Novel Small Molecule Inhibitors of Caspase-3 Block Cellular and Biochemical Features of Apoptosis

Scott, Clay W.; Sobotka-Briner, Cindy; Wilkins, Deidre; Jacobs, Robert T.; Folmer, James J.; Frazee, William J.; Bhat, Ratan V.; Ghanekar, Smita V.; Aharony, David

doi:10.1124/jpet.102.039651

Cited by 58 publications

(41 citation statements)

References 30 publications

(30 reference statements)

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“…Caspase-3 has been one of the more intensely studied enzymes in terms of potential target in neurodegenerative diseases and stroke. 1,9,37 However, as it is true for other cellular pathways of relevance in therapy for a potential therapeutic intervention, more than one isolated protein has to be targeted to increase the therapeutic value. Accordingly, the identification of new potential drugs for the treatment of diseases characterised by excessive apoptosis should be evaluated and developed.…”

Section: Discussionmentioning

confidence: 99%

“…[5][6][7][8] In this macromolecular complex, apoptosomeassociated caspase-9 is activated and then, in turn, activate effector caspases. To identify molecules that could ameliorate disease-associated apoptosis, drug discovery efforts have initially targeted caspase activity 9,10 rather than activation. Nevertheless, protein-protein interactions upstream of caspase activation can be also relevant points of intervention for the development of modulators of apoptosis pathways.…”

Section: Introductionmentioning

confidence: 99%

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Small molecule inhibitors of Apaf-1-related caspase- 3/-9 activation that control mitochondrial-dependent apoptosis

et al. 2005

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Apoptosis is a biological process relevant to human disease states that is strongly regulated through protein-protein complex formation. These complexes represent interesting points of chemical intervention for the development of molecules that could modulate cellular apoptosis. The apoptosome is a holoenzyme multiprotein complex formed by cytochrome c-activated Apaf-1 (apoptotic proteaseactivating factor), dATP and procaspase-9 that link mitochondria disfunction with activation of the effector caspases and in turn is of interest for the development of apoptotic modulators. In the present study we describe the identification of compounds that inhibit the apoptosome-mediated activation of procaspase-9 from the screening of a diversityoriented chemical library. The active compounds rescued from the library were chemically optimised to obtain molecules that bind to both recombinant and human endogenous Apaf-1 in a cytochrome c-noncompetitive mechanism that inhibits the recruitment of procaspase-9 by the apoptosome. These newly identified Apaf-1 ligands decrease the apoptotic phenotype in mitochondrial-mediated models of cellular apoptosis. Keywords: apoptosis; apoptosome; Apaf-1; caspasa-3; caspasa-9; combinatorial libraries; inhibitor; molecular recognition; peptoid; protein-protein interactions; small molecule Abbreviations: Apaf-1, apoptotic protease-activating factor; DEVDase, hydrolysis of Ac-DEVD-afc; DTT, dithiothreitol; FCS, fetal-calf serum; MEFs, mouse embryo fibroblasts; MMP, mitochondrial membrane potential; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Ni-NTA, (Ni 2 þ -nitrilotriacetate)-agarose; peptoid f1a, 5 0 -6 0 carboxyfluorescein-labelled peptoid 1a; PBS, phosphate-buffered saline; PGA, poly-(L-glutamic acid); rApaf-1, recombinant Apaf-1

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Small molecule inhibitors of Apaf-1-related caspase- 3/-9 activation that control mitochondrial-dependent apoptosis

et al. 2005

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“…A similar difference between caspase-3 inhibition and whole-cell antiapoptotic potency was shown for isatin sulfonamides 7 and anilinoquinazolines. 8 Such a difference could be due to many factors, including poor cell membrane permeability, metabolic transformation inside the cells, protein binding, and so forth. 7,8 At least for isatin sulfonamides, it was shown that cytosol binding is responsible for lower potency in cell-based assay versus isolated enzyme assay.…”

Section: Antiapoptotic Effect Of Cd-001-0011mentioning

confidence: 99%

“…This makes caspase-3 an interesting therapeutic target, and the search for caspase-3 inhibitors is an ongoing endeavor many pharmaceutical companies are embracing in the search for effective drugs. [6][7][8][9] As an alternative to the 2 extremes of drug discovery-purely rational drug design and blind screening campaigns-in silico computational docking has resulted in the design and synthesis of novel small-molecule inhibitors of cysteinyl proteases, including caspases. Different classes of the reversible and irreversible protease inhibitors discovered through these efforts are described in Schirmeister and Kaeppler.…”

Section: Caspases Are Found Practically In All Organisms Frommentioning

confidence: 99%

Screening for Caspase-3 Inhibitors: A New Class of Potent Small-Molecule Inhibitors of Caspase-3

Okun

Malarchuk²,

Dubrovskaya³

et al. 2006

SLAS Discovery

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From the authors'650,000 compound collection, they have selected approximately 15,000 potential small-molecule protease inhibitors, which were subjected to high-throughput screening against caspase-3. The screening yielded a series of hits that belong to 11 different scaffolds. Based on the structure of one of the hits, a new class of the small-molecule inhibitors with a double electrophilic warhead, 8-sulfonyl-pyrrolo[3,4-c]quinoline-1,3-diones (SPQ), was synthesized and tested in follow-up mechanistic and antiapoptosis assays. Mechanistic analysis of a representative compound of this class, CD-001-0011, showed that the compound exhibited a high potency (IC 50 = 130 nM), was reversible though noncompetitive, and had a broad selectivity profile to other caspases belonging to groups I to III. The compound was effective in preventing staurosporineinduced apoptosis in a few cell lines and retinoic acid-induced apoptosis in zebrafish. (Journal of Biomolecular Screening 2006:277-285)

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“…First, the sub-micromolar Csp-6 activity of compound 1 was reproduced using the combination of detergent and reducing agent (DTT/CHAPS) reported by Scott et al, 18 but the corresponding concentration-response curve exhibited a Hill coefficient of 3.3 ( Fig. 2A and Table 1).…”

Section: Evaluation Of Csp-6 Inhibitorsmentioning

confidence: 99%

A Label-Free LC/MS/MS-Based Enzymatic Activity Assay for the Detection of Genuine Caspase Inhibitors and SAR Development

et al. 2013

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The resurgence of interest in caspases (Csp) as therapeutic targets for the treatment of neurodegenerative diseases prompted us to examine the suitability of published nonpeptidic Csp-3 and Csp-6 inhibitors for our medicinal chemistry programs. To support this effort, fluorescence-based Csp-2, Csp-3, and Csp-6 enzymatic assays were optimized for robustness against apparent enzyme inhibition caused by redox-cycling or aggregating compounds. The data obtained under these improved conditions challenge the validity of previously published data on Csp-3 and Csp-6 inhibitors for all but one series, namely, the isatins. Furthermore, in this series, it was observed that the nature of the rhodamine-labeled substrate, typically used to measure caspase activity, interfered with the pharmacological sensitivity of the Csp-2 assay. As a result, a liquid chromatography/tandem mass spectrometry-based assay that eliminates label-dependent assay interference was developed for Csp-2 and Csp-3. In these label-free assays, the activity values of the Csp-2 and Csp-3 reference inhibitors were in agreement with those obtained with the fluorogenic substrates. However, isatin 10a was 50-fold less potent in the label-free Csp-2 assay compared with the rhodamine-based fluorescence format, thus proving the need for an orthogonal readout to validate inhibitors in this class of targets highly susceptible to artifactual inhibition.

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Novel Small Molecule Inhibitors of Caspase-3 Block Cellular and Biochemical Features of Apoptosis

Abstract: Caspase-3 is an intracellular cysteine protease, activated as part of the apoptotic response to cell injury.

Cited by 58 publications

References 30 publications

Small molecule inhibitors of Apaf-1-related caspase- 3/-9 activation that control mitochondrial-dependent apoptosis

Small molecule inhibitors of Apaf-1-related caspase- 3/-9 activation that control mitochondrial-dependent apoptosis

Screening for Caspase-3 Inhibitors: A New Class of Potent Small-Molecule Inhibitors of Caspase-3

A Label-Free LC/MS/MS-Based Enzymatic Activity Assay for the Detection of Genuine Caspase Inhibitors and SAR Development

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