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call loadScript javascripts\jsmol\core\package.js call loadScript javascripts\jsmol\core\core.z.js -- required by ClazzNode call loadScript javascripts\jsmol\J\awtjs2d\WebOutputChannel.js Jmol JavaScript applet jmolApplet0_object__7764218167065047__ initializing getValue debug = null getValue logLevel = null getValue allowjavascript = null AppletRegistry.checkIn(jmolApplet0_object__7764218167065047__) call loadScript javascripts\jsmol\core\corestate.z.js viewerOptions: { "name":"jmolApplet0_object","applet":true,"documentBase":"https://www.ebi.ac.uk/chebi/searchId.do?chebiId=CHEBI%3ACHEBI%3A109540","platform":"J.awtjs2d.Platform","fullName":"jmolApplet0_object__7764218167065047__","display":"jmolApplet0_canvas2d","signedApplet":"true","appletReadyCallback":"Jmol._readyCallback","statusListener":"[J.appletjs.Jmol.MyStatusListener object]","codeBase":"https://www.ebi.ac.uk/chebi/javascripts/jsmol/","syncId":"7764218167065047","bgcolor":"#000" } (C) 2012 Jmol Development Jmol Version: 13.2.7 $Date: 2013-10-01 11:35:15 -0500 (Tue, 01 Oct 2013) $ java.vendor: j2s java.version: 0.0 os.name: j2s Access: ALL memory: 0.0/0.0 processors available: 1 useCommandThread: false appletId:jmolApplet0_object (signed) starting HoverWatcher_1 getValue emulate = null defaults = "Jmol" getValue boxbgcolor = null getValue bgcolor = #000 backgroundColor = "#000" getValue ANIMFRAMECallback = null getValue APPLETREADYCallback = Jmol._readyCallback APPLETREADYCallback = "Jmol._readyCallback" getValue ATOMMOVEDCallback = null getValue CLICKCallback = null getValue ECHOCallback = null getValue ERRORCallback = null getValue EVALCallback = null getValue HOVERCallback = null getValue LOADSTRUCTCallback = null getValue MEASURECallback = null getValue MESSAGECallback = null getValue MINIMIZATIONCallback = null getValue PICKCallback = null getValue RESIZECallback = null getValue SCRIPTCallback = null getValue SYNCCallback = null getValue STRUCTUREMODIFIEDCallback = null getValue doTranslate = null language=en_US getValue popupMenu = null getValue script = null Jmol applet jmolApplet0_object__7764218167065047__ ready call loadScript javascripts\jsmol\core\corescript.z.js call loadScript javascripts\jsmol\J\script\FileLoadThread.js starting QueueThread0_2 script 1 started starting HoverWatcher_3 starting HoverWatcher_4 The Resolver thinks Mol DIC - Ideal conformer Mrv1927 09202111403D starting HoverWatcher_5 Time for openFile(DIC - Ideal conformer Mrv1927 09202111403D 17 18 0 0 0 0 999 V2000 0.6980 -2.0320 0.1010 C 0 0 0 0 0 0 0 0 0 0 0 0 1.8090 -2.8790 0.0680 C 0 0 0 0 0 0 0 0 0 0 0 0 3.1090 -2.3680 -0.0920 C 0 0 0 0 0 0 0 0 0 0 0 0 3.2570 -0.9790 -0.2180 C 0 0 0 0 0 0 0 0 0 0 0 0 2.1410 -0.1290 -0.1850 C 0 0 0 0 0 0 0 0 0 0 0 0 0.8630 -0.6580 -0.0260 C 0 0 0 0 0 0 0 0 0 0 0 0 4.5950 -0.3690 -0.3890 C 0 0 0 0 0 0 0 0 0 0 0 0 4.7430 0.8410 -0.4990 O 0 0 0 0 0 0 0 0 0 0 0 0 4.3100 -3.2170 -0.1340 C 0 0 0 0 0 0 0 0 0 0 0 0 5.5060 -2.6270 -0.2900 C 0 0 0 0 0 0 0 0 0 0 0 0 5.6510 -1.2290 -0.4150 O 0 0 0 0 0 0 0 0 0 0 0 0 4.1440 -4.9340 0.0190 Cl 0 0 0 0 0 0 0 0 0 0 0 0 6.9900 -3.5310 -0.3510 Cl 0 0 0 0 0 0 0 0 0 0 0 0 -0.2970 -2.4500 0.2260 H 0 0 0 0 0 0 0 0 0 0 0 0 1.6240 -3.9450 0.1710 H 0 0 0 0 0 0 0 0 0 0 0 0 2.2490 0.9480 -0.2820 H 0 0 0 0 0 0 0 0 0 0 0 0 0.0000 0.0000 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0 1 2 2 0 0 0 0 1 6 1 0 0 0 0 1 14 1 0 0 0 0 2 3 1 0 0 0 0 2 15 1 0 0 0 0 3 4 2 0 0 0 0 3 9 1 0 0 0 0 4 5 1 0 0 0 0 4 7 1 0 0 0 0 5 6 2 0 0 0 0 5 16 1 0 0 0 0 6 17 1 0 0 0 0 7 8 2 0 0 0 0 7 11 1 0 0 0 0 9 10 2 0 0 0 0 9 12 1 0 0 0 0 10 11 1 0 0 0 0 10 13 1 0 0 0 0 M END): 13 ms reading 17 atoms ModelSet: haveSymmetry:false haveUnitcells:false haveFractionalCoord:false 1 model in this collection. Use getProperty "modelInfo" or getProperty "auxiliaryInfo" to inspect them. Default Van der Waals type for model set to Babel 17 atoms created ModelSet: not autobonding; use forceAutobond=true to force automatic bond creation Script completed Jmol script terminated
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| InChI=1S/C9H4Cl2O2/c10-7-5-3-1-2-4-6(5)9(12)13-8(7)11/h1-4H |
| SUGXUUGGLDCZKB-UHFFFAOYSA-N |
| ClC1=C(Cl)C2=C(C=CC=C2)C(=O)O1 |
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serine protease inhibitor
Any protease inhibitor that restricts the action of a serine protease.
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geroprotector
Any compound that supports healthy aging, slows the biological aging process, or extends lifespan.
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View more via ChEBI Ontology
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3,4-dichloro-1H-isochromen-1-one
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3,4-DCI
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ChEBI
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3,4-dichloro-1H-2-benzopyran-1-one
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IUPAC
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3,4-dichloro-2-benzopyran-1-one
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ChEBI
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3,4-dichloroisochromen-1-one
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PDBeChem
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51050-59-0
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CAS Registry Number
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ChemIDplus
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Sukeda M, Shiota K, Kondo M, Nagasawa T, Nakao M, Somamoto T (2021)
Innate cell-mediated cytotoxicity of CD8+ T cells against the protozoan parasite Ichthyophthirius multifiliis in the ginbuna crucian carp, Carassius auratus langsdorfii.
Developmental and comparative immunology 115, 103886 [PubMed:33045272]
[show Abstract]
Cytotoxic T cells are known to have the ability to kill microbe-infected host cells, which makes them essential in the adaptive immunity processes of various vertebrates. In this study, we demonstrated innate cell-mediated cytotoxicity of CD8+ T cells against protozoan parasites found in the ginbuna crucian carp. When isolated effector cells such as CD8+, CD4+ (CD4-1+), or CD8- CD4- (double-negative, DN), from naïve ginbuna crucian carp were co-incubated with target parasites (Ichthyophthirius multifiliis), CD8+ cells from the kidney and gill showed the highest cytotoxic activity. On the other hand, DN cells, which include macrophages and CD4- CD8- lymphocytes, showed the lowest cytotoxic activity against I. multifiliis. Additionally, the cytotoxic activity of CD8+ cells was found to significantly decrease in the presence of a membrane separating the effector cells from I. multifiliis. Furthermore, the serine protease inhibitor 3,4-dichloroisocoumarin and perforin inhibitor concanamycin A significantly inhibited the cytotoxic activity of CD8+ cells. These results demonstrate that CD8+ T cells of ginbuna crucian carp can kill extracellular parasites in a contact-dependent manner via serine proteases and perforin. Therefore, we conclude that CD8+ T cells play an essential role in anti-parasite innate immunity of teleost fish. | Al-Omar MS, Jabir M, Karsh E, Kadhim R, Sulaiman GM, Taqi ZJ, Khashan KS, Mohammed HA, Khan RA, Mohammed SAA (2021)
Gold Nanoparticles and Graphene Oxide Flakes Enhance Cancer Cells' Phagocytosis through Granzyme-Perforin-Dependent Biomechanism.
Nanomaterials (Basel, Switzerland) 11, 1382 [PubMed:34073808]
[show Abstract]
The study aimed to investigate the roles of gold nanoparticles (GNPs) and graphene oxide flakes (GOFs) as phagocytosis enhancers against cancer cells. The nanomaterials were characterized through SEM and UV-VIS absorptions. The GNPs and GOFs increased the macrophages' phagocytosis ability in engulfing, thereby annihilating the cancer cells in both in vitro and in vivo conditions. The GNPs and GOFs augmented serine protease class apoptotic protein, granzyme, passing through the aquaporin class protein, perforin, with mediated delivery through the cell membrane site for the programmed, calibrated, and conditioned cancer cells killing. Additionally, protease inhibitor 3,4-dichloroisocoumarin (DCI) significantly reduced granzyme and perforin activities of macrophages. The results demonstrated that the GOFs and GNPs increased the activation of phagocytic cells as a promising strategy for controlling cancer cells by augmenting the cell mortality through the granzyme-perforin-dependent mechanism. | Sassetti E, Durante Cruz C, Tammela P, Winterhalter M, Augustyns K, Gribbon P, Windshügel B (2019)
Identification and Characterization of Approved Drugs and Drug-Like Compounds as Covalent Escherichia coli ClpP Inhibitors.
International journal of molecular sciences 20, E2686 [PubMed:31159170]
[show Abstract]
The serine protease Caseinolytic protease subunit P (ClpP) plays an important role for protein homeostasis in bacteria and contributes to various developmental processes, as well as virulence. Therefore, ClpP is considered as a potential drug target in Gram-positive and Gram-negative bacteria. In this study, we utilized a biochemical assay to screen several small molecule libraries of approved and investigational drugs for Escherichia coli ClpP inhibitors. The approved drugs bortezomib, cefmetazole, cisplatin, as well as the investigational drug cDPCP, and the protease inhibitor 3,4-dichloroisocoumarin (3,4-DIC) emerged as ClpP inhibitors with IC50 values ranging between 0.04 and 31 µM. Compound profiling of the inhibitors revealed cefmetazole and cisplatin not to inhibit the serine protease bovine α-chymotrypsin, and for cefmetazole no cytotoxicity against three human cell lines was detected. Surface plasmon resonance studies demonstrated all novel ClpP inhibitors to bind covalently to ClpP. Investigation of the potential binding mode for cefmetazole using molecular docking suggested a dual covalent binding to Ser97 and Thr168. While only the antibiotic cefmetazole demonstrated an intrinsic antibacterial effect, cDPCP clearly delayed the bacterial growth recovery time upon chemically induced nitric oxide stress in a ClpP-dependent manner. | Ye X, Linton JM, Schork NJ, Buck LB, Petrascheck M (2014)
A pharmacological network for lifespan extension in Caenorhabditis elegans.
Aging cell 13, 206-215 [PubMed:24134630]
[show Abstract]
One goal of aging research is to find drugs that delay the onset of age-associated disease. Studies in invertebrates, particularly Caenorhabditis elegans, have uncovered numerous genes involved in aging, many conserved in mammals. However, which of these encode proteins suitable for drug targeting is unknown. To investigate this question, we screened a library of compounds with known mammalian pharmacology for compounds that increase C. elegans lifespan. We identified 60 compounds that increase longevity in C. elegans, 33 of which also increased resistance to oxidative stress. Many of these compounds are drugs approved for human use. Enhanced resistance to oxidative stress was associated primarily with compounds that target receptors for biogenic amines, such as dopamine or serotonin. A pharmacological network constructed with these data reveal that lifespan extension and increased stress resistance cluster together in a few pharmacological classes, most involved in intercellular signaling. These studies identify compounds that can now be explored for beneficial effects on aging in mammals, as well as tools that can be used to further investigate the mechanisms underlying aging in C. elegans. | Ašler IL, Kovačić F, Marchetti-Deschmann M, Allmaier G, Stefanić Z, Kojić-Prodić B (2013)
Inhibition of extracellular lipase from Streptomyces rimosus with 3,4-dichloroisocoumarin.
Journal of enzyme inhibition and medicinal chemistry 28, 1094-1104 [PubMed:22994701]
[show Abstract]
Kinetic characterization of lipase inhibition was performed by activity measurement and mass spectrometry (MS), for the first time with serine-protease inhibitor 3,4-dichloroisocoumarin (DCI). Inhibition of Streptomyces rimosus extracellular lipase (SrLip), a member of the SGNH superfamily, by means of DCI follows the mechanism of two-step irreversible inhibition. The dissociation constant of the noncovalent E•I complex and first-order rate constant for inactivation were determined by incubation (Ki* = 26.6 ± 2.8 µM, k2 = 12.2 ± 0.6 min-1) or progress curve (Ki* = 6.5 ± 1.5 µM, k2 = 0.11 ± 0.01 min-1) method. Half-times of reactivation for lipase inhibited with 10-fold molar excess of DCI were determined by activity measurement (t1/2 = 11.3 ± 0.2 h), matrix-assisted laser desorption/ionization (MALDI, t1/2 = 13.5 ± 0.4 h), and electro-spray ionization (ESI, t1/2 = 12.2 ± 0.5 h) MS. The active SrLip concentration was determined by incubating the enzyme with near equimolar concentrations of DCI, followed by activity and MS measurement. | Xue Y, Ha Y (2012)
Catalytic mechanism of rhomboid protease GlpG probed by 3,4-dichloroisocoumarin and diisopropyl fluorophosphonate.
The Journal of biological chemistry 287, 3099-3107 [PubMed:22130671]
[show Abstract]
Rhomboid proteases have many important biological functions. Unlike soluble serine proteases such as chymotrypsin, the active site of rhomboid protease, which contains a Ser-His catalytic dyad, is submerged in the membrane and surrounded by membrane-spanning helices. Previous crystallographic analyses of GlpG, a bacterial rhomboid protease, and its complex with isocoumarin have provided insights into the mechanism of the membrane protease. Here, we studied the interaction of GlpG with 3,4-dichloroisocoumarin and diisopropyl fluorophosphonate, both mechanism-based inhibitors for the serine protease, and describe the crystal structure of the covalent adduct between GlpG and diisopropyl fluorophosphonate, which mimics the oxyanion-containing tetrahedral intermediate of the hydrolytic reaction. The crystal structure confirms that the oxyanion is stabilized by the main chain amide of Ser-201 and by the side chains of His-150 and Asn-154. The phosphorylation of the catalytic Ser-201 weakens its interaction with His-254, causing the catalytic histidine to rotate away from the serine. The rotation of His-254 is accompanied by further rearrangement of the side chains of Tyr-205 and Trp-236 within the substrate-binding groove. The formation of the tetrahedral adduct is also accompanied by opening of the L5 cap and movement of transmembrane helix S5 toward S6 in a direction different from that predicted by the lateral gating model. Combining the new structural data with those on the isocoumarin complex sheds further light on the plasticity of the active site of rhomboid membrane protease. | Ašler IL, Pigac J, Vujaklija D, Luić M, Štefanić Z (2011)
Crystallization and preliminary X-ray diffraction studies of a complex of extracellular lipase from Streptomyces rimosus with the inhibitor 3,4-dichloroisocoumarin.
Acta crystallographica. Section F, Structural biology and crystallization communications 67, 1378-1381 [PubMed:22102236]
[show Abstract]
A recombinant lipase (triacylglycerol acylhydrolase; EC 3.1.1.3) from the bacterium Streptomyces rimosus was inhibited by the serine protease inhibitor 3,4-dichloroisocoumarin and crystallized by the hanging-drop vapour-diffusion method at 291 K. The crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a = 38.1, b = 78.7, c = 56.6 Å, β = 104.5° and probably two molecules in the asymmetric unit. Diffraction data were collected to 1.7 Å resolution using synchrotron radiation on the XRD beamline of the Elettra synchrotron, Trieste, Italy. | Cole LB, Kilpatrick JM, Chu N, Babu YS (1998)
Structure of 3,4-dichloroisocoumarin-inhibited factor D.
Acta crystallographica. Section D, Biological crystallography 54, 711-717 [PubMed:9757085]
[show Abstract]
Factor D (D) is a serine protease essential in the activation of the alternative complement pathway. Only a few of the common serine protease inhibitors inhibit D, binding covalently to the serine hydroxyl of the catalytic triad. 3,4-Dichloroisocoumarin (DCI) is a mechanism-based inhibitor which inhibits most serine proteases and many esterases, including D. The structure of the enzyme:inhibitor covalent adduct of D with DCI, DCI:D, to a resolution of 1.8 A is described, which represents the first structural analysis of D with a mechanism-based inhibitor. The side chain of the ring-opened DCI moiety of the protein adduct undergoes chemical modification in the buffered solution, resulting in the formation of an alpha-hydroxy acid moiety through the nucleophilic substitution of both Cl atoms. The inhibited enzyme is similar in overall structure to the native enzyme, as well as to a variety of isocoumarin-inhibited trypsin and porcine pancreatic elastase (PPE) structures, yet notable differences are observed in the active site and binding mode of these small-molecule inhibitors. One region of the active site (residues 189-195) is relatively conserved between factor D, trypsin, and elastase with respect to amino-acid sequence and to conformation. Another region (residues 214-220) reflects the amino-acid substitutions and conformational flexibility between these enzymes. The carbonyl O atom of the DCI moiety was found to be oriented away from the oxyanion hole, which greatly contributes to the stability of the DCI:D adduct. The comparisons of the active sites between native factor D, DCI-inhibited factor D, and various inhibited trypsin and elastase (PPE) molecules are providing the chemical bases directing our design of novel, small-molecule pharmaceutical agents capable of modulating the alternative complement pathway. | Hameed A, Aslam U, Ying AJ (1998)
3,4-Dichloroisocoumarin serine protease inhibitor induces DNA fragmentation and apoptosis in susceptible target cells.
Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.) 219, 132-137 [PubMed:9790170]
[show Abstract]
3,4-Dichloroisocoumarin (DCI) inhibition of serine proteases generates reactive intermediates that have been theorized to affect apoptosis. To examine this possibility various target cells were treated with different concentrations of DCI and assessed for intracellular nuclear DNA fragmentation and apoptosis. DCI treatment caused oligonucleosomal DNA fragmentation in cell lines expressing high levels of protease activity (LAK cells, NK-92, CTLL-2, L929, 3T3). This DNA breakdown characteristic of apoptosis occurred in a dose-dependent fashion within 4-6 hr of treatment and was confirmed by electron microscopy. In cell lines expressing low levels of protease activity (unstimulated human peripheral blood mononuclear (PBMN) cells, YAC-1 cells), DCI effectively inhibited protease activity without inducing oligonucleosomal DNA fragmentation. ZN2+ ions significantly inhibited DCI-induced DNA degradation. The mixture of DCI and BLT esterase active NK cell lysate triggered DNA fragmentation in isolated YAC-1 nuclei. Degree of DNA fragmentation in YAC-1 nuclei was proportional to the level of BLT esterase activity. Cell lysate protease activity, initially inhibited by DCI acylation, was restored by hydroxylamine deacylation, thus preventing DCI-mediated DNA fragmentation. Our results suggest that DCI treatment of cells expressing high levels of protease activity generates toxic levels of acyl-enzyme intermediates. These intermediates may trigger nuclear DNA breakdown and apoptosis by activating endogenous endonucleases. This effect may compromise the analysis of apoptosis in experimental systems using high concentrations of DCI for extended periods. | Orlowski M, Cardozo C, Eleuteri AM, Kohanski R, Kam CM, Powers JC (1997)
Reactions of [14C]-3,4-dichloroisocoumarin with subunits of pituitary and spleen multicatalytic proteinase complexes (proteasomes).
Biochemistry 36, 13946-13953 [PubMed:9374874]
[show Abstract]
Exposure to [14C]-3,4-dichloroisocoumarin (DCI) of multicatalytic proteinase complexes (MPC) isolated from bovine pituitary and spleen leads to label incorporation into several beta-type subunits, to rapid inactivation of the chymotrypsin-like (ChT-L) activity, and to a slower inactivation of other activities of the MPC. The pituitary and spleen MPCs differ in that the first contains almost exclusively the X, Y, and Z subunits, whereas in the latter these subunits are largely replaced by LMP2, LMP7, and MECL1. Preincubation with two peptidyl aledhyde inhibitors of the ChT-L activity protected the X subunit in the pituitary MPC and unexpectedly the LMP2 subunit in the spleen MPC from label incorporation, despite the greater amino acid sequence homology of the LMP7 subunit to that of the X subunit. Losses in the yield of amino acids in both subunits, shown by amino acid sequencing, and lability of the DCI-protein bond indicated formation of an acyl derivative by reaction of DCI with the threonine OH group. Brief exposure to [14C]-DCI led to preferential incorporation of label into the LMP2 and X subunits, consistent with the high inactivation rate constants of the ChT-L activity. Z-LLF-CHO, an inhibitor of ChT-L activity, but not Z-GPFL-CHO, an inhibitor of the branched chain amino acid preferring component, prevented incorporation of radioactivity into the X subunits, whereas both inhibitors prevented label incorporation into LMP2, indicating differences in susceptibility to inhibition between the two components. These and other data are consistent with involvement of the X and LMP2 subunits in expression of the ChT-L activity in the pituitary and spleen MPC, respectively, and suggest the catalytic functions of two other beta-subunits. | Olson ST, Swanson R, Patston PA, Björk I (1997)
Apparent formation of sodium dodecyl sulfate-stable complexes between serpins and 3,4-dichloroisocoumarin-inactivated proteinases is due to regeneration of active proteinase from the inactivated enzyme.
The Journal of biological chemistry 272, 13338-13342 [PubMed:9148956]
[show Abstract]
Protein proteinase inhibitors of the serpin family were recently reported to form SDS-stable complexes with inactive serine proteinases modified at the catalytic serine with 3, 4-dichloroisocoumarin (DCI) that resembled the complexes formed with the active enzymes (Christensen, S., Valnickova, Z., Thogersen, I. B. , Pizzo, S. V., Nielsen, H. R., Roepstorff, P., and Enghild, J. J. (1995) J. Biol. Chem. 270, 14859-14862). The discordance between these findings and other reports that similar active site modifications of serine proteinases block the ability of serpins to form SDS-stable complexes prompted us to investigate the mechanism of complex formation between serpins and DCI-inactivated enzymes. Both neutrophil elastase and beta-trypsin inactivated by DCI appeared to form SDS-stable complexes with the serpin, alpha1-proteinase inhibitor (alpha1PI), as reported previously. However, several observations suggested that such complex formation resulted from a reaction not with the DCI enzyme but rather with active enzyme regenerated from the DCI enzyme by a rate-limiting hydrolysis reaction. Thus (i) complex formation was blocked by active site-directed peptide chloromethyl ketone inhibitors; (ii) the kinetics of complex formation indicated that the reaction was not second order but rather showed a first-order dependence on DCI enzyme concentration and zero-order dependence on inhibitor concentration; and (iii) complex formation was accompanied by stoichiometric release of a peptide having the sequence SIPPE corresponding to cleavage at the alpha1PI reactive center P1-P1' bond. Quantitation of kinetic constants for DCI and alpha1PI inactivation of human neutrophil elastase and trypsin and for reactivation of the DCI enzymes showed that the observed complex formation could be fully accounted for by alpha1PI preferentially reacting with active enzyme regenerated from DCI enzyme during the reaction. These results support previous findings of the critical importance of the proteinase catalytic serine in the formation of SDS-stable serpin-proteinase complexes and are in accord with an inhibitory mechanism in which the proteinase is trapped at the acyl intermediate stage of proteolysis of the serpin as a substrate. | Guesdon F, Ikebe T, Stylianou E, Warwick-Davies J, Haskill S, Saklatvala J (1995)
Interleukin 1-induced phosphorylation of MAD3, the major inhibitor of nuclear factor kappa B of HeLa cells. Interference in signalling by the proteinase inhibitors 3,4-dichloroisocoumarin and tosylphenylalanyl chloromethylketone.
The Biochemical journal 307 ( Pt 1), 287-295 [PubMed:7717987]
[show Abstract]
The regulation of the inhibitor of nuclear factor kappa B (I kappa B) by interleukin 1 (IL1) was investigated in HeLa cells. Two forms of I kappa B were resolved by ion-exchange chromatography. The major form (75%) was identified as MAD3 by specific antisera. IL1 generated rapidly (6 min) an electrophoretically retarded form of MAD3 that was stable in acid and was converted into the unmodified form by phosphatase 2A. It thus corresponded to a phosphorylation of the protein on serine or threonine. IL1 also caused the disappearance of MAD3 from the cells, which was complete 15 min after stimulation and coincided with a 46% reduction of cellular I kappa B activity. Newly-synthesized MAD3 accumulated to pre-stimulation levels between 60 and 90 min after stimulation and this coincided with the down-regulation of the phosphorylating activity. The serine proteinase inhibitors 3,4-dichloroisocoumarin (DCI) and tosylphenylalanyl chloromethylketone (TPCK) prevented phosphorylation and disappearance of MAD3. At the same concentrations (10-100 microM), they also increased basal phosphorylation of the small heat shock protein (hsp27) and prevented the IL1- and phorbol 12-myristate 13-acetate-induced increases of its phosphorylation. The inhibitors were thus interfering with protein kinases when blocking degradation of MAD3. Recombinant MAD3 phosphorylated in vitro by protein kinase C was not electrophoretically retarded, suggesting that MAD3 was phosphorylated by another kinase in IL1-stimulated cells. Our results suggest that the IL1-induced phosphorylation of MAD3 on serine or threonine leads to its degradation. DCI and TPCK blocked phosphorylation mechanisms and it could not be concluded that serine proteinases were involved in the breakdown of MAD3. | Anees M, Steven FS (1994)
Inhibition of a tumour protease with 3,4-dichloroisocoumarin, pentamidine-isethionate and guanidino derivatives.
Journal of enzyme inhibition 8, 213-221 [PubMed:7539490]
[show Abstract]
Guanidinobenzoatase (GB) is a cell surface proteolytic enzyme capable of degrading fibronectin, and is associated with tumour cells and cells capable of migration. The location of active GB in sections has been demonstrated with 9-aminoacridine (9-AA), a competitive inhibitor of GB. 3,4-Dichloroisocoumarin (3,4-DCI) and pentamidine isethionate (PI) are inhibitors of trypsin-like enzymes. It has now been demonstrated that 3,4-DCI, PI, and guanidino derivative compounds are significant inhibitors of GB, on the surfaces of lung squamous cell carcinoma cells in frozen sections and free GB in solution. Dexamethasone acetate (DMA) and medroxy-progesterone (MP) did not show any significant inhibition of GB activity. These molecules lack a reactive chloride or guanidino groups and are thought to react at the nuclear level, rather than directly on this cell surface protease. Kinetic studies have shown that 3,4-DCI, PI and guanidino derivatives are reversible competitive inhibitors of GB, as determined in vitro on the purified enzyme. The inhibition resulting with 3,4-DCI was a time-dependent process. It is suggested that these inhibitors interact with GB by binding to its active site, resulting in the formation of enzyme-inhibiter complexes (GB-I). The GB-I complexes can be dissociated with SDS treatment, resulting in the regain of GB activity. | Cardozo C, Vinitsky A, Hidalgo MC, Michaud C, Orlowski M (1992)
A 3,4-dichloroisocoumarin-resistant component of the multicatalytic proteinase complex.
Biochemistry 31, 7373-7380 [PubMed:1510927]
[show Abstract]
The multicatalytic proteinase complex (MPC) exhibits three proteolytic activities designated as trypsin-like, chymotrypsin-like, and peptidylglutamyl-peptide hydrolyzing (PGPHA). Evidence based on inhibitor and specificity studies indicates that each of the three activities is associated with a different component of the complex. Inactivation of the three activities by the serine proteinase inhibitor, 3,4-dichloroisocoumarin (DCI), reveals the presence of an additional DCI-resistant component that cleaves natural peptides including neurotensin, dynorphin, angiotensin II, the oxidized B-chain of insulin, and also proinsulin at a rate greater than that of the native uninhibited complex. Examination of the reaction products of neurotensin (NT) and proinsulin degradation showed cleavage of the Ile12-Leu13 bond in NT and cleavage of the Leu44-Ala45 and Val39-Gly40 bonds within the connecting peptide (C-chain) of bovine proinsulin, suggesting preferential cleavage of bonds on the carboxyl side of branched chain amino acids. Although resistant to inhibition by DCI, the component was sensitive to inhibition by the isocoumarin derivatives, 7-amino-4-chloro-3-[3-(isothioureido)propoxy]isocoumarin and 4-chloro-7-guanidino-3-(2-phenylethoxy)isocoumarin. Degradation of NT was activated by leupeptin, chymostatin, and antipain indicating that binding of these aldehyde inhibitors at one site can stimulate proteolytic activity at a different site of the complex. The DCI-resistant component seems to constitute a major component of the complex active in degradation of natural peptides and proteins. | Pereira ME, Nguyen T, Wagner BJ, Margolis JW, Yu B, Wilk S (1992)
3,4-dichloroisocoumarin-induced activation of the degradation of beta-casein by the bovine pituitary multicatalytic proteinase complex.
The Journal of biological chemistry 267, 7949-7955 [PubMed:1560024]
[show Abstract]
The breakdown of beta-casein (caseinolytic activity) by the bovine pituitary multicatalytic proteinase complex (MPC) is initiated by a fourth active site different from the previously described chymotrypsin-like activity (cleavage of Cbz-Gly-Gly-Leu-p-nitroanilide, where Cbz is benzyloxycarbonyl), trypsin-like activity (cleavage of Cbz-D-Ala-Leu-Arg-2-naphthylamide), and peptidylglutamyl peptide bond-hydrolyzing (PGP) activity (cleavage of Cbz-Leu-Leu-Glu-2-naphthylamide) (Yu, B., Pereira, M. E., and Wilk, S. (1991) J. Biol. Chem. 266, 17396-17400). 3,4-Dichloroisocoumarin, a serine proteinase inhibitor, stimulated the caseinolytic activity of bovine pituitary or lens MPC, 3-18-fold under conditions under which the other three catalytic activities were inactivated. Addition of hydroxylamine to the modified enzyme did not reverse the effects of the inhibitor. A form of the proteinase exhibiting only 2-4% of control chymotrypsin-like, trypsin-like, and PGP activities degraded beta-casein with no accumulation of intermediate peptides. 3,4-Dichloroisocoumarin, by reacting with the chymotrypsin-like, trypsin-like, and/or PGP-active sites, may promote a conformational change of MPC, rendering the caseinolytic active site accessible to the substrate. Once bound to the active site, beta-casein is rapidly degraded either by the caseinolytic component itself or by a cooperative interaction with catalytic centers that are not affected by the serine proteinase inhibitor. These results imply that the caseinolytic component does not belong to the class of serine proteinases. Other proteins tested were not degraded by the 3,4-dichloroisocoumarin-treated enzyme, suggesting that the conformation of beta-casein may be more adequate for degradation by the caseinolytic component. | Rusbridge NM, Beynon RJ (1990)
3,4-Dichloroisocoumarin, a serine protease inhibitor, inactivates glycogen phosphorylase b.
FEBS letters 268, 133-136 [PubMed:2384149]
[show Abstract]
3,4-Dichloroisocoumarin (3,4-DCI) is a highly reactive, mechanism-based inhibitor of serine proteases. We show here that glycogen phosphorylase b is also inactivated by this inhibitor, in a mechanism that parallels the inactivation of serine proteases, but involving multiple sites of covalent modification. Such a process may compromise studies in which 3,4-DCI is used to arrest proteolysis of a second native protein which may itself be modified. | Harper JW, Hemmi K, Powers JC (1985)
Reaction of serine proteases with substituted isocoumarins: discovery of 3,4-dichloroisocoumarin, a new general mechanism based serine protease inhibitor.
Biochemistry 24, 1831-1841 [PubMed:3893537]
[show Abstract]
The mechanism-based inactivations of a number of serine proteases, including human leukocyte (HL) elastase, cathepsin G, rat mast cell proteases I and II, several human and bovine blood coagulation proteases, and human factor D by substituted isocoumarins and phthalides which contain masked acyl chloride or anhydride moieties, are reported. 3,4-Dichloroisocoumarin, the most potent inhibitor investigated here, inactivated all the serine proteases tested but did not inhibit papain, leucine aminopeptidase, or beta-lactamase. 3,4-Dichloroisocoumarin was fairly selective toward HL elastase (kobsd/[I] = 8920 M-1 s-1); the inhibited enzyme was quite stable to reactivation (kdeacyl = 2 X 10(-5) s-1), while enzymes inhibited by 3-acetoxyisocoumarin and 3,3-dichlorophthalide regained full activity upon standing. The rate of inactivation was decreased dramatically in the presence of reversible inhibitors or substrates, and ultraviolet spectral measurements indicate that the isocoumarin ring structure is lost upon inactivation. Chymotrypsin A gamma is totally inactivated by 1.2 equiv of 3-chloroisocoumarin or 3,4-dichloroisocoumarin, and approximately 1 equiv of protons is released upon inactivation. These results indicate that these compounds react with serine proteases to release a reactive acyl chloride moiety which can acylate another active site residue. These are the first mechanism-based inhibitors reported for many of the enzymes tested, and 3,4-dichloroisocoumarin should find wide applicability as a general serine protease inhibitor. |
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