Detection of caspase activation in situ by fluorochrome-labeled caspase inhibitors

Apoptosis is dependent on the activation of a group of proteolytic enzymes called caspases. Caspase activation can be detected by immunoblotting using caspase-specific antibodies or by caspase activity measurement employing pro-fluorescent substrates that become fluorescent upon cleavage by the caspase. Most of these methods require the preparation of cell extracts and, therefore, are not suitable for the detection of active caspases within the living cell. Using FAM-VAD-FMK, we have developed a simple and sensitive assay for the detection of caspase activity in living cells. FAM-VAD-FMK is a carboxyfluorescein (FAM) derivative of benzyloxycarbonyl-valine-alanine-aspartic acid-fluoromethyl ketone (zVAD-FMK), which is a potent broad-spectrum inhibitor of caspases. FAM-VAD-FMK enters the cell and irreversibly binds to activated caspases. Cells containing bound FAM-VAD-FMK can be analyzed by flow cytometry, fluorescence microscopy, or a fluorescence plate reader. Using FAM-VAD-FMK, we have measured caspase activation in live non-adherent and adherent cells. We show that FAM-VAD-FMK labeled Jurkat and HeLa cells that had undergone apoptosis following treatment with camptothecin or staurosporine. Non-stimulated negative control cells were not stained. Pretreatment with the general caspase inhibitor zVAD-FMK blocked caspase-specific staining in induced Jurkat and HeLa cells. Pretreatment of staurosporine-induced Jurkat cells with FAM-VAD-FMK inhibited affinity labeling of caspase-3, -6, and -7, blocked caspase-specific cell staining, and led to the inhibition of apoptosis. In contrast, the fluorescent control inhibitor FAM-FA-FMK had no effect. Measurement of caspase activation in 96-well plates showed a 3- to 5-fold increase in FAM-fluorescence in staurosporine-treated cells compared to control cells. In summary, we show that FAM-VAD-FMK is a versatile and specific tool for detecting activated caspases in living cells.     

Synthesis and evaluation of novel prodrugs of caspase inhibitors

A novel type of caspase inhibitor prodrug that improves systemic exposure after oral administration in rats has been designed. Such a prodrug, based on a 6,6a-dihydrofuro[3,2-d]oxazol-5(3aH)-one motif, has the advantage of rapidly liberating the active inhibitor without producing any cleavage by-product. Prodrugs 6-8, are synthesised in a high yielding one-step transformation from the active parents with high diastereomeric excess.     

A class of allosteric caspase inhibitors identified by high-throughput screening

Caspase inhibition is a promising approach for treating multiple diseases. Using a reconstituted assay and high-throughput screening, we identified?a group?of nonpeptide caspase inhibitors. These inhibitors share common chemical scaffolds, suggesting the same mechanism of action. They can inhibit apoptosis in various cell types induced by multiple stimuli; they can also inhibit caspase-1-mediated interleukin generation in macrophages, indicating potential anti-inflammatory application. While these compounds inhibit all the tested caspases, kinetic analysis indicates they do not compete for the catalytic sites of the enzymes. The cocrystal structure of one of these compounds with caspase-7 reveals that it binds to the dimerization interface of the caspase, another common structural element shared by all active caspases. Consistently, biochemical analysis demonstrates that the compound abates caspase-8 dimerization. Based on these kinetic, biochemical, and structural analyses, we suggest that these compounds are allosteric caspase inhibitors that function through binding to the dimerization interface of caspases.     

Oxamyl dipeptide caspase inhibitors developed for the treatment of stroke

Structural modifications were made to a previously described acyl dipeptide caspase inhibitor, leading to the oxamyl dipeptide series. Subsequent SAR studies directed toward the warhead, P2, and P4 regions of this novel peptidomimetic are described herein.     

Natural inhibitors targeting osteoclast-mediated bone resorption

Human cathepsin K, matrix metalloproteinase 9, and alpha(V)beta(3) integrin are the key regulators in osteoclast-mediated bone resorption. In this paper, we found natural inhibitors 1-10 for them by enzyme inhibition assays. Inhibitors 1-7, 8-9, and 10 are novel inhibitors of human cathepsin K, matrix metalloproteinase 9, and alpha(V)beta(3), respectively.     

Chemical caspase inhibitors enhance cell culture viabilities and protein titer

Mammalian cell cultures are integral to the production of therapeutic and diagnostic proteins. A common problem encountered in culturing these cell lines, however, is a loss in viability at later stages of the cell culture process. In this study the effects of three newly synthesized chemical caspase inhibitors were investigated for their capacity to inhibit cell death. Findings show that these protease inhibitors were successful in prolonging cellular viabilities among anchorage-dependent CHO-K1 and HEK-293 cells lines. Cells treated with one inhibitor, 7312, performed as well or better when compared with the commercially known caspase inhibitor, zVAD.fmk. Suspension CHO cells producing an IgG were used to investigate the capacity of 7312 to improve protein production. Treatment of cells with 7312 increased integrated cell densities by 33% with treated cells having a higher maximum cell density and higher viability. As a result, monoclonal antibody titers increased by 20% and higher in spinner flask experiments. Increasing productivity in mammalian cell cultures has key implications for the pharmaceutical and biotechnology sectors, which are presently focused on developing methods to enhance cell performance in bioreactor environments.     

Yeast techniques for modeling drugs targeting Bcl-2 and caspase family members

Development of drugs targeting Bcl-2 relatives and caspases, for treating diseases including cancer and inflammatory disorders, often involves measuring interactions with recombinant target molecules, and/or monitoring cancer cell killing in vitro. Here, we present yeast-based methods for evaluating drug-mediated inhibition of Bcl-2 relatives or caspases. Active Bax and caspases kill Saccharomyces cerevisiae, and pro-survival Bcl-2 proteins can inhibit Bax-induced yeast death. By measuring the growth or adenosine triphosphate content of transformants co-expressing Bax with pro-survival Bcl-2 relatives, we found that the Bcl-2 antagonist drugs ABT-737 or ABT-263 abolished Bcl-2 or Bcl-x_L function and reduced Bcl-w activity, but failed to inhibit Mcl-1, A1 or the poxvirus orthologs DPV022 and SPPV14. Using this technique, we also demonstrated that adenoviral E1B19K was resistant to these agents. The caspase inhibitor Q-VD-OPh suppressed yeast death induced by caspases 1 and 3. Yeast engineered to express human apoptotic regulators enable simple, automatable assessment of the activity and specificity of candidate drugs targeting Bcl-2 relatives or caspases.     

Peptides of arachnid venoms with insecticidal activity targeting sodium channels

Arachnids have a venom apparatus and secrete a complex chemical mixture of low molecular mass organic molecules, enzymes and polypeptide neurotoxins designed to paralyze or kill their prey. Most of these toxins are specific for membrane voltage-gated sodium channels, although some may also target calcium or potassium channels and other membrane receptors. Scorpions and spiders have provided the greatest number of the neurotoxins studied so far, for which, a good number of primary and 3D structures have been obtained. Structural features, comprising a folding that determines a similar spatial distribution of charged and hydrophobic side chains of specific amino acids, are strikingly common among the toxins from spider and scorpion venoms. Such similarities are, in turn, the key feature to target and bind these proteins to ionic channels. The search for new insecticidal compounds, as well as the study of their modes of action, constitutes a current approach to rationally design novel insecticides. This goal tends to be more relevant if the resistance to the conventional chemical products is considered. A promising alternative seems to be the biotechnological approach using toxin-expressing recombinant baculovirus. Spider and scorpion toxins having insecticidal activity are reviewed here considering their structures, toxicities and action mechanisms in sodium channels of excitable membranes.     

The IAP family：endogenous caspase inhibitors with multiple biological activities

IAPs (inhibitors of apoptosis) are a family of proteins containing one or more characteristic BIR domains.These proteins have multiple biological activities that include binding and inhibiting caspases,regulating cell cycle progression,and modulating receptor-mediated signal transduction.Our recent studies found the IAP family members XIAP and c-IAP1 are ubiquitinated and degraded in proteasomes in response to apoptotic stimuli in T cells,and their degradation appears to be important for T cells to commit to death.In addition to three BIR domains,each of these IAPs also contains a RING finger domain. We found this region confers ubiquitin protease ligase(E3) activity to IAPs,and is responsible for the auto-ubiquitination and degradation of IAPs after an apoptotic stimulus.Given the fact that IAPs can bind a variety of proteins,such as caspases and TRAFs,it will be of interest to characterize potential substrates of the E3 activity of IAPs and the effects of ubiquitination by IAPs on signal transduction,cell cycle,and apoptosis.     

Study of caspase inhibitors for limiting death in mammalian cell culture

Apoptosis in mammalian cell culture is associated with decreased bioproduct yields and can be inhibited through altering the intracellular signaling pathways mediating programmed cell death. In this study, we evaluated the capacity to inhibit caspases to maintain high viable cell numbers in CHO and 293 cultures. Two genetic caspase inhibitors, XIAP and CrmA, were examined along with a mutant of each, XIAP-BIR123NC, which contains three BIR domains but lacks the RING finger, and CrmA-DQMD, which has CrmA's pseudosubstrate site replaced with that of another caspase inhibitor, p35. Stable CHO pooled and 293 clonal cell lines expressing each protein were exposed to apoptotic insults, including spent medium, Sindbis virus, and etoposide. For each insult the mutated protein resulted in higher viabilities than its wild-type counterpart. However, the mutants provided different levels of protection, depending on the insult considered. CrmA-DQMD was the preferred inhibitor for spent medium-induced apoptosis, whereas XIAP-BIR123NC conferred better protection for etoposide-induced death. Addition of Z-VAD.fmk to the genetically engineered cells enhanced viabilities in the presence of spent medium or etoposide; however, the largest increases in viability were experienced by the control cells, indicating an overlap in caspase inhibition between the genetic and chemical inhibitors. Finally, parental 293 cells were treated with caspase-8 and -9 inhibitors, Z-IETD.fmk and Z-LEHD.fmk, in concert with spent medium or etoposide exposure. Spent medium-induced death was delayed more readily with the caspase-8 inhibitors, CrmA-DQMD and Z-IETD.fmk, and etoposide-induced death was stalled more so with XIAP-BIR123NC and Z-LEHD.fmk. These results suggest that the apoptosis pathways induced and the level of protection afforded by a particular caspase inhibitor may vary with the insult considered.     

Synthesis and evaluation of novel dipeptidyl benzoyloxymethyl ketones as caspase inhibitors

We describe novel peptide-based caspase inhibitors. Potent and comparatively selective compounds containing a dipeptide scaffold and a substituted oxymethyl ketone as a warhead were developed. The newly synthesized compounds were tested for inhibition in in vitro enzymatic assays of caspases-1, -3, -6, -8, and -9. The benzyloxycarbonyl-phenylglycyl-aspartyl benzoyloxymethyl ketone (Z-Phg-Asp-CH2OCO-Ph, coded as HU44) was the most potent inhibitor of caspase-1 and caspase-3. Of several analogs of HU44 that were made, the beta-Asp methyl ester (2) is an effective inhibitor against caspase-3 and caspase-8, and less effective against caspase-1. These compounds did not inhibit caspase-6 and caspase-9 significantly.     

Novel non-peptide inhibitors targeting death receptor-mediated apoptosis

We have previously reported that ECH, (2R, 3R, 4S)-2,3-epoxy-4-hydroxy-5-hydroxymethyl-6-(1E)-propenyl-cyclohex-5-en-1-one inhibits Fas-mediated apoptosis by blocking self-activation of pro-caspase-8 in the death-inducing signaling complex (DISC). A series of ECH derivatives were asymmetrically synthesized via key synthetic intermediates obtained from lipase-catalyzed kinetic resolution. Inhibitory activities of the derivatives towards death receptor-mediated apoptosis both in type I and type II cells were investigated, revealing that novel non-peptide inhibitors, RKTS-33 and RKTS-34, are effective as ECH.     

Acyl dipeptides as reversible caspase inhibitors. Part 2: further optimization

A new structural class of broad spectrum caspase inhibitors was optimized for its activity against caspases 1, 3, 6, 7, and 8. The most potent compound had low nanomolar broad spectrum activity, in particular, single digit nanomolar inhibitory activity against caspase 8.     

Differential effects of caspase inhibitors on endotoxin-induced myocardial dysfunction and heart apoptosis

Endotoxin is one of the major factors causing myocardial depression and death during sepsis in humans. Recently, it was reported that endotoxin may induce cardiomyocyte apoptosis. Also, multiple caspase activation has been implicated in endotoxin-induced apoptosis in several organ systems. In this study, we investigated whether endotoxin would increase myocardial caspase activities and evaluated the effects of in vivo administration (3 mg/kg) of the broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone(z-VAD.fmk), the caspase-3-like inhibitor benzyloxycarbonyl-Asp-Glu-Val-Asp-chloromethylketone (z-DEVD.cmk), and the caspase-1-like inhibitor acetyl-Tyr-Val-Ala-Asp-chloromethylketone (Ac-YVAD. fmk), on endotoxin-induced myocardial dysfunction and apoptosis. Endotoxin administration (10 mg/kg iv) induced myocardial contractile dysfunction that was associated with caspase activity increases and nuclear apoptosis. Broad-spectrum z-VAD.fmk and z-DEVD.cmk improved endotoxin-induced myocardial dysfunction and reduced caspase activation and nuclear apoptosis when given immediately and 2 h after endotoxin. In contrast, no effects of Ac-YVAD.fmk were observed on myocardial function and caspase-induced apoptosis. Administration of caspase inhibitors 4 h after endotoxin treatment was not able to protect the rat heart from myocardial dysfunction and nuclear apoptosis. These observations provide evidence that in our model, caspase activation plays a role in endotoxin-induced myocardial apoptosis. Caspase inhibition strategy may represent a therapeutic approach to endotoxin-induced myocardial dysfunction.     

Antiviral activity of caspase inhibitors: effect on picornaviral 2A proteinase

Peptide-based fluoromethyl ketones have been considered for many years to be highly specific caspase inhibitors distinctly blocking the progress of apoptosis in a variety of systems. Here we demonstrate that these compounds can significantly reduce rhinovirus multiplication in cell culture. In their methylated forms they block eIF4GI cleavage in vivo and in vitro and inhibit the activity of picornaviral 2A proteinases.