L-<i>trans</i>-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L

Barrett, Alan J.; Kembhavi, A A; Brown, M A; Kirschke, Heidrun; Knight, Christopher G.; Tamai, Masamitsu; Hanada, Kazunori

doi:10.1042/bj2010189

Cited by 1,016 publications

(672 citation statements)

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“…Papain (EC 3.4.22.2, from papaya latex, type III, Sigma) was repurified by covalent chromatography on an agarose mercurial column [9] leading to a 96070 active enzyme as determined by titration with E-64 [10].…”

Section: Methodsmentioning

confidence: 99%

Mechanism of inhibition of papain by chicken egg white cystatin

et al. 1989

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N-terminally truncated forms of chicken egg white cystatin and its cyanogen bromide fragments were isolated and assayed for inhibition of papain. Truncated forms beginning with Gly-9 and Ala-10 had a 5000-fold lower affinity for papain than the two isoelectric forms (pi=6.5 and 5.6) of the full-length inhibitor (K~--6 pM and 7 pM) or a truncated form beginning with Leu-7 (/~ = 6 pM), indicating the outstanding importance of one or two residues preceding conserved Gly-9 for binding. A weak inhibition of papain (K~ = 900 nM) was exhibited by the intermediate cyanogen bromide fragment (residues 30-89) containing the chicken cystatin QLVSG variation of the QWAG segment which is conserved in almost all members of the cystatin superfamily. The obtained affinity data provide independent evidence for the validity of the proposed docking model of a chicken cystatin-papain complex [( ) EMBO J. 7, 2593[( -2599.

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Section: Methodsmentioning

confidence: 99%

Mechanism of inhibition of papain by chicken egg white cystatin

et al. 1989

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“…Pepstatin A (PEP) and E-64 are known to be largely specific towards aspartyl (Barrett, 1977b) and cysteine proteinases (Hanada et al, 1978;Barrett et al, 1982), respectively. The inhibition pattern obtained with these inhibitors without added thiol activator is presented in Fig.…”

Section: Class-specific Inhibition Of Proteolytic Activitymentioning

confidence: 99%

Inhibition of cysteine and aspartyl proteinases in the alfalfa weevil midgut with biochemical and plant-derived proteinase inhibitors

Wilhite

Elden

Brzin

et al. 2000

Insect Biochemistry and Molecular Biology

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"Inhibition of cysteine and aspartyl proteinases in the alfalfa weevil midgut with biochemical and plant-derived proteinase inhibitors" (2000). AbstractProteolytic activities in alfalfa weevil (Hypera postica) larval midguts have been characterized. Effects of pH, thiol activators, low-molecular weight inhibitors, and proteinase inhibitors (PIs) on general substrate hydrolysis by midgut extracts were determined. Hemoglobinolytic activity was highest in the acidic to mildly acidic pH range, but was maximal at pH 3.5. Addition of thiolactivators dithiothreitol (DTT), 2-mercaptoethanol (2-ME), or l-cysteine had little effect on hemoglobin hydrolysis at pH 3.5, but enhanced azocaseinolytic activity two to three-fold at pH 5.0. The broad cysteine PI E-64 reduced azocaseinolytic activity by 64% or 42% at pH 5 in the presence or absence of 5 mM l-cysteine, respectively. Inhibition by diazomethyl ketones, Z-Phe-Phe-CHN 2 and Z-Phe-Ala-CHN 2 , suggest that cathepsins L and B are present and comprise approximately 70% and 30% of the cysteine proteolytic activity, respectively. An aspartyl proteinase component was identified using pepstatin A, which inhibited 32% (pH 3.5, hemoglobin) and 50% (pH 5, azocasein) of total proteolytic activity. This activity was completely inhibited by an aspartyl proteinase inhibitor from potato (API), and is consistent with the action of a cathepsin D-like enzyme. Hence, genes encoding PIs with specificity toward cathepsins L, B and D could potentially be effective for control of alfalfa weevil using transgenic plants.

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“…2,3,13,14,16,17 Other cathepsin B inhibitor types include nitroxoline derivatives, 6,18 redox-reactive compounds, 19 1,2,4-thiadiazoles, 20 aziridinyl peptides, 21 and cystatin-derived azapeptides. 22 To enhance the selectivity of the broad spectrum cathepsin inhibitor E-64 (II, Figure S1), 23 further epoxysuccinyl derivatives, for example, the highly potent and cathepsin B-selective CA-074 and CA-030 (III and IV, Figure S1) have been developed, binding exclusively to the S1′ and S2′ pockets and exploiting interactions with the positively charged histidine residues of the occluding loop. 24−27 Noteworthy, such epoxide dipeptides with a free Cterminal proline exhibited stronger cathepsin B inactivation at lower pH values than under neutral conditions.…”

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confidence: 99%

Cathepsin B Inhibitors: Combining Dipeptide Nitriles with an Occluding Loop Recognition Element by Click Chemistry

Schmitz

Bartz

et al. 2016

ACS Med. Chem. Lett.

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An active site mapping of human cathepsin B with dipeptide nitrile inhibitors was performed for a combinatorial approach by introducing several points of diversity and stepwise optimizing the inhibitor structure. To address the occluding loop of cathepsin B by a carboxylate moiety, click chemistry to generate linker-connected molecules was applied. Inhibitor 17 exhibited K i values of 41.3 nM, 27.3 nM, or 19.2 nM, depending on the substrate and pH of the assay. Kinetic data were discussed with respect to the conformational selection and induced fit models. KEYWORDS:Copper-catalyzed azide−alkyne cycloaddition, cysteine proteases, human cathepsin B, nitrile inhibitors C athepsin B is a papain-like cysteine protease that is ubiquitously expressed and involved in various physiological processes. 1 In nontumor cells, it is localized within endosomes/lysosomes, while in transformed cells it adopts a peripheral distribution in the cytoplasm and is, moreover, associated with the plasma membrane. 2,3 At the surface of invasive tumor cells, cathepsin B was found to bind to annexin II or caveolin-1, the structural protein of caveolae. Caveolae serve as a platform for a proteolytic cascade for the degradation of the extracellular matrix (ECM), executed by several proteases, including cathepsin B. 2−4 Cathepsin B affects the ECM either directly by extracellular proteolytic degradation of its components or indirectly via activation of other proteases. Partially degraded ECM components can be internalized to intracellular endosomal/lysosomal vesicles where the proteolysis is continued. This ECM breakdown remodels the tumor environment, promotes tumor invasion, and enables angiogenesis and metastasis. Cathepsin B also contributes to angiogenesis by degrading metalloprotease inhibitors and releasing growth factors that are bound to ECM components. 2,5,6 The crucial roles of cathepsin B at multiple points of the tumor development have been established in several in vitro and in vivo models and cathepsin B was proposed to be a prognostic marker in patients with various types of cancer. 1,2,7 Cathepsin B shows endopeptidase and exopeptidase activity, a unique feature for cysteine cathepsins, arousing from a flexible structural element, referred to as the occluding loop. The occluding loop is a 19−20 amino acid section that blocks the active site cleft at the end of the primed site. This event leaves only space for two amino acid residues of the substrate C-terminal of the scissile bond, that is, in the P1′ and P2′ positions. In this socalled closed conformation, two salt bridges, His110-Asp22 and Arg116-Asp224, hold the occluding loop over the primed subsites of the substrate binding cleft, preventing extended binding of large endopeptidase substrates and conferring an exopeptidase activity to cathepsin B. Mutations of His110 and Asp22 or the deletion of 12 amino acids of the occluding loop to enforce an open conformation reduced the exopeptidase activity of cathepsin B in favor of its endopeptidase activity. 8,9 His110...

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L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L

Cited by 1,016 publications

References 37 publications

Mechanism of inhibition of papain by chicken egg white cystatin

Mechanism of inhibition of papain by chicken egg white cystatin

Inhibition of cysteine and aspartyl proteinases in the alfalfa weevil midgut with biochemical and plant-derived proteinase inhibitors

Cathepsin B Inhibitors: Combining Dipeptide Nitriles with an Occluding Loop Recognition Element by Click Chemistry

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