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At least 19 recordsLinked to original sources
PubMed2001

Attenuation of bleomycin-induced pneumopathy in mice by a caspase inhibitor.

Caspases have been implicated in the effector process of apoptosis in several systems including the Fas-Fas ligand pathway. We previously demonstrated that excessive apoptosis of lung epithelial cells and the Fas-Fas ligand pathway were essential in the pathogenesis of bleomycin-induced pneumopathy in mice. Therefore, the purpose of this study was to investigate whether a caspase inhibitor could prevent the development of this model. The expression of caspase-1 and caspase-3 was upregulated on lung epithelial cells, alveolar macrophages, and infiltrating inflammatory cells in this model. We demonstrated that a broad-spectrum caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, decreased the caspase-1- and caspase-3-like activity, the number of apoptotic cells, the pathological grade of lung inflammation and fibrosis, and the hydroxyproline content in lung tissues in this model. We conclude that caspase inhibitors could be a new therapeutic approach against lung injury and pulmonary fibrosis.

Administration, Inhalation↗
PubMed2001

Inhibition of distant caspase homologues by natural caspase inhibitors.

Caspases play an important role in the ability of animal cells to kill themselves by apoptosis. Caspase activity is regulated in vivo by members of three distinct protease inhibitor families, two of which, baculovirus p35 and members of the inhibitor of apoptosis (IAP) family, are thought to be caspase specific. However, caspases are members of the clan of cysteine proteases designated CD, which also includes animal and plant legumains, and the bacterial proteases clostripain, gingipain-R and gingipain-K. Since these proteases have been proposed to have a common mechanism and evolutionary origin, we hypothesized that the caspase inhibitors may also regulate these other proteases. We tested this hypothesis by examining the effect of the natural caspase inhibitors on other members of protease clan CD. The IAP family proteins were found to have only a slight inhibitory effect on gingipain-R. The cowpox viral cytokine-response modifier A (CrmA) serpin had no effect on any of the proteases tested but a single point mutation of CrmA (Asp-->Lys) resulted in strong inhibition of gingipain-K. More substantial, with respect to the hypothesis, was the strong inhibition of gingipain-K by wild-type p35. The site in p35, required for inhibition of gingipain-K, was mapped to Lys94, seven residues C-terminal to the caspase inhibitory site. Our data indicate that the virally encoded caspase inhibitors have adopted a mechanism that allows them to regulate disparate members of clan CD proteases.

Amino Acid Sequence↗
PubMed1999

Caspase inhibitors.

Caspases are the key effector molecules of the physiological death process known as apoptosis, although some are involved in activation of cytokines, rather than cell death. They exist in most of our cells as inactive precursors (zymogens) that kill the cell once activated. Caspases can be controlled in two ways. The processing and activation of a caspase can be regulated by molecules such as FADD, APAF-1, Bcl-2 family members, FLIP and IAPs. Active caspases can be controlled by a variety of inhibitors that directly interact with the protease. This review describes the later direct caspase inhibitors that have been identified, products of both viral and cellular genes, and artificial caspase inhibitors that have been developed both as research tools and as pharmaceutical agents to inhibit cell death in vivo.

Animals↗
PubMed2003

First clinical trial of a novel caspase inhibitor: anti-apoptotic caspase inhibitor, IDN-6556, improves liver enzymes.

OBJECTIVE: To evaluate the safety of IDN-6556, a novel anti-apoptotic pan-caspase inhibitor, administered in single and multiple ascending doses in normal volunteers and patients with hepatic dysfunction. MATERIALS AND METHODS: IDN-6556 was administered as a 30-minute intravenous infusion in rising doses to 3 groups: Group A, normal volunteers, given as a single infusion, Group B, normal volunteers, given q.i.d. for 7 days, Group C, patients with hepatic impairment (elevated transaminases, alanine transaminase, ALT and aspartate transaminase, AST), given q.i.d. for 7 days. RESULTS: The drug was well tolerated up to 10 mg/kg/infusion for a single dose, and 1.5 mg/kg/infusion q.i.d. for 7 days, with the dose-limiting adverse event of phlebitis or inflammation at the site of the infusion. This toxicity was predicted from animal studies. Clinically and statistically meaningful dose-related falls in transaminases were seen in all but 1 of the hepatic impaired patients. Two-way ANOVA analyses of the changes for all the IDN-6556 groups combined versus placebo were: ALT absolute change: p < 0.0001 and % change: p = 0.012, AST absolute and % changes: p < 0.0001. After discontinuation of the drug (after 7 days of dosing), the transaminases rapidly returned to the pre-treatment levels. CONCLUSIONS: Following intravenous administration of a novel anti-apoptotic caspase inhibitor, adverse events were mild-to-moderate in severity, resolved in a few days and did not result in any subject terminating treatment prematurely. The effects in hepatic impaired patients appear to be consistent with both the administration and subsequent abrupt withdrawal of an effective hepatoprotective drug that delays cell death in hepatocytes.

Adult↗
PubMed2003

The caspase inhibitor IDN-6556 prevents caspase activation and apoptosis in sinusoidal endothelial cells during liver preservation injury.

Cold ischemia (CI)-warm reperfusion (WR) liver injury remains a problem in liver transplantation. CI-WR initially causes sinusoidal endothelial cell (SEC) apoptosis through a caspase-dependent mechanism. We previously showed that the caspase inhibitor IDN-1965 prevents CI-WR-induced SEC apoptosis. However, this agent required to be administered to the donor, preservation solution, and recipient for efficacy. Here, we show that a second-generation caspase inhibitor, IDN-6556, effectively prevents CI-WR-induced SEC injury when added only to University of Wisconsin (UW) cold storage media. Rat livers were stored in UW solution for 24 hours at 4 degrees C and reperfused for 1 hour at 37 degrees C. Apoptosis was quantitated using terminal deoxynucleotide transferasemediated deoxyuridine triphosphate nick end labeling (TUNEL) assay and caspase 3 activation determined by biochemical measurement and immunohistochemical analysis. Pan-caspase inhibitors (IDN-8066, IDN-7503, IDN-7436, IDN-1965, and IDN-6556) were applied at preischemic, cold preservation, or reperfusion periods. TUNEL-positive SEC and caspase 3-like activity in the liver was increased by CI-WR. Three caspase inhibitors (IDN-8066, IDN-1965, and IDN-6556) effectively attenuated SEC apoptosis and caspase 3 activation. The most potent inhibitor, IDN-6556, reduced SEC apoptosis and caspase 3 activity by 55% and 94%, respectively. Prevention of SEC apoptosis by IDN-6556 was not reduced when this agent was administered only during the cold preservation period. When added to the preservation solution, the caspase inhibitor IDN-6556 appears to be a feasible therapeutic agent against ischemia-reperfusion injury in liver transplantation.

Adenosine↗
PubMed1998

Calpain inhibitors, but not caspase inhibitors, prevent actin proteolysis and DNA fragmentation during apoptosis.

Apoptosis, or programmed cell death, involves a cascade of regulatory events leading to the activation of specific proteases. However, the key substrates for these proteases remain to be identified. We previously demonstrated that levels of five unidentified polypeptides were specifically increased in neurons from embryonic chicken ciliary ganglia undergoing apoptosis by trophic deprivation. Here we show by microsequencing of two of these polypeptides that they are fragments of actin. One of them represents cleavage of actin at the site of interaction with DNase I. The same actin fragments are also found at early stages of apoptosis in chicken and rat dorsal root ganglion neurons, chicken spinal motoneurons and rat thymocytes. Actin fragmentation may play a role in the apoptotic process, since calpain inhibitors I and II both inhibit neuronal death and suppress actin fragmentation. In contrast, caspase (ICE family) inhibitors, though effective in delaying neuronal death, do not prevent actin cleavage or DNA fragmentation. These results indicate a key role for calpain-like proteases in neuronal programmed cell death and suggest that actin fragmentation in the cell is correlated with subsequent DNA fragmentation.

Actins↗
PubMed1999

A caspase inhibitor fully protects rats against lethal normothermic liver ischemia by inhibition of liver apoptosis.

Apoptosisis activated during the early phase of reperfusion after liver ischemia and after liver transplantation in animals. However, the molecular basis of ischemia-induced cell death remains poorly understood. In this study we show that hepatocytes from ischemic liver lobes undergo apoptosis after reperfusion. In vivo pretreatment of rats with a specific inhibitor of caspases abrogates the apoptotic response in ischemic liver lobes. Inhibition of apoptosis can be accounted for by total inhibition of caspase activation as assessed in an enzymatic assay and by specific affinity labeling. Treatment with a caspase inhibitor fully protects rats from death induced by ischemia/reperfusion. These findings indicate that liver injury after ischemia/reperfusion can be prevented by inhibition of caspases. Thus, caspase inhibitors may have important therapeutic implications in liver ischemic diseases and after liver transplantation.

Animals↗
PubMed2005

A generalized caspase inhibitor disrupts early mammalian development.

The role and mechanism of cell death in early mammalian embryos is not well understood. In mouse embryos collected after fertilization and maintained in vitro until blastula formation, two instances of cell death are observed: the polar bodies and one or two cells near the equator, at the junction of the inner cell mass to the prototrophoblast. Inhibitors of caspases do not block the death of the polar bodies. Inhibitors of caspases 3, 7 and 8 do not affect post-cavitation death, but the pan-caspase inhibitor zVAD-FMK, when applied at the 1-2 cell stage, causes an expansion of post-cavitation death and ultimately malformation or death of the embryo. Our results indicate that the early deaths are not caspase-dependent and that there is a role for caspase activity in early embryos, which is not related to cell death.

Amino Acid Chloromethyl Ketones↗
PubMed2006

Synthesis of novel caspase inhibitors for characterization of the active caspase proteome in vitro and in vivo.

Caspases are cysteine proteases that are essential for cytokine maturation and apoptosis. To facilitate the dissection of caspase function in vitro and in vivo, we have synthesized irreversible caspase inhibitors with biotin attached via linker arms of various lengths (12a-d) and a 2,4-dinitrophenyl labeled inhibitor (13). Affinity labeling of apoptotic extracts followed by blotting reveals that these affinity probes detect active caspases. Using the strong affinity of avidin for biotin, we have isolated affinity-labeled caspase 6 from apoptotic cytosolic extracts of cells overexpressing procaspase 6 by treatment with 12c, which contains biotin attached to the N(epsilon)-lysine of the inhibitor by a 22.5 A linker arm, followed by affinity purification on monomeric avidin-sepharose beads. Compound 13 has proven sufficiently cell permeable to rescue cells from apoptotic execution. These novel caspase inhibitors should provide powerful probes for the study of the active caspase proteome during apoptosis both in vitro and in vivo.

Affinity Labels↗
PubMed2003

MX1013, a dipeptide caspase inhibitor with potent in vivo antiapoptotic activity.

1. Caspases play a critical role in apoptosis, and are considered to be key targets for the design of cytoprotective drugs. As part of our antiapoptotic drug-discovery effort, we have synthesized and characterized Z-VD-fmk, MX1013, as a potent, irreversible dipeptide caspase inhibitor. 2. MX1013 inhibits caspases 1, 3, 6, 7, 8, and 9, with IC50 values ranging from 5 to 20 nm. MX1013 is selective for caspases, and is a poor inhibitor of noncaspase proteases, such as cathepsin B, calpain I, or Factor Xa (IC50 values >10 microm). 3. In several cell culture models of apoptosis, including caspase 3 processing, PARP cleavage, and DNA fragmentation, MX1013 is more active than tetrapeptide- and tripeptide-based caspase inhibitors, and blocked apoptosis at concentrations as low as 0.5 microm. 4. MX1013 is more aqueous soluble than tripeptide-based caspase inhibitors such as Z-VAD-fmk. 5. At a dose of 1 mg kg-1 i.v., MX1013 prevented liver damage and the lethality caused by Fas death receptor activation in the anti-Fas mouse-liver apoptosis model, a widely used model of liver failure. 6. At a dose of 20 mg kg-1 (i.v. bolus) followed by i.v. infusion for 6 or 12 h, MX1013 reduced cortical damage by approximately 50% in a model of brain ischemia/reperfusion injury. 7. At a dose of 20 mg kg-1 (i.v. bolus) followed by i.v. infusion for 12 h, MX1013 reduced heart damage by approximately 50% in a model of acute myocardial infarction. 8. Based on these studies, we conclude that MX1013, a dipeptide pan-caspase inhibitor, has a good combination of in vitro and in vivo properties. It has the ability to protect cells from a variety of apoptotic insults, and is systemically active in three animal models of apoptosis, including brain ischemia.

Amino Acid Chloromethyl Ketones↗
PubMed2002

Use of fluorescently labeled caspase inhibitors as affinity labels to detect activated caspases.

Activation of caspases is the key event of apoptosis and different approaches were developed to assay it. To detect their activation in situ, we applied fluorochrome labeled inhibitors of caspases (FLICA) as affinity labels of active centers of these enzymes. The FLICA ligands are fluorescein or sulforhodamine conjugated peptide-fluoromethyl ketones that covalently bind to enzymatic centers of caspases with 1:1 stoichiometry. The specificity of FLICA towards individual caspases is provided by the peptide sequence of amino acids. Exposure of live cells to FLICA results in uptake of these ligands and their binding to activated caspases; unbound FLICA is removed by cell rinse. Cells labeled with FLICA can be examined by fluorescence microscopy or subjected to quantitative analysis by cytometry. Intracellular binding sites of FLICA are consistent with known localization of caspases. Covalent binding of FLICA allowed us to identify the labeled proteins by immunoblotting: the proteins that bound individual FLICAs had molecular weight between 17 and 22 kDa, which corresponds to large subunits of the caspases. Detection of caspases activation by FLICA can be combined with other markers of apoptosis or cell cycle for multiparametric analysis. Because FLICA are caspase inhibitors they arrest the process of apoptosis preventing cell disintegration. The stathmo-apoptotic method was developed, therefore, that allows one to assay cumulative apoptotic index over long period of time and estimate the rate of cell entry into apoptosis for large cell populations. FLICA offers a rapid and convenient assay of caspases activation and can also be used to accurately estimate the incidence of apoptosis.

Affinity Labels↗
PubMedDate not supplied

Differential modulation of interleukin-2-and interleukin-4-mediated early activation of normal human B lymphocytes by the caspase inhibitor zVAD-fmk.

Caspases are a group of cysteine-related proteases that control the process of apoptosis and may also be involved in the control of lymphocyte activation. We show here that the broad-spectrum caspase inhibitor benzyloxycarbonyl (Cbz)-Val-Ala-Asp (Ome)-fluoromethylketone (zVAD-fmk) prevents the proliferation of resting human B tonsilar lymphocytes mediated by the B cell mitogen SAC or the combination of anti-mu Ab and IL-2. zVAD-fmk inhibits IL-2-induced phosphorylation of the retinoblastoma protein, and cyclin D2 expression. However, neither the IL-2-mediated proliferation of cycling activated B cells nor that of lymphoma cells were inhibited by zVAD-fmk, suggesting that only the early steps of SAC- or IL-2-mediated B cell activation were sensitive to the inhibitory properties of zVAD-fmk. Our data also demonstrated that the inhibitory effect of zVAD-fmk was not observed when B cells were activated with IL-4 in the presence of either anti-mu Ab or anti-CD40 Ab. Thus, our results suggest that caspase activation is required for the IL-2-mediated entry of primary B lymphocytes into the cell cycle and show that caspase activation plays different roles in IL-2- and IL-4-mediated B cell proliferation.

Amino Acid Chloromethyl Ketones↗
PubMed2000

Abrogation of bleomycin-induced epithelial apoptosis and lung fibrosis by captopril or by a caspase inhibitor.

Angiotensin-converting enzyme is involved in apoptosis of alveolar epithelial cells (Wang R, Zagariya A, Ang E, Ibarra-Sunga O, and Uhal BD. Am J Physiol Lung Cell Mol Physiol 277: L1245-L1250, 1999). This study tested the ability of the angiotensin-converting enzyme inhibitor captopril or the caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone (ZVAD-fmk) to block alveolar epithelial cell apoptosis and lung fibrosis in vivo in response to bleomycin (Bleo). Male Wistar rats received 8 U/kg of Bleo (bleomycin sulfate) or vehicle intratracheally. Subgroups of Bleo-treated rats received captopril, ZVAD-fmk, or vehicle alone. Lung collagen was assessed by picrosirius red or hydroxyproline assay at 1, 7, and 14 days post-Bleo, and apoptosis was detected by in situ end labeling (ISEL). Bleo increased alveolar septal and peribronchial collagen by 100 and 133%, respectively (both P < 0.01), by day 14 but not earlier. In contrast, ISEL was increased in alveolar and airway cells at all time points. Captopril or ZVAD-fmk inhibited collagen accumulation by 91 and 85%, respectively (P < 0. 01). Both agents also inhibited ISEL in alveoli by 99 and 81% and in airways by 67 and 63%, respectively. These data suggest that the efficacy of captopril to inhibit experimental lung fibrogenesis is related to inhibition of apoptosis. They also demonstrate the antifibrotic potential of a caspase inhibitor.

Amino Acid Chloromethyl Ketones↗
PubMed2002

In vivo role of caspases in excitotoxic neuronal death: generation and analysis of transgenic mice expressing baculoviral caspase inhibitor, p35, in postnatal neurons.

Caspases, a family of cysteine proteases, are thought to be critical mediators of apoptosis. To examine the role of neuronal caspases in excitotoxic neurodegeneration in vivo, we have generated transgenic mice expressing the baculovirus protein p35, a potent viral caspase inhibitor, using the neuron-specific calmodulin dependent kinase-II alpha (CaMKII-alpha) promoter. The expression of p35 was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry. We analyzed caspase activation and cell death by employing an experimental paradigm, in which the excitotoxin kainate (KA) was injected into CA1 of hippocampus and the distribution of the caspase-generated actin fragment was detected immunohistochemically. While kainate treatment led to selective neuronal death in the CA1, CA3 and CA4 of non-transgenic control mice, we observed restricted caspase activation only in the CA3 sector. The transgenic expression of p35 consistently inhibited the kainate-induced caspase activation, but failed to influence the death of neurons to any extent. In addition, we observed concomitant early calpain activation in the specific areas where neurons underwent degeneration in both the transgenic and non-transgenic mice. These results indicate that p35-inhibitable caspases play rather minor roles in the kainate-induced excitotoxicity and that the relative contribution of calpain is likely to be greater than that of caspases.

Animals↗
PubMed2002

The prevention of the staurosporine-induced apoptosis by Bcl-X(L), but not by Bcl-2 or caspase inhibitors, allows the extensive differentiation of human neuroblastoma cells.

Staurosporine is one of the best apoptotic inducers in different cell types including neuroblastomas. In this study we have compared the efficiency and final outcome of three different anti-apoptotic strategies in staurosporine-treated SH-SY5Y human neuroblastoma cells. At staurosporine concentrations up to 500 nm, z-VAD.fmk a broad-spectrum, noncompetitive inhibitor of caspases, reduced apoptosis in SH-SY5Y cells. At higher concentrations, z-VAD.fmk continued to inhibit caspases and the apoptotic phenotype but not cell death which seems to result from oxidative damage. Stable over-expression of Bcl-2 in SH-SY5Y protected cells from death at doses of staurosporine up to 1 microm. At higher doses, cytochrome c release from mitochondria occurred, caspases were activated and cells died by apoptosis. Therefore, we conclude that Bcl-2 increased the threshold for apoptotic cell death commitment. Over-expression of Bcl-X(L) was far more effective than Bcl-2. Bcl-X(L) transfected cells showed a remarkable resistance staurosporine-induced cytochrome c release and associated apoptotic changes and survived for up to 15 days in 1 microm staurosporine. In these conditions, SH-SY5Y displayed a remarkable phenotype of neuronal differentiation as assessed by neurite outgrowth and expression of neurofilament, Tau and MAP-2 neuronal specific proteins.

Amino Acid Chloromethyl Ketones↗
PubMed2006

AAV-mediated delivery of the caspase inhibitor XIAP protects against cisplatin ototoxicity.

HYPOTHESIS: Delivery of the gene encoding X-linked inhibitor of apoptosis (XIAP) using an adeno-associated viral (AAV) vector can protect against cisplatin-mediated ototoxicity. BACKGROUND: Cisplatin is a widely used chemotherapeutic agent with significant ototoxic side effects. One possible mechanism of toxicity is apoptotic death of many cochlear cell types. Acute treatment with inhibitors of caspases- enzymes critical for apoptosis- has been shown to prevent hearing loss in vivo, but is too short-acting for therapeutic use. Gene therapy provides a specific and chronic means of delivering potential therapeutic gents. Introducing an anti-apoptotic gene into the cochlea could provide long-term prophylaxis against the ototoxic effects of cisplatin. METHOD: Two groups of rats were treated with unilateral injection into the round window of AAV harboring a gene encoding either XIAP or green fluorescent protein (GFP). After at least two months of gene expression, auditory-brainstem-response (ABR) threshold shifts and outer-hair-cell (OHC) number were measured in these two groups of animals after 72-hour treatment with cisplatin. RESULTS: Consistent with previous reports, uninjected and AAV.GFP-injected ears displayed profound ABR threshold elevations and OHC loss after cisplatin treatment. Ears that had been injected with AAV encoding XIAP, however, were significantly protected from these effects: cisplatin-induced ABR-threshold shift and hair-cell loss were attenuated by as much as 78% and 45%, respectively, when compared with contralateral (untreated) ears. CONCLUSION: XIAP delivery to the cochlea can protect against the audiometric changes and hair-cell loss associated with cisplatin ototoxicity. The efficacy, specificity, and duration of the protective effects make this a potentially attractive therapeutic paradigm.

Animals↗
PubMed2006

Exchange of P/V genes between two non-cytopathic simian virus 5 variants results in a recombinant virus that kills cells through death pathways that are sensitive to caspase inhibitors.

The paramyxovirus Simian virus 5 (SV5) is largely non-cytopathic in human epithelial and fibroblast cells. WF-PIV has been described previously as a naturally occurring SV5 variant that encodes P and V proteins differing from the wild-type (WT) SV5 proteins in eight and five amino acid positions, respectively. In this study, it is shown that WF-PIV is like WT SV5 by being largely non-cytopathic in A549 lung epithelial cells. However, substitution of the WF-PIV P/V gene into the background of WT SV5 resulted in a hybrid virus (P/V-WF) that induced apoptotic cell death not seen with either of the parental viruses. The kinetics of HeLa cell killing and induction of apoptosis by the P/V-WF chimera differed from those of the previously described P/V-CPI- chimera by being slower and less extensive. HeLa cell killing by the P/V-WF chimera was effectively reduced by inhibitors of caspase-9, but not of caspase-8. These results demonstrate that an exchange of P/V genes from two non-cytopathic SV5 variants can produce apoptosis-inducing chimeras, and that the role of the SV5 P/V gene products in limiting apoptosis can be dependent on expression in the context of a native viral genome.

Apoptosis↗