Structural studies of cysteine proteases and their inhibitors (original) (raw)
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The cystatins: Protein inhibitors of cysteine proteinases
FEBS Letters, 1991
The iast decade has witnessed enormous progress of protein inhibitors of cysteine proteinases concerning their structures, functions and evolutionary relationships. Although they differ in their molecular properties and biological distribution, they are structurally related proteins. All three inhibitory families, the stetins, the cystatins and the kininogens, are members of the same superfamily. Recently determined crystal structures of chicken cystatin and human stefin B established a new mechanism of interaction between cysteine proteinases and their inhibitors which is fundamentally different from the standard mechanism for serine proteinases and their inhibitors.
Mechanisms Applied by Protein Inhibitors to Inhibit Cysteine Proteases
International Journal of Molecular Sciences, 2021
Protein inhibitors of proteases are an important tool of nature to regulate and control proteolysis in living organisms under physiological and pathological conditions. In this review, we analyzed the mechanisms of inhibition of cysteine proteases on the basis of structural information and compiled kinetic data. The gathered structural data indicate that the protein fold is not a major obstacle for the evolution of a protease inhibitor. It appears that nature can convert almost any starting fold into an inhibitor of a protease. In addition, there appears to be no general rule governing the inhibitory mechanism. The structural data make it clear that the “lock and key” mechanism is a historical concept with limited validity. However, the analysis suggests that the shape of the active site cleft of proteases imposes some restraints. When the S1 binding site is shaped as a pocket buried in the structure of protease, inhibitors can apply substrate-like binding mechanisms. In contrast, w...
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1999
Papain from Carica papaya, an easily available cysteine protease, is the best-studied representative of this family of enzymes. The three dimensional structure of papain is very similar to that of other cysteine proteases of either plant (actinidin, caricain, papaya protease IV) or animal (cathepsins B, K, L, H) origin. As abnormalities in the activities of mammalian cysteine proteases accompany a variety of diseases, there has been a long-lasting interest in the development of potent and selective inhibitors for these enzymes. A covalent inhibitor of cysteine proteases, designed as a combination of epoxysuccinyl and peptide moieties, has been modeled in the catalytic pocket of papain. A number of its configurations have been generated and relaxed by constrained simulated annealing-molecular dynamics in water. A clear conformational variability of this inhibitor is discussed in the context of a conspicuous conformational diversity observed earlier in several solid-state structures of other complexes between cysteine proteases and covalent inhibitors. The catalytic pockets S2 and even more so S3, as defined by the pioneering studies on the papain^ZPACK, papain^E64c and papain^leupeptin complexes, appear elusive in view of the evident flexibility of the present inhibitor and in confrontation with the obvious conformational scatter seen in other examples. This predicts limited chances for the development of selective structure-based inhibitors of thiol proteases, designed to exploit the minute differences in the catalytic pockets of various members of this family. A simultaneous comparison of the three published proenzyme structures suggests the enzyme's prosegment binding loopp rosegment interface as a new potential target for selective inhibitors of papain-related thiol proteases. ß 1999 Elsevier Science B.V. All rights reserved.
Modes of inhibition of cysteine proteases
2004
Cysteine proteases are involved in many physiological processes and their hyperactivity may lead to severe diseases. Nature has developed various strategies to protect cells and whole organisms against undesired proteolysis. One of them is the control of proteolytic activity by inhibition. This paper presents the mechanisms underlying the action of proteinaceous inhibitors of cysteine proteinases and covers propeptides binding backwards relative to the substrate or distorting the protease catalytic centre similarly to serpins, the p35 protein binding covalently to the enzyme, and cystatins that are exosite binding inhibitors. The paper also discusses tyropins and chagasins that, although unrelated to cystatins, inhibit cysteine proteinases by a similar mechanism, as well as inhibitors of the apoptosis protein family that bind in a direction opposite to that of the substrate, similarly to profragments. Special attention is given to staphostatins, a novel family of inhibitors acting in an unusual manner.
1995
Peptide segments derived from consensus sequences of the inhibitory site of cystatins, the natural inhibitors of cysteine proteinases, were used to develop new substrates and inhibitors of papain and rat liver cathepsins B, H, and L. Papain hydrolyzed Abz-QVVAGA-EDDnp and Abz-LVGGA-EDDnp at about the same rate, with specificity constants in the 107 M 1 sec-i range; cathepsin L also hydrolyzes both substrates with specificity constants in the 105 M i sec-~ range due to lower koat values, with the Km'S being identical to those with papain. Only Abz-LVGGA-EDDnp was rapidly hydrolyzed by cathepsin B, and to a lesser extent by cathepsin H. Peptide substrates that alternate these two building blocks (LVGGQVVAGAPWK and QVVAGALVGGAPWK) discriminate the activities of cathepsins B and L and papain. Cathepsin L was highly selective for cleavage at the G-G bond of the LVGG fragment in both peptides. Papain and cathepsin B cleaved either the LVGG fragment or the QVVAG fragment, depending on their position within the peptide. While papain was more specific for the segment located C-terminally, cathepsin B was specific for that in N-terminal position. Peptidyl diazomethylketone inhibitors based on these two sequences also reacted differently with papain and cathepsins. GlcA-QVVA-CHN2 was a potent inhibitor of papain and reacted with papain 60 times more rapidly (k+o = 1,100,000 M -1 sec 1) than with cathepsin L, and 220 times more rapidly than with cathepsin B. Cathepsins B and L were preferentially inhibited by Z-RLVG-CHN2. Thus cystatin-derived peptides provide a valuable flamework for designing sensitive, selective substrates and inhibitors of cysteine proteinases. KEY WORDS: Cysteine proteinase; cystatin; diazomethylketone; peptidyl fluorogenic substrate. 1Laboratoire d'Enzymologie et Chimie des Prot6ines, CNRS-URA 1334, Universit6 Franqois Rabelais, Facult6 de M6decine de Tours,
Design of a new selective cysteine protease inactivator and its mechanistic implications
Bioorganic & Medicinal Chemistry Letters, 1995
Cbz-Phe-epoxide was designed as a selective inactivator of cysteine proteases. It exhibits a time-and concentration-dependent inactivation of cysteine proteases, while showing no activity towards serine proteases. The inhibition is irreversible, correlated with loss of the five active-site thiol, and its rate is at least 104 faster than the rate of a model reaction in solution. These results support the proposed active-site directed, protonationdependent, mechanism-based mode of inactivation of cysteine proteases by the new inhibitor. Proteolytic enzymes can be divided into four families, based on their active site residues and their mechanism of catalysis. 1 Two of these families, namely the serine-and cysteine proteases share many characteristics; They both utilize a covalent nucleophilic catalysis, and involve formation of an acyl cnzyme and two tetrahedral intermediates along the catalytic pathway. It was recently suggested that x~ bile in scrine proteases the nucleophilic attack of the serine alkoxide precedes the protonation of the leaving amino group (thus, leading to a negatively charged tetrahedral intermediate), the corresponding attack of the cysteine thiolate in cysteine p,'oteases is subsequent to or concomitant with protonation of the substrate (leading to a ncutral tetrahedral intermediate). 2 This may explain the selectivity of peptidyl diazomethanes as inhibitors of cysteine proteases. 3'4 This subtle distinction (if found to be correct) between serine-and cysteine proteases may serve as the basis for the design of selective inhibitors for cysteine protcases. Furthermore. if a compound designed to challange the proposed mechanistic difference exhibits such selectivity towards cysteine proteases-it would support the suggested modification in cysteine protease catalytic mechanism. While epoxides are stable compounds, 5 they become highly electrophilic upon protonation. This principle was used recently in the development of an inhibitor for carboxypeptidase A, a metallo protease in which Zn +2 can activate the epoxidic moiety. 6 It is probably also a dominant factor in making E-64 and its derivatives efficient general inbibitors of most cysteine proteases, 7 though it was recently suggested that protonation of the epoxide in this case is carried out by a water molecule rather than by the active site histidine, g Weak inhibition of the cysteine protease cathepsin B was observed, upon its incubation with epoxides derived from allyl amine. 9 Other epoxides were also demonstrated to inactivate a variety of enzymes, in which activation of the epoxide is mechanistically feasible. 1° N-protected-or-amino epoxides (Figure 1), derived from the corresponding natural or-amino acids, are good analogs of the corresponding substrates of either serine or cysteine proteases and therefore are expected to bind "normally" in their respective binding sites. However, if the suggested mechanistic difference between serine-and cysteine proteases (initial nucleophilic attack in the former vs. initial protonation in the latter) is
Vito Turk – 30 Years of Research on Cysteine Proteases and Their Inhibitors
Biological Chemistry, 2003
Max Planck Institute in Martinsried near Munich turned out to be crucial for the future research in Ljubljana. Vito's contacts with Robert, and particularly with Wolfram Bode, were essential for the major progress in the understanding of molecular mechanisms of cathepsin-cystatin interactions. The crystal structure of chicken cystatin and the famous elephant trunk model (Bode et al., 1988) were confirmed by the crystal structure of the stefin B/papain complex (Stubbs et al., 1990). A further outcome of this relationship was the crystal structure of the first cathepsin (cathepsin B), determined by Musil et al. (1991). This work was also done in collaboration with another of Vito's old friends and colleagues, Nobuhiko Katunuma from Japan. Finally Vito managed to establish a crystallography group in his Department in Ljubljana, headed by Dušan Turk, who was trained in this field in Robert Huber's laboratory in early 1990s. Although initially spread between Ljubljana and Munich (crystallization and com
Cysteine Proteases: Modes of Activation and Future Prospects as Pharmacological Targets
Frontiers in pharmacology, 2016
Proteolytic enzymes are crucial for a variety of biological processes in organisms ranging from lower (virus, bacteria, and parasite) to the higher organisms (mammals). Proteases cleave proteins into smaller fragments by catalyzing peptide bonds hydrolysis. Proteases are classified according to their catalytic site, and distributed into four major classes: cysteine proteases, serine proteases, aspartic proteases, and metalloproteases. This review will cover only cysteine proteases, papain family enzymes which are involved in multiple functions such as extracellular matrix turnover, antigen presentation, processing events, digestion, immune invasion, hemoglobin hydrolysis, parasite invasion, parasite egress, and processing surface proteins. Therefore, they are promising drug targets for various diseases. For preventing unwanted digestion, cysteine proteases are synthesized as zymogens, and contain a prodomain (regulatory) and a mature domain (catalytic). The prodomain acts as an endo...