RESUMO
Previous studies have shown that caspase inhibitors are effective at protecting against anti-Fas antibody (alpha-Fas)-mediated liver injury/lethality. The purpose of these experiments was to characterize more fully the efficacy of a broad-spectrum, irreversible caspase inhibitor, IDN-1965 (N-[(1,3-dimethylindole-2-carbonyl)valinyl]-3-amino-4-oxo-5-fluoropentanoic acid), in this model and the role of caspase inhibition in long-term protection. The ED(50) for IDN-1965 by i.p. administration, based on alanine aminotransferase activities, was 0.14 mg/kg. The caspase inhibitor was also efficacious when administered intravenously and orally (ED(50) values of 0.04 and 1.2 mg/kg, respectively). Histologically, marked reduction in Fas-induced apoptosis with IDN-1965 (1 mg/kg, i.p.) was apparent at 6 h. Also, caspase 3-like activities were decreased in a dose-dependent manner, but the inhibition of caspase activity was transient. Immunohistochemical studies demonstrated that IDN-1965 greatly reduced the activation of caspase 3. In survival studies, a single i.p. treatment of 1 mg/kg IDN-1965 or continuous i.p. infusion via osmotic pumps completely blocked lethality measured up to 7 days after alpha-Fas administration. IDN-1965 was also effective in inhibiting liver injury when administered as long as 3 h after or 1 h before alpha-Fas administration. Lastly, Western blot analysis demonstrated that processing of caspases 3, 6, and 8, as well as Bid (a protein responsible for the release of mitochondrial cytochrome C and amplification of the apoptotic cascade) was inhibited by IDN-1965. In conclusion, the broad-spectrum caspase inhibitor IDN-1965 is markedly effective at inhibiting Fas-mediated apoptosis by multiple routes of administration. The therapeutic potential of caspase inhibitors appears promising for the treatment of apoptosis-mediated liver injury based on potency and postinsult efficacy.
Assuntos
Apoptose/efeitos dos fármacos , Inibidores de Caspase , Doença Hepática Induzida por Substâncias e Drogas/patologia , Inibidores de Cisteína Proteinase/farmacologia , Indóis/farmacologia , Oligopeptídeos/farmacologia , Alanina Transaminase/metabolismo , Animais , Western Blotting , Imuno-Histoquímica , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Receptor fas/genéticaRESUMO
The mechanism by which membrane-bound Bcl-2 inhibits the activation of cytoplasmic procaspases is unknown. Here we characterize an intracellular, membrane-associated form of procaspase-3 whose activation is controlled by Bcl-2. Heavy membranes isolated from control cells contained a spontaneously activatable caspase-3 zymogen. In contrast, in Bcl-2 overexpressing cells, although the caspase-3 zymogen was still associated with heavy membranes, its spontaneous activation was blocked. However, Bcl-2 expression had little effect on the levels of cytoplasmic caspase activity in unstimulated cells. Furthermore, the membrane-associated caspase-3 differed from cytosolic caspase-3 in its responsiveness to activation by exogenous cytochrome c. Our results demonstrate that intracellular membranes can generate active caspase-3 by a Bcl-2-inhibitable mechanism, and that control of caspase activation in membranes is distinct from that observed in the cytoplasm. These data suggest that Bcl-2 may control cytoplasmic events in part by blocking the activation of membrane-associated procaspases.
Assuntos
Caspases/metabolismo , Precursores Enzimáticos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Apoptose , Caspase 3 , Inibidores de Caspase , Linhagem Celular , Cumarínicos/metabolismo , Grupo dos Citocromos c/farmacologia , Ativação Enzimática , Precursores Enzimáticos/antagonistas & inibidores , Humanos , Hidrólise , Oligopeptídeos/metabolismo , Frações Subcelulares/metabolismoRESUMO
A series of 7-(di)alkyl and spirocyclic substituted azepinones were generated and incorporated as conformationally restricted dipeptide surrogates in mercaptoacyl dipeptides. Clear structure-activity relationships with respect to both angiotensin-converting enzyme (ACE) and neutral endopeptidase (NEP) activity in vitro were observed. The best in this series, compound 1g, a geminally dimethylated C-7-substituted azepinone, demonstrated excellent blood pressure lowering in animal models. Compound 1g (BMS-189921) is characterized by a good duration of activity and excellent oral efficacy in models relevant to ACE or NEP inhibition, and its activity is comparable to that of the clinically efficacious agent omapatrilat. Consequently this inhibitor has been advanced clinically for the treatment of hypertension and congestive heart failure.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Azepinas/química , Dipeptídeos/química , Neprilisina/antagonistas & inibidores , Peptidil Dipeptidase A/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Inibidores da Enzima Conversora de Angiotensina/química , Animais , Macaca fascicularis , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Inibidores de Proteases/química , RatosRESUMO
A systematic study of interleukin-1 beta converting enzyme (ICE, caspase-1) and caspase-3 (CPP32, apopain) inhibitors incorporating a P2-P3 conformationally constrained dipeptide mimetic is reported. Depending on the nature of the P4 substituent, highly selective inhibitors of both Csp-1 or Csp-3 were obtained.
Assuntos
Inibidores de Caspase , Inibidores de Cisteína Proteinase/síntese química , Inibidores de Cisteína Proteinase/farmacologia , Oligopeptídeos/síntese química , Oligopeptídeos/farmacologia , Caspase 3 , Inibidores de Cisteína Proteinase/química , Dipeptídeos/síntese química , Dipeptídeos/química , Dipeptídeos/farmacologia , Humanos , Conformação Molecular , Oligopeptídeos/química , Relação Estrutura-AtividadeRESUMO
The cysteine protease CPP32 has been expressed in a soluble form in Escherichia coli and purified to >95% purity. The three-dimensional structure of human CPP32 in complex with the irreversible tetrapeptide inhibitor acetyl-Asp-Val-Ala-Asp fluoromethyl ketone was determined by x-ray crystallography at a resolution of 2.3 A. The asymmetric unit contains a (p17/p12)2 tetramer, in agreement with the tetrameric structure of the protein in solution as determined by dynamic light scattering and size exclusion chromatography. The overall topology of CPP32 is very similar to that of interleukin-1beta-converting enzyme (ICE); however, differences exist at the N terminus of the p17 subunit, where the first helix found in ICE is missing in CPP32. A deletion/insertion pattern is responsible for the striking differences observed in the loops around the active site. In addition, the P1 carbonyl of the ketone inhibitor is pointing into the oxyanion hole and forms a hydrogen bond with the peptidic nitrogen of Gly-122, resulting in a different state compared with the tetrahedral intermediate observed in the structure of ICE and CPP32 in complex with an aldehyde inhibitor. The topology of the interface formed by the two p17/p12 heterodimers of CPP32 is different from that of ICE. This results in different orientations of CPP32 heterodimers compared with ICE heterodimers, which could affect substrate recognition. This structural information will be invaluable for the design of small synthetic inhibitors of CPP32 as well as for the design of CPP32 mutants.
Assuntos
Caspases , Cisteína Endopeptidases/química , Inibidores de Cisteína Proteinase/química , Clorometilcetonas de Aminoácidos/química , Sítios de Ligação , Caspase 3 , Cristalografia por Raios X , Cisteína Endopeptidases/ultraestrutura , Escherichia coli , Humanos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Oligopeptídeos/química , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas RecombinantesRESUMO
Neuronal apoptosis occurs during nervous system development and after pathological insults to the adult nervous system. Inhibition of CED3/ICE-related proteases has been shown to inhibit neuronal apoptosis in vitro and in vivo, indicating a role for these cysteine proteases in neuronal apoptosis. We have studied the activation of the CED3/ICE-related protease CPP32 in two in vitro models of mouse cerebellar granule neuronal cell death: K+/serum deprivation-induced apoptosis and glutamate-induced necrosis. Pretreatment of granule neurons with a selective, irreversible inhibitor of CED3/ICE family proteases, ZVAD-fluoromethylketone, specifically inhibited granule neuron apoptosis but not necrosis, indicating a selective role for CED3/ICE proteases in granule neuron apoptosis. Extracts prepared from apoptotic, but not necrotic, granule neurons contained a protease activity that cleaved the CPP32 substrate Ac-DEVD-aminomethylcoumarin. Induction of the protease activity was prevented by inhibitors of RNA or protein synthesis or by the CED3/ICE protease inhibitor. Affinity labeling of the protease activity with an irreversible CED3/ICE protease inhibitor, ZVK(biotin)D-fluoromethylketone, identified two putative protease subunits, p20 and p18, that were present in apoptotic but not necrotic granule neuron extracts. Western blotting with antibodies to the C terminus of the large subunit of mouse CPP32 (anti-CPP32) identified p20 and p18 as processed subunits of the CPP32 proenzyme. Anti-CPP32 specifically inhibited the DEVD-amc cleaving activity, verifying the presence of active CPP32 protease in the apoptotic granule neuron extracts. Western blotting demonstrated that the CPP32 proenzyme was expressed in granule neurons before induction of apoptosis. These results demonstrate that the CED3/ICE homolog CPP32 is processed and activated during cerebellar granule neuron apoptosis. CPP32 activation requires macromolecular synthesis and CED3/ICE protease activity. The lack of CPP32 activation during granule neuron necrosis suggests that proteolytic processing and activation of CED3/ICE proteases are specific biochemical markers of apoptosis.
Assuntos
Apoptose/fisiologia , Caspases , Córtex Cerebelar/enzimologia , Cisteína Endopeptidases/metabolismo , Precursores Enzimáticos/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Caspase 3 , Células Cultivadas , Córtex Cerebelar/citologia , Cumarínicos/farmacologia , Meios de Cultura Livres de Soro/farmacologia , Cicloeximida/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Dipeptídeos/farmacologia , Ativação Enzimática/efeitos dos fármacos , Ácido Glutâmico/farmacologia , Cetonas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Necrose , Inibidores da Síntese de Ácido Nucleico/farmacologia , Oligopeptídeos/farmacologia , Potássio/farmacologia , Inibidores da Síntese de Proteínas/farmacologiaRESUMO
The human proto-oncogene bcl-2 and its Caenorhabditis elegans homologue ced-9 inhibit programmed cell death. In contrast, members of the human interleukin-1beta converting enzyme (ICE) family of cysteine proteases and their C. elegans homologue CED-3 promote the death program. Genetic experiments in C. elegans have shown that ced-9 is formally a negative regulator of ced-3 function, but neither those studies nor others have determined whether CED-9 or Bcl-2 proteins act biochemically upstream or downstream of CED-3/ICE proteases. CPP32, like all known members of the CED-3/ICE family, is synthesized as a proenzyme that is subsequently processed into an active protease with specificity for cleavage at Asp-X peptide bonds. In this report, we demonstrate that the CPP32 proenzyme is proteolytically processed and activated in Jurkat cells induced to die by Fas ligation. CPP32 activation is blocked by cell-permeable inhibitors of aspartate-directed, cysteine proteases, suggesting that pro-CPP32 is cleaved by active CPP32 or by other ICE family members. Heterologous expression of Bcl-2 in Jurkat cells prevents Fas-induced cell death as well as proteolytic processing and activation of CPP32. Thus, Bcl-2 acts at or upstream of the CPP32 activation step to inhibit apoptosis induced by Fas stimulation.
Assuntos
Caspases , Cisteína Endopeptidases/efeitos dos fármacos , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Receptor fas/metabolismo , Sequência de Aminoácidos , Apoptose , Caspase 1 , Caspase 3 , Linhagem Celular , Ativação Enzimática , Humanos , Hidrólise , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-bcl-2RESUMO
Angiotensin-converting enzyme (ACE), the receptor for an important new class of antihypertensive drugs, is now one of the better studied zinc metallopeptidases. The development of several classes of tightly binding competitive inhibitors of ACE has led to increased understanding of the structure and function of this enzyme while also yielding important new drugs for the diagnosis and treatment of hypertensive disease. Peptides from snake venom provided the first proof of the therapeutic utility of ACE inhibitors, and a tripeptide sequence, Phe-Ala-Pro, was used as a model for sidechain interactions with ACE in the rational design of simpler nonpeptidic inhibitors such as captopril and enalapril. These and more recently developed ACE inhibitors can be classified according to their structural analogy to dipeptides or tripeptides and according to the nature of their zinc-binding ligands, such as sulfhydryl, ketone, carboxylate, or hydroxyphosphinyl, that contribute greatly to their binding to ACE. Several newer ACE inhibitors have increased potency and/or improved pharmacokinetic properties due to modifications such as substitution of the proline ring or replacement of the methyl side chain analogous to Ala by an aminobutyl residue analogous to Lys. The availability of structurally diverse ACE inhibitors with great potency and specificity provides a powerful biochemical tool for purification, localization, and characterization of ACE in different tissues, and for distinguishing related zinc metallopeptidases with similar properties.