise a P2-Gly↓Ala(Ser) sequence motif, where P2 distinguishes the specificity of staphopain A (Leu) from that of both staphopains B and C (Phe/Tyr). However, we show that at the same time the overall specificity of staphopains is relaxed, insofar as multiple substrates that diverge from the sequences described above are also efficiently hydrolyzed.
Subject(s)
Bacterial Proteins/metabolism , Cysteine Endopeptidases/metabolism , Cysteine Proteases/metabolism , Staphylococcus aureus , Amino Acid Motifs , Animals , Bacterial Proteins/isolation & purification , Binding, Competitive , Birds , Catalytic Domain , Cysteine Endopeptidases/isolation & purification , Humans , Hydrolysis , Kinetics , Molecular Sequence Data , Protein Binding , Small Molecule Libraries , Staphylococcal Infections/microbiology , Staphylococcus aureus/enzymology , Staphylococcus aureus/pathogenicity , Substrate Specificity , VirulenceABSTRACT
ß-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 ß-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 ß-tryptase. To further improve the affinity/selectivity profile of this lead structure, we have now investigated ß-homo-3-aminomethylphenylalanine as S1 ligand. In contrast to the corresponding ß-homo amino acids derived from lysine or arginine, we demonstrate that this particular basic ß-homo amino acid is a privileged S1 ligand for the development of ß-tryptase inhibitors. Besides affinity, selectivity and reduced basicity, these novel cyclotheonamide E4 analogs show excellent stability in human plasma and serum.
Subject(s)
Arginine/chemistry , Peptides, Cyclic/chemistry , Peptides, Cyclic/pharmacology , Phenylalanine/analogs & derivatives , Serine Proteinase Inhibitors/chemistry , Tryptases/antagonists & inhibitors , Biomimetic Materials/chemistry , Drug Stability , Humans , Ligands , Peptides, Cyclic/blood , Peptides, Cyclic/chemical synthesis , Phenylalanine/chemistry , Serine Proteinase Inhibitors/blood , Serine Proteinase Inhibitors/chemical synthesis , Serine Proteinase Inhibitors/pharmacology , Structure-Activity Relationship , Tryptases/metabolismABSTRACT
The papain-like cysteine cathepsins expressed by Leishmania play a key role in the life cycle of these parasites, turning them into attractive targets for the development of new drugs. We previously demonstrated that two compounds of a series of peptidomimetic aziridine-2,3-dicarboxylate [Azi(OBn)(2)]-based inhibitors, Boc-(S)-Leu-(R)-Pro-(S,S)-Azi(OBn)(2) (compound 13b) and Boc-(R)-Leu-(S)-Pro-(S,S)-Azi(OBn)(2) (compound 13e), reduced the growth and viability of Leishmania major and the infection rate of macrophages while not showing cytotoxicity against host cells. In the present study, we characterized the mode of action of inhibitors 13b and 13e in L. major. Both compounds targeted leishmanial cathepsin B-like cysteine cathepsin cysteine proteinase C, as shown by fluorescence proteinase activity assays and active-site labeling with biotin-tagged inhibitors. Furthermore, compounds 13b and 13e were potent inducers of cell death in promastigotes, characterized by cell shrinkage, reduction of mitochondrial transmembrane potential, and increased DNA fragmentation. Transmission electron microscopic studies revealed the enrichment of undigested debris in lysosome-like organelles participating in micro- and macroautophagy-like processes. The release of digestive enzymes into the cytoplasm after rupture of membranes of lysosome-like vacuoles resulted in the significant digestion of intracellular compartments. However, the plasma membrane integrity of compound-treated promastigotes was maintained for several hours. Taken together, our results suggest that the induction of cell death in Leishmania by cysteine cathepsin inhibitors 13b and 13e is different from mammalian apoptosis and is caused by incomplete digestion in autophagy-related lysosome-like vacuoles.
Subject(s)
Autophagy , Aziridines/pharmacology , Cysteine Proteinase Inhibitors/pharmacology , Leishmania major/drug effects , Lysosomes/metabolism , Vacuoles/drug effects , Animals , Leishmania major/ultrastructure , Mice , Mice, Inbred BALB C , Microscopy, Electron, Transmission , Vacuoles/ultrastructureSubject(s)
Peptides, Cyclic/pharmacology , Serine Proteinase Inhibitors/pharmacology , Tryptases/metabolism , Animals , Asthma/drug therapy , Cattle , Crystallography, X-Ray , Humans , Models, Molecular , Peptides, Cyclic/chemistry , Serine Proteinase Inhibitors/chemistry , Trypsin/chemistry , Trypsin/metabolismSubject(s)
Cathepsins/antagonists & inhibitors , Cathepsins/chemistry , Cysteine Endopeptidases/chemistry , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Peptides/chemistry , Peptides/pharmacology , Succinic Acid/chemistry , Amino Acid Sequence , Binding Sites , Cathepsin L , Cathepsins/metabolism , Crystallography, X-Ray , Cysteine Endopeptidases/metabolism , Enzyme Inhibitors/chemical synthesis , Humans , Models, Molecular , Molecular Sequence Data , Peptides/chemical synthesis , Protein ConformationABSTRACT
The 27-mer peptide CP1B-[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 CP1B-[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.
Subject(s)
Calcium-Binding Proteins/pharmacology , Calpain/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , Amino Acid Sequence , Calcium-Binding Proteins/chemical synthesis , Calcium-Binding Proteins/chemistry , Cysteine Proteinase Inhibitors/chemical synthesis , Cysteine Proteinase Inhibitors/chemistry , Drug Design , Humans , Kinetics , Lactams/chemistry , Models, Molecular , Molecular Sequence Data , Peptides/pharmacology , Protein Conformation , Structure-Activity Relationship , Substrate SpecificityABSTRACT
Migration, proliferation and differentiation of keratinocytes are important processes during tissue regeneration and wound healing of the skin. Here, we focussed on proteases that contribute to extracellular matrix (ECM) remodeling as a prerequisite of keratinocyte migration. In particular, we assessed the significance of the mammalian cysteine peptidase cathepsin B for human keratinocytes during regeneration from scratch wounding. We describe the construction of a scratch apparatus that allows applying scratches of defined length, width and depth to cultured cells in a reproducible fashion. The rationale for our approach derived from our previous work where we have shown that HaCaT keratinocytes secrete cathepsin B into the extracellular space during spontaneous and induced migration. Here, we observed rapid removal of type IV collagen from underneath lamellipodial extensions of keratinocytes at the advancing fronts of regenerating monolayers, indicating that proteolytic ECM remodeling starts upon initiation of keratinocyte migration. Furthermore, we verified our previous results with HaCaT cells by using normal human epidermal keratinocytes (NHEK) and show that non-cell-permeant cathepsin B-specific inhibitors delayed full regeneration of the monolayers from scratch wounding in both cell systems, HaCaT and NHEK. Application of a single dose of cathepsin B inhibitor directly after scratch wounding of keratinocytes demonstrated that cathepsin B is essential during initial stages of wound healing, while its contribution to the subsequent processes of proliferation and differentiation of keratinocytes was of less significance. This notion was supported by our observation that the cathepsin B inhibitors used in this study did not affect proliferation rates of keratinocytes of regenerating cultures. Thus, we conclude that cathepsin B is indeed involved in ECM remodeling after its secretion from migrating keratinocytes. Cathepsin B might directly cleave ECM constituents or it may initiate proteolytic cascades that involve other proteases with the ability to degrade ECM components. Because cathepsin B is important for enabling migration of both, HaCaT cells and NHEK, our results support the notion that HaCaT keratinocytes represent an excellent cell culture model for analysis of human epidermal skin keratinocyte migration.
Subject(s)
Cathepsin B/metabolism , Epidermis/pathology , Epidermis/physiology , Keratinocytes/enzymology , Keratinocytes/pathology , Regeneration , Cathepsin B/antagonists & inhibitors , Cell Death/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Epidermis/drug effects , Epidermis/enzymology , Extracellular Matrix/drug effects , Extracellular Matrix/enzymology , Humans , Keratinocytes/drug effects , Protein Processing, Post-Translational/drug effects , Regeneration/drug effects , Reproducibility of ResultsABSTRACT
The notion that human cysteine cathepsins contribute only to general protein turnover within the lysosomes has changed in the last decade in a substantial manner. A continuously growing number of data accumulated in different fields of life sciences revealed that these enzymes are involved in a variety of pivotal physiological processes. To investigate these particular fraction of proteolytical activity of the human degradome even in a complex cellular environment, chemical probes that covalently label the corresponding proteases proved to be versatile tools. (2S,3S)-Oxirane-2,3-dicarboxylic acid provides an ideal platform for the design of such probing systems. Depending on the complexity of the attached recognition elements, either the activity of the entire group of human cysteine cathepsins or individual members can be detected.
Subject(s)
Cathepsins/chemistry , Cysteine Endopeptidases/chemistry , Dicarboxylic Acids/chemistry , Ethylene Oxide/analogs & derivatives , Proteomics/methods , Cathepsins/antagonists & inhibitors , Cathepsins/physiology , Cysteine Endopeptidases/physiology , Ethylene Oxide/chemistry , Humans , Structure-Activity Relationship , Substrate Specificity/physiologyABSTRACT
beta-Tryptases are mast cell-derived serine proteases that are enzymatically active in the form of an oligomer consisting of four subunits each with trypsin-like activity. The active-site clefts, which are directed toward the central pore of the tetramer, form spatial arrays of four negatively charged S1 binding pockets. Therefore, dibasic inhibitors of appropriate geometry can bind in a bivalent fashion to neighboring subunits. We have recently identified a potent bivalent inhibitor (K(i)=18 nM), based on the bifunctional scaffold cyclo-(-D-Asp-L-Asp-) and the arginine mimetic dl-3-aminomethyl-phenylalanine methyl ester as a ligand for S1 pockets that takes advantage of the this unique tetrameric geometry. To generate an affinity matrix, the bivalent ligand was modified and immobilized on a Sepharose matrix by use of the PEG derivative Jeffamine ED 900 as spacer. This matrix selectively recognizes and binds beta-tryptase from crude protein mixtures and thus is useful as a geometry-driven means of isolating and purifying human mast cell tryptases.
Subject(s)
Chromatography, Affinity/methods , Enzyme Inhibitors/chemical synthesis , Serine Endopeptidases/isolation & purification , Dipeptides/chemical synthesis , Dipeptides/metabolism , Enzyme Inhibitors/chemistry , Humans , Ligands , Models, Molecular , Peptides, Cyclic/chemical synthesis , Peptides, Cyclic/metabolism , Phenylalanine/analogs & derivatives , Phenylalanine/chemical synthesis , Phenylalanine/metabolism , TryptasesABSTRACT
The crystal structure of the inhibitor NS-134 in complex with bovine cathepsin B reveals that functional groups attached to both sides of the epoxysuccinyl reactive group bind to the part of active-site cleft as predicted. The -Leu-Pro-OH side binds to the primed binding sites interacting with the His110 and His111 residues with its C-terminal carboxy group, whereas the -Leu-Gly-Meu (-Leu-Gly-Gly-OMe) part (Meu, methoxycarbonylmethyl) binds along the non-primed binding sites. Comparison with the propeptide structures of cathepsins revealed that the binding of the latter part is least similar to the procathepsin B structure; this result, together with the two-residue shift in positioning of the Leu-Gly-Gly part, suggests that the propeptide structures of the cognate enzymes may not be the best starting point for the design of reverse binding inhibitors.
Subject(s)
Cathepsin B/chemistry , Crystallography, X-Ray/methods , Leucine/analogs & derivatives , Oligopeptides/chemistry , Pyrroles/chemistry , Animals , Binding Sites , Cathepsin B/antagonists & inhibitors , Cathepsin B/isolation & purification , Cattle , Cysteine Proteinase Inhibitors/chemistry , Dipeptides/chemistry , Epoxy Compounds/chemistry , Kidney/enzymology , Leucine/chemistry , Molecular Conformation , Molecular Structure , Oligopeptides/chemical synthesis , Protein Binding , Protein Conformation , Protein Structure, Quaternary , Pyridines/chemistry , Pyrroles/chemical synthesisABSTRACT
The natural product miraziridine A isolated from the marine sponge Theonella aff. mirabilis unifies within one molecule three structurally privileged elements: (i) (2R,3R)-aziridine-2,3-dicarboxylic acid, (ii) (3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid (statine), and (iii) (E)-(S)-4-amino-7-guanidino-hept-2-enoic acid (vinylogous arginine). The alignment of them realized in the tetrapetide allows for a simultaneous inhibition of the proteolytic activity of trypsin-like serine proteases, papain-like cysteine proteases, and pepsin-like aspartyl proteases. Therefore, this unique compound represents a blueprint for the design of protease class-spanning inhibitors.
Subject(s)
Aziridines/pharmacology , Cathepsin B/antagonists & inhibitors , Cathepsins/antagonists & inhibitors , Endopeptidases/classification , Oligopeptides/pharmacology , Pepsin A/antagonists & inhibitors , Protease Inhibitors/pharmacology , Trypsin/metabolism , Animals , Aziridines/chemistry , Aziridines/isolation & purification , Cathepsin L , Cysteine Endopeptidases , Molecular Structure , Oligopeptides/chemistry , Oligopeptides/isolation & purification , Porifera/chemistry , Protease Inhibitors/chemistry , Protease Inhibitors/isolation & purificationABSTRACT
The conformational features of a conjugate of the C-terminus of human gastrin (HG[11-17]), the shortest gastrin sequence retaining biological function, with beta-cyclodextrin ([Nle(15)]-HG[11-17]-betaCD) were determined by NMR spectroscopy in an aqueous solution of dodecylphosphocholine (DPC) micelles. The peptide-betaCD conjugate displays a binding affinity and activation profile comparable to those of HG[11-17] at the cholecysokinin 2 (CCK(2)) receptor, the G protein-coupled receptor responsible for the gastrointestinal function of gastrin. The structure of the peptide consisted of a well-defined beta-turn between Gly(13) and Asp(16) of gastrin. The structural preferences of [Nle(15)]-HG[11-17]-betaCD in DPC micelles and the 5-doxylstearate-induced relaxation of the (1)H NMR resonances support a membrane-associated receptor recognition mechanism. Addition of [Nle(15)]-HG[11-17]-betaCD to the third extracellular loop domain of the CCK(2) receptor, CCK(2)-R(352-379), generated a number of intermolecular nuclear Overhauser enhancements (NOEs) and chemical shift perturbations. NOE-restrained MD simulations of the [Nle(15)]-HG[11-17]-betaCD-CCK(2)-R complex produced a topological orientation in which the C-terminus was located in a shallow hydrophobic pocket near the confluence of TM2 and -3. Despite the steric bulk and physicochemical properties of betaCD, the [Nle(15)]-HG[11-17]-betaCD-CCK(2)-R complex is similar to the CCK-8-CCK(2)-R complex determined previously, providing insight into the mode of ligand binding and the role of electrostatic interactions.