RESUMO
A total of 2,245 extracts, derived from 449 marine fungi cultivated in five types of media, were screened against the C(4) plant enzyme pyruvate phosphate dikinase (PPDK), a potential herbicide target. Extracts from several fungal isolates selectively inhibited PPDK. Bioassay-guided fractionation of one isolate led to the isolation of the known compound unguinol, which inhibited PPDK with a 50% inhibitory concentration of 42.3 +/- 0.8 muM. Further kinetic analysis revealed that unguinol was a mixed noncompetitive inhibitor of PPDK with respect to the substrates pyruvate and ATP and an uncompetitive inhibitor of PPDK with respect to phosphate. Unguinol had deleterious effects on a model C(4) plant but no effect on a model C(3) plant. These results indicate that unguinol inhibits PPDK via a novel mechanism of action which also translates to an herbicidal effect on whole plants.
Assuntos
Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Fungos/metabolismo , Herbicidas/isolamento & purificação , Herbicidas/farmacologia , Compostos Heterocíclicos com 3 Anéis/isolamento & purificação , Compostos Heterocíclicos com 3 Anéis/farmacologia , Piruvato Ortofosfato Diquinase/antagonistas & inibidores , DNA Fúngico/química , DNA Fúngico/genética , DNA Ribossômico/química , DNA Ribossômico/genética , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , Digitaria/efeitos dos fármacos , Fungos/classificação , Fungos/isolamento & purificação , Hordeum/efeitos dos fármacos , Cinética , Dados de Sequência Molecular , Filogenia , Ligação Proteica , RNA Ribossômico 18S/genética , Análise de Sequência de DNARESUMO
Marine organism derived extracts, previously identified as containing compounds that inhibited the C4 acid cycle enzyme pyruvate P(i) dikinase (PPDK), were assessed for their ability to exhibit an effect on the C4 plants Digitaria ciliaris and Echinochloa crus-galli. Oxygen electrode studies revealed that over half of these extracts inhibited C4 acid driven photosynthesis in leaf slices. Seventeen extracts had a deleterious effect on C4 plants in vivo within 24 h, whereas 36 caused an observable phytotoxic response in one or both of the C4 plants used for in vivo testing. None of the extracts inhibited PPDK metabolism of pyruvate via a directly competitive mechanism, instead hindering the enzyme by either mixed or uncompetitive means. This screening strategy, using a suite of assays, led to the isolation and identification of the herbicidal marine natural product ilimaquinone.
Assuntos
Inibidores Enzimáticos/farmacologia , Herbicidas/farmacologia , Proteínas de Plantas/antagonistas & inibidores , Piruvato Ortofosfato Diquinase/antagonistas & inibidores , Quinonas/farmacologia , Sesquiterpenos/farmacologia , Animais , Moluscos/química , Fotossíntese/efeitos dos fármacos , Plantas/efeitos dos fármacos , Poríferos/química , Piruvato Quinase , Quinonas/isolamento & purificação , Sesquiterpenos/isolamento & purificação , Estrelas-do-Mar/química , Urocordados/químicaRESUMO
Plants using the C(4) photosynthetic pathway are highly represented among the world's worst weeds, with only 4 C(4) species being agriculturally productive (maize, sorghum, millet, and sugar cane). With the C(4) acid cycle operating as a biochemical appendage of C(3) photosynthesis, the additional enzymes involved in C(4) photosynthesis represent an attractive target for the development of weed-specific herbicides. The rate-limiting enzyme of this metabolic pathway is pyruvate orthophosphate dikinase (PPDK). PPDK, coupled with phosphoenolpyruvate carboxylase and nicotinamide adenine dinucleotide-malate dehydrogenase, was used to develop a microplate-based assay to detect inhibitors of enzymes of the C(4) acid cycle. The resulting assay had a Z' factor of 0.61, making it a high-quality assay able to reliably identify active test samples. Organic extracts of 6679 marine macroscopic organisms were tested within the assay, and 343 were identified that inhibited the 3 enzyme-coupled reaction. A high confirmation rate was achieved, with 95% of these hit extracts proving active again upon retesting. Sequential addition of phosphoenolpyruvate and oxaloacetate to the assay facilitated identification of 83 extracts that specifically inhibited PPDK.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Herbicidas/farmacologia , Plantas/efeitos dos fármacos , Plantas/enzimologia , Piruvato Ortofosfato Diquinase/antagonistas & inibidores , Dimetil Sulfóxido/farmacologia , Inibidores Enzimáticos/química , Herbicidas/química , Malato Desidrogenase/antagonistas & inibidores , Malato Desidrogenase/metabolismo , Estrutura Molecular , Ácido Oxálico/farmacologia , Fosfoenolpiruvato Carboxilase/antagonistas & inibidores , Fosfoenolpiruvato Carboxilase/metabolismo , Extratos Vegetais/metabolismo , Piruvato Ortofosfato Diquinase/metabolismo , Especificidade da Espécie , Fatores de TempoRESUMO
Sperm beta-acrosin activity is inhibited by suramin, a polysulfonated naphthylurea compound with therapeutic potential as a combined antifertility agent and microbicide. A kinetic analysis of enzyme inhibition suggests that three and four molecules of suramin bind to one molecule of ram and boar beta-acrosins respectively. Surface charge distribution models of boar beta-acrosin based on its crystal structure indicate several positively charged exosites that represent potential 'docking' regions for suramin. It is hypothesised that the spatial arrangement and distance between these exosites determines the capacity of beta-acrosin to bind suramin.