A caspase active site probe reveals high fractional inhibition needed to block DNA fragmentation

被引:23
作者
Méthot, N [1 ]
Vaillancourt, JP [1 ]
Huang, JQ [1 ]
Colucci, J [1 ]
Han, YX [1 ]
Ménard, S [1 ]
Zamboni, R [1 ]
Toulmond, S [1 ]
Nicholson, DW [1 ]
Roy, S [1 ]
机构
[1] Merck Frosst Ctr Therapeut Res, Merck Res Labs, Dept Biochem & Mol Biol, Montreal, PQ H9H 3L1, Canada
关键词
D O I
10.1074/jbc.M400247200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Apoptotic markers consist of either caspase substrate cleavage products or phenotypic changes that manifest themselves as a consequence of caspase-mediated substrate cleavage. We have shown recently that pharmacological inhibitors of caspase activity prevent the appearance of two such apoptotic manifestations, alphaII-spectrin cleavage and DNA fragmentation, but that blockade of the latter required a significantly higher concentration of inhibitor. We investigated this phenomenon through the use of a novel radiolabeled caspase inhibitor, [I-125] M808, which acts as a caspase active site probe. [I-125] M808 bound to active caspases irreversibly and with high sensitivity in apoptotic cell extracts, in tissue extracts from several commonly used animal models of cellular injury, and in living cells. Moreover, [I-125] M808 detected active caspases in septic mice when injected intravenously. Using this caspase probe, an active site occupancy assay was developed and used to measure the fractional inhibition required to block apoptosis- induced DNA fragmentation. In thymocytes, occupancy of up to 40% of caspase active sites had no effect on DNA fragmentation, whereas inhibition of half of the DNA cleaving activity required between 65 and 75% of active site occupancy. These results suggest that a high and persistent fractional inhibition will be required for successful caspase inhibition-based therapies.
引用
收藏
页码:27905 / 27914
页数:10
相关论文
共 44 条
[1]   Ways of dying: multiple pathways to apoptosis [J].
Adams, JM .
GENES & DEVELOPMENT, 2003, 17 (20) :2481-2495
[2]   Mitochondrial membrane potential and apoptosis peripheral blood monocytes in severe human sepsis [J].
Adrie, C ;
Bachelet, M ;
Vayssier-Taussat, M ;
Russo-Marie, F ;
Bouchaert, I ;
Adib-Conquy, M ;
Cavaillon, JM ;
Pinsky, MR ;
Dhainaut, JF ;
Polla, BS .
AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE, 2001, 164 (03) :389-395
[3]   Differential induction of apoptosis in lymphoid tissues during sepsis: Variation in onset, frequency, and the nature of the mediators [J].
Ayala, A ;
Herdon, CD ;
Lehman, DL ;
Ayala, CA ;
Chaudry, IH .
BLOOD, 1996, 87 (10) :4261-4275
[4]   Co-localization of the cysteine protease caspase-3 with apoptotic myocytes after in vivo myocardial ischemia and reperfusion in the rat [J].
Black, SC ;
Huang, JQ ;
Rezaiefar, P ;
Radinovic, S ;
Eberhart, A ;
Nicholson, DW ;
Rodger, IW .
JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY, 1998, 30 (04) :733-742
[5]   A unified model for apical caspase activation [J].
Boatright, KM ;
Renatus, M ;
Scott, FL ;
Sperandio, S ;
Shin, H ;
Pedersen, IM ;
Ricci, JE ;
Edris, WA ;
Sutherlin, DP ;
Green, DR ;
Salvesen, GS .
MOLECULAR CELL, 2003, 11 (02) :529-541
[6]   A Novel nonpeptidic caspase-3/7 inhibitor, (S)-(+)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin reduces myocardial ischemic injury [J].
Chapman, JG ;
Magee, WP ;
Stukenbrok, HA ;
Beckius, GE ;
Milici, AJ ;
Tracey, WR .
EUROPEAN JOURNAL OF PHARMACOLOGY, 2002, 456 (1-3) :59-68
[7]   Caspase inhibition by Z-VAD increases the survival of grafted bone marrow cells and improves functional outcome after MCAo in rats [J].
Chen, JL ;
Li, Y ;
Wang, L ;
Lu, M ;
Chopp, M .
JOURNAL OF THE NEUROLOGICAL SCIENCES, 2002, 199 (1-2) :17-24
[8]   Overexpression of Bcl-2 in the intestinal epithelium improves survival in septic mice [J].
Coopersmith, CM ;
Chang, KC ;
Swanson, PE ;
Tinsley, KW ;
Stromberg, PE ;
Buchman, TG ;
Karl, IE ;
Hotchkiss, RS .
CRITICAL CARE MEDICINE, 2002, 30 (01) :195-201
[9]  
Cursio R, 2000, Transpl Int, V13 Suppl 1, pS568, DOI 10.1007/s001470050405
[10]   A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD [J].
Enari, M ;
Sakahira, H ;
Yokoyama, H ;
Okawa, K ;
Iwamatsu, A ;
Nagata, S .
NATURE, 1998, 391 (6662) :43-50