RESUMO
The phosphotriesterase of the bacterium Brevundimonas diminuta (BdPTE) is a naturally occurring enzyme that catalyzes the hydrolysis of organophosphate (OP) nerve agents as well as pesticides and offers a potential treatment of corresponding intoxications. While BdPTE mutants with improved catalytic efficiencies against several OPs have been described, unexpectedly, less efficient breakdown of an OP was observed upon application in an animal model compared with in vitro measurements. Here, we describe detailed inhibition studies with the high-activity BdPTE mutant 10-2C3(C59M/C227A) by human plasma components, indicating that this enzyme is inhibited by serum albumin. The inhibitory activity is mediated by depletion of crucial zinc ions from the BdPTE active site, either via the known high-affinity zinc binding site of albumin or via chemical complex formation with its free thiol side chain at position Cys34. Albumin pre-charged with zinc ions or carrying a chemically blocked Cys34 side chain showed significantly reduced inhibitory activity; in fact, the combination of both measures completely abolished BdPTE inhibition. Consequently, the available zinc ion concentration in blood plays an important role for BdPTE activity in vivo and should be taken into account for therapeutic development and application of a catalytic OP scavenger.
Assuntos
Albuminas/farmacologia , Proteínas de Bactérias/farmacologia , Inibidores Enzimáticos/farmacologia , Intoxicação por Organofosfatos/tratamento farmacológico , Hidrolases de Triester Fosfórico/metabolismo , Hidrolases de Triester Fosfórico/uso terapêutico , Compostos de Sulfidrila/metabolismo , Albuminas/metabolismo , Proteínas de Bactérias/metabolismo , Caulobacteraceae/química , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Inibidores Enzimáticos/metabolismo , Modelos Animais , Compostos Organofosforados/metabolismo , Compostos de Sulfidrila/sangueRESUMO
The recent attacks with the nerve agent sarin in Syria reveal the necessity of effective countermeasures against highly toxic organophosphorus compounds. Multiple studies provide evidence that a rapid onset of antidotal therapy might be life-saving but current standard antidotal protocols comprising reactivators and competitive muscarinic antagonists show a limited efficacy for several nerve agents. We here set out to test the newly developed phosphotriesterase (PTE) mutant C23AL by intravenous (i.v.), intramuscular (i.m.; model for autoinjector) and intraosseous (i.o.; model for intraosseous insertion device) application in an in vivo guinea pig model after VX challenge (â¼2LD50). C23AL showed a Cmax of 0.63µmolL(-1) after i.o. and i.v. administration of 2mgkg(-1) providing a stable plasma profile up to 180min experimental duration with 0.41 and 0.37µmolL(-1) respectively. The i.m. application of C23AL did not result in detectable plasma levels. All animals challenged with VX and subsequent i.o. or i.v. C23AL therapy survived although an in part substantial inhibition of erythrocyte, brain and diaphragm AChE was detected. Theoretical calculation of the time required to hydrolyze in vivo 96.75% of the toxic VX enantiomer is consistent with previous studies wherein similar activity of plasma containing catalytic scavengers of OPs resulted in non-lethal protection although accompanied with a variable severity of cholinergic symptoms. The relatively low C23AL plasma level observed immediately after its i.v. or i.o load, point at a possible volume of distribution greater than the guinea pig plasma content, and thus underlines the necessity of in vivo experiments in antidote research. In conclusion the i.o. application of PTE is efficient and resulted in comparable plasma levels to the i.v. application at a given time. Thus, i.o. vascular access systems could improve the post-exposure PTE therapy of nerve agent poisoning.
Assuntos
Antídotos/administração & dosagem , Mutação , Agentes Neurotóxicos/toxicidade , Intoxicação por Organofosfatos/tratamento farmacológico , Compostos Organotiofosforados/toxicidade , Fragmentos de Peptídeos/administração & dosagem , Hidrolases de Triester Fosfórico/administração & dosagem , Animais , Animais não Endogâmicos , Antídotos/metabolismo , Antídotos/farmacocinética , Antídotos/uso terapêutico , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacocinética , Proteínas de Bactérias/uso terapêutico , Medula Óssea , Cobaias , Inativação Metabólica , Injeções Intralesionais , Injeções Intramusculares , Injeções Intravenosas , Injeções Subcutâneas , Masculino , Agentes Neurotóxicos/análise , Agentes Neurotóxicos/metabolismo , Intoxicação por Organofosfatos/sangue , Intoxicação por Organofosfatos/etiologia , Intoxicação por Organofosfatos/metabolismo , Compostos Organotiofosforados/administração & dosagem , Compostos Organotiofosforados/antagonistas & inibidores , Compostos Organotiofosforados/metabolismo , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacocinética , Fragmentos de Peptídeos/uso terapêutico , Hidrolases de Triester Fosfórico/genética , Hidrolases de Triester Fosfórico/farmacocinética , Hidrolases de Triester Fosfórico/uso terapêutico , Proteólise , Pseudomonas/enzimologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/uso terapêutico , ToxicocinéticaRESUMO
Highly toxic organophosphorus compounds that irreversibly inhibit the enzyme acetycholinesterase (AChE), including nerve agents like tabun, sarin, or soman, still pose a credible threat to civilian populations and military personnel. New therapeutics that can be used as a pretreatment or after poisoning with these compounds, complementing existing treatment schemes such as the use of atropine and AChE reactivating oximes, are currently the subject of intense research. A prominent role among potential candidates is taken by enzymes that can detoxify nerve agents by hydrolysis. Diisopropyl fluorophosphatase (DFPase) from the squid Loligo vulgaris is known to effectively hydrolyze DFP and the range of G-type nerve agents including sarin and soman. In the present work, DFPase was PEGylated to increase biological half-life, and to lower or avoid an immunogenic reaction and proteolytic digest. Addition of linear polyethylene glycol (PEG) chains was achieved using mPEG-NHS esters and conjugates were characterized by electrospray ionization--time of flight--mass specrometry (ESI-ToF-MS). PEGylated wildtype DFPase and a mutant selective for the more toxic stereoisomers of the agents were tested in vivo with rats that were challenged with a subcutaneous 3x LD(50) dose of soman. While wildtype DFPase prevented death only at extremely high doses, the mutant was able keep the animals alive and to minimize or totally avoid symptoms of poisoning. The results serve as a proof of principle that engineered variants of DFPase are potential candidates for in vivo use if substrate affinity can be improved or the turnover rate enhanced to lower the required enzyme dose.
Assuntos
Antídotos/uso terapêutico , Substâncias para a Guerra Química/intoxicação , Inibidores da Colinesterase/intoxicação , Loligo/enzimologia , Hidrolases de Triester Fosfórico/uso terapêutico , Soman/intoxicação , Animais , Antídotos/química , Loligo/genética , Masculino , Espectrometria de Massas , Mutação , Hidrolases de Triester Fosfórico/química , Hidrolases de Triester Fosfórico/genética , Polietilenoglicóis/química , Ratos , Ratos WistarRESUMO
Organophosphorus (OP) pesticide poisoning causes significant morbidity and mortality, particularly in the developing world, with upwards of 3 million people poisoned each year. Although OP poisoning is not common in developed countries, recently greater attention has been given to these chemicals because of their similarity to chemical warfare agents. Despite the agricultural use of OP pesticides for roughly 60 years, no new therapies have been developed since the 1960s. A promising field of novel antidotes for OP poisoning, OP hydrolases, has recently garnered increased support. These bacterial enzymes have demonstrated tremendous prophylactic and antidotal efficacy against a few different OP classes in animal models. These studies, as well as the limitations and challenges of therapeutic development of these enzymes, are discussed.
Assuntos
Antídotos/uso terapêutico , Intoxicação por Organofosfatos , Praguicidas/intoxicação , Monoéster Fosfórico Hidrolases/uso terapêutico , Intoxicação/tratamento farmacológico , Antídotos/farmacocinética , Catálise , Glutationa Transferase/uso terapêutico , Humanos , Monoéster Fosfórico Hidrolases/farmacocinética , Hidrolases de Triester Fosfórico/farmacocinética , Hidrolases de Triester Fosfórico/uso terapêuticoRESUMO
INTRODUCTION AND DEVELOPMENT: Organophosphorus compounds are worldwide employed as insecticides and are yearly responsible of several millions of poisonings. The chemical structure of most of the warfare nerve agents also corresponds with an organophosphorus compound. Organophosphorus insecticides and warfare nerve agents exert their main toxicological effects through inhibition of acetylcholinesterase. Current treatments of patients poisoned with organophosphorus compounds include atropine (in order to protect muscarinic receptors), oximes (in order to accelerate the reactivation of the inhibited acetylcholinesterase) and benzodiazepines (in order to avoid convulsions). The administration of phosphotriesterases (enzymes involved in the detoxication of organophosphorus compounds through hydrolysis) is a very effective treatment against poisonings by organophosphorus insecticides and warfare nerve agents. There are experimental preventive treatments based on the simultaneous administration of carbamates and certain antimuscarinic drugs, different from atropine, which notably improve the efficacy of the classical treatments applied after poisonings by warfare nerve agents. CONCLUSIONS: The treatments based in the administration of phosphotriesterases might be the response to the call of the World Health Organization for searching new treatments with capability to reduce the high mortality recorded in the cases of poisonings by organophosphorus compounds. These treatments can be applied in a preventive way without the intrinsic neurotoxicity associated to the preventive treatments based on carbamates and antimuscarinic drugs. Therefore, these treatments are specially interesting for people susceptible to suffer severe exposures, i.e. sprayers in the farms.
Assuntos
Substâncias para a Guerra Química/intoxicação , Inseticidas/intoxicação , Síndromes Neurotóxicas , Neurotoxinas/intoxicação , Intoxicação por Organofosfatos , Antídotos , Atropina/uso terapêutico , Benzodiazepinas/uso terapêutico , Guerra Química , Substâncias para a Guerra Química/química , Humanos , Inseticidas/química , Antagonistas Muscarínicos/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Síndromes Neurotóxicas/tratamento farmacológico , Síndromes Neurotóxicas/mortalidade , Síndromes Neurotóxicas/prevenção & controle , Neurotoxinas/química , Compostos Organofosforados/química , Oximas/uso terapêutico , Hidrolases de Triester Fosfórico/uso terapêuticoRESUMO
Organophosphorus compounds (OPs) are being used as insecticides and warfare agents. OP insecticides represent an important problem of public health, causing around 200,000 deaths annually. The World Health Organization has pointed to the necessity to introduce new medical practices that improve the results of classical treatments. Many studies have shown that the administration of phosphotriesterases (enzymes that detoxify OPs through hydrolysis) is a promising treatment of persons poisoned with OPs. Such an enzyme-based treatment might introduce important improvements in the treatment of patients having ingested large amounts of OPs. Phosphotriesterases might also be suitable for prophylactic treatment of persons at risk to be severely exposed. The new experimental treatments do not exhibit the intrinsic neurotoxicity of the classical prophylaxis based on carbamates and antimuscarinic drugs. Experimental data suggest that might be time to initiate clinical trials in order to study the efficacy of phosphotriesterases in the therapy and prophylaxis of OP intoxication.