RESUMO
Aza-peptide Michael acceptors are a novel class of inhibitors that are potent and specific for caspases-2, -3, -6, -7, -8, -9, and -10. The second-order rate constants are in the order of 10(6) M(-1) s(-1). The aza-peptide Michael acceptor inhibitor 18t (Cbz-Asp-Glu-Val-AAsp-trans-CH=CH-CON(CH(2)-1-Naphth)(2) is the most potent compound and it inhibits caspase-3 with a k(2) value of 5620000 M(-1) s(-1). The inhibitor 18t is 13700, 190, 6.4, 594, 37500, and 173-fold more selective for caspase-3 over caspases-2, -6, -7, -8, -9, and -10, respectively. Aza-peptide Michael acceptors designed with caspase specific sequences are selective and do not show any cross reactivity with clan CA cysteine proteases such as papain, cathepsin B, and calpains. High-resolution crystal structures of caspase-3 and caspase-8 in complex with aza-peptide Michael acceptor inhibitors demonstrate the nucleophilic attack on C2 and provide insight into the selectivity and potency of the inhibitors with respect to the P1' moiety.
Assuntos
Compostos Aza/síntese química , Inibidores de Caspase , Oligopeptídeos/síntese química , Compostos Aza/química , Caspase 10 , Caspase 2 , Caspase 3 , Caspase 6 , Caspase 7 , Caspase 9 , Caspases/química , Cristalografia por Raios X , Cinética , Modelos Moleculares , Oligopeptídeos/química , Relação Estrutura-AtividadeRESUMO
AIMS: Due to pralidoxime chloride's (2-PAM) positive charge, it's penetration through the blood brain barrier (BBB) and reactivation of organophosphate (OP) inhibited central nervous system (CNS) acetylcholinesterase (AChE) is poor. The results of CNS inhibited AChE are seizures. Pro-2-PAM (1-methyl-1,6-dihydropyridine-2-carbaldoxime), a pro-drug of 2-PAM, due to higher hydrophobicity, penetrates the BBB better but must be oxidized to 2-PAM, the active form of the oxime to reactivate CNS AChE in order to abrogate seizures. In this study, we characterize the in vivo mechanism of pro-2-PAM oxidation. MAIN METHODS: A high pressure liquid chromatography (HPLC) assay was developed to quantify the conversion of pro-2-PAM to 2-PAM. NADPH oxidase activity was measured by a photo-luminescence assay using lucigenin substrate. Upon analysis, the rate of NADPH induced oxidation suggested that an alternate mechanism may be involved. Therefore, various enzyme co-factors of oxidation-reduction enzyme systems were evaluated, including nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), flavin adenine dinucleotide (FAD), riboflavin 5'-phosphate (FMN), and riboflavin. Next, a spectrophotometric assay was developed to measure the conversion of pro-2-PAM to 2-PAM in the presence of riboflavin. KEY FINDINGS: In guinea pig brain homogenate, diphenyleneiodonium (DPI), a specific NADPH oxidase inhibitor, reduced pro-2-PAM to 2-PAM conversion to less than 25%. In contrast, riboflavin, FAD, and FMN rapidly oxidized all pro-2-PAM to 2-PAM in an in vitro assay. Riboflavin oxidized pro-2-PAM reactivated diisopropylfluorophosphate (DFP) inhibited AChE. SIGNIFICANCE: The present study shows that pro-2-PAM was rapidly oxidized by riboflavin to 2-PAM, which reactivated organophosphate (OP)-inhibited AChE.
Assuntos
Acetilcolinesterase/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , NADPH Oxidases/metabolismo , Compostos de Pralidoxima/metabolismo , Animais , Cobaias , Oniocompostos/farmacologia , Oxirredução , Pró-Fármacos , Ratos , Riboflavina/farmacologia , Espectrofotometria/métodosRESUMO
Small ubiquitin-related modifier (SUMO) is implicated in the regulation of numerous biological processes including transcription, protein localization, and cell cycle control. Protein modification by SUMO is found in Plasmodium falciparum; however, its role in the regulation of the parasite life cycle is poorly understood. Here we describe functional studies of a SUMO-specific protease (SENP) of P. falciparum, PfSENP1 (PFL1635w). Expression of the catalytic domain of PfSENP1 and biochemical profiling using a positional scanning substrate library demonstrated that this protease has unique cleavage sequence preference relative to the human SENPs. In addition, we describe a class of small molecule inhibitors of this protease. The most potent lead compound inhibited both recombinant PfSENP1 activity and P. falciparum replication in infected human blood. These studies provide valuable new tools for the study of SUMOylation in P. falciparum.
Assuntos
Hidrazinas/farmacologia , Ácidos Ftálicos/farmacologia , Plasmodium falciparum/enzimologia , Inibidores de Proteases/farmacologia , Proteínas de Protozoários/química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Cisteína Endopeptidases , Endopeptidases/metabolismo , Humanos , Hidrazinas/química , Dados de Sequência Molecular , Ácidos Ftálicos/química , Inibidores de Proteases/química , Proteínas de Protozoários/classificação , Proteínas de Protozoários/genética , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
The US Army utilizes pralidoxime (2-PAM) for the reactivation of OP-inhibited AChE. While 2-PAM effectively reactivates acetylcholinesterase (AChE) in the body, it does not cross the blood-brain barrier (BBB) at therapeutically relevant levels. To address this problem of central nervous system AChE reactivation, novel sugar-oxime conjugates were utilized. These 'sugar-oximes' would potentially be transported across the BBB because they contain a sugar moiety which would be recognized by the facilitative glucose transporters. Eight previously reported, but understudied sugar-oximes, as well as six novel sugar-oximes were synthesized, and their ability to reactivate both human red blood cell AChE and plasma butyrylcholinesterase poisoned with DFP, paraoxon, sarin and VX were tested. The results show that the novel sugar-oxime 13c was more active than the other compounds with a reactivation potential similar to 2-PAM. The sugar-oxime 8b had low toxicity with a LD(50) of 1,590 mg/kg from a single IM dose in the guinea pig and >2,000 mg/kg IP in the mouse. Histopathological analysis showed that there were no apparent differences in hippocampus, heart, liver, kidney sciatic nerve, or skeletal muscle between treated and untreated animals. These results show that sugar-oximes can be effective reactivators and suggest that high treatment doses may be possible.
Assuntos
Acetilcolinesterase/metabolismo , Barreira Hematoencefálica/metabolismo , Butirilcolinesterase/metabolismo , Reativadores da Colinesterase/metabolismo , Reativadores da Colinesterase/farmacologia , Oximas/metabolismo , Oximas/farmacologia , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Carboidratos/química , Inibidores da Colinesterase/toxicidade , Reativadores da Colinesterase/síntese química , Reativadores da Colinesterase/toxicidade , Ativação Enzimática/efeitos dos fármacos , Cobaias , Humanos , Camundongos , Organofosfatos/toxicidade , Oximas/síntese química , Oximas/toxicidade , Testes de ToxicidadeRESUMO
Novel therapeutics to overcome the toxic effects of organophosphorus (OP) chemical agents are needed due to the documented use of OPs in warfare (e.g. 1980-1988 Iran/Iraq war) and terrorism (e.g. 1995 Tokyo subway attacks). Standard OP exposure therapy in the United States consists of atropine sulfate (to block muscarinic receptors), the acetylcholinesterase (AChE) reactivator (oxime) pralidoxime chloride (2-PAM), and a benzodiazepine anticonvulsant to ameliorate seizures. A major disadvantage is that quaternary nitrogen charged oximes, including 2-PAM, do not cross the blood brain barrier (BBB) to treat brain AChE. Therefore, we have synthesized and evaluated pro-2-PAM (a lipid permeable 2-PAM derivative) that can enter the brain and reactivate CNS AChE, preventing seizures in guinea pigs after exposure to OPs. The protective effects of the pro-2-PAM after OP exposure were shown using (a) surgically implanted radiotelemetry probes for electroencephalogram (EEG), (b) neurohistopathology of brain, (c) cholinesterase activities in the PNS and CNS, and (d) survivability. The PNS oxime 2-PAM was ineffective at reducing seizures/status epilepticus (SE) in diisopropylfluorophosphate (DFP)-exposed animals. In contrast, pro-2-PAM significantly suppressed and then eliminated seizure activity. In OP-exposed guinea pigs, there was a significant reduction in neurological damage with pro-2-PAM but not 2-PAM. Distinct regional areas of the brains showed significantly higher AChE activity 1.5h after OP exposure in pro-2-PAM treated animals compared to the 2-PAM treated ones. However, blood and diaphragm showed similar AChE activities in animals treated with either oxime, as both 2-PAM and pro-2-PAM are PNS active oximes. In conclusion, pro-2-PAM can cross the BBB, is rapidly metabolized inside the brain to 2-PAM, and protects against OP-induced SE through restoration of brain AChE activity. Pro-2-PAM represents the first non-invasive means of administering a CNS therapeutic for the deleterious effects of OP poisoning by reactivating CNS AChE.
Assuntos
Acetilcolinesterase/metabolismo , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/enzimologia , Sistema Nervoso Periférico/efeitos dos fármacos , Sistema Nervoso Periférico/enzimologia , Compostos de Pralidoxima/farmacologia , Pró-Fármacos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Encéfalo/patologia , Encéfalo/fisiopatologia , Sistema Nervoso Central/patologia , Sistema Nervoso Central/fisiopatologia , Reativadores da Colinesterase/farmacologia , Relação Dose-Resposta a Droga , Eletroencefalografia , Ativação Enzimática/efeitos dos fármacos , Cobaias , Hipocampo/patologia , Isoflurofato/intoxicação , Masculino , Neurônios/efeitos dos fármacos , Neurônios/patologia , Sistema Nervoso Periférico/patologia , Sistema Nervoso Periférico/fisiopatologia , Pele , Soman/intoxicação , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/enzimologia , Estado Epiléptico/patologia , Estado Epiléptico/fisiopatologia , Análise de SobrevidaRESUMO
Caspase-3 is a prototypic executioner caspase that plays a central role in apoptosis. Aza-peptide epoxides are a novel class of irreversible inhibitors that are highly specific for clan CD cysteine proteases. The five crystal structures of caspase-3-aza-peptide epoxide inhibitor complexes reported here reveal the structural basis for the mechanism of inhibition and the specificities at the S1' and the S4 subsites. Unlike the clan CA cysteine proteases, the catalytic histidine in caspase-3 plays a critical role during protonation and subsequent ring opening of the epoxide moiety and facilitates the nucleophilic attack by the active site cysteine. The nucleophilic attack takes place on the C3 carbon atom of the epoxide and results in an irreversible alkylation of the active site cysteine residue. A favorable network of hydrogen bonds involving the oxyanion hole, catalytic histidine, and the atoms in the prime site of the inhibitor enhance the binding affinity and specificity of the aza-peptide epoxide inhibitors toward caspase-3. The studies also reveal that subtle movements of the N-terminal loop of the beta-subunit occur when the P4 Asp is replaced by a P4 Ile, whereas the N-terminal loop and the safety catch Asp179 are completely disordered when the P4 Asp is replaced by P4 Cbz group.