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Eusynstyelamides A−C (1−3) were isolated from the Great Barrier Reef ascidian Eusynstyela latericius, together with the known metabolites homarine and trigonelline. The structures of 1−3, with relative configurations, were elucidated by interpretation of their spectroscopic data (NMR, MS, UV, IR, and CD). The NMR data of 1were found to be virtually identical to that reported for eusynstyelamide (4), isolated from E. misakiensis, indicating that a revision of the structure of 4is needed. Eusynstyelamides A−C exhibited inhibitory activity against neuronal nitric oxide synthase (nNOS), with IC50values of 41.7, 4.3, and 5.8 μM, respectively, whereas they were found to be nontoxic toward the three human tumor cell lines MCF-7 (breast), SF-268 (CNS), and H-460 (lung). Compounds 1and 2displayed mild inhibitory activity toward Staphylococcus aureus(IC505.6 and 6.5 mM, respectively) and mild inhibitory activity toward the C4plant regulatory enzyme pyruvate phosphate dikinase (PPDK) (IC50values of 19 and 20 mM, respectively).

Covering: up to the end of 2005

Plants using the C4photosynthetic pathway are highly represented among the world’s worst weeds, with only 4 C4species being agriculturally productive (maize, sorghum, millet, and sugar cane). With the C4acid cycle operating as a biochemical appendage of C3photosynthesis, the additional enzymes involved in C4photosynthesis 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 C4acid 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. (Journal of Biomolecular Screening2005:67-75)

Plants using the C4photosynthetic pathway are highly represented among the world’s worst weeds, with only 4 C4species being agriculturally productive (maize, sorghum, millet, and sugar cane). With the C4acid cycle operating as a biochemical appendage of C3photosynthesis, the additional enzymes involved in C4photosynthesis 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 C4acid 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. (Journal of Biomolecular Screening2005:67-75)

Saxiphilin is a soluble protein of unknown function which binds the neurotoxin, saxitoxin (STX), with high affinity. Molecular characterization of saxiphilin from the North American bullfrog, Rana catesbeiana, has previously shown that it is a member of the transferrin family. In this study we surveyed various animal species to investigate the phylogenetic distribution of saxiphilin, as detected by the presence of soluble [3H]STX binding activity in plasma, haemolymph or tissue extracts. We found that saxiphilin activity is readily detectable in a wide variety of arthropods, fish, amphibians, and reptiles. The pharmacological characteristics of [3H]STX binding activity in phylogenetically diverse species indicates that a protein homologous to bullfrog saxiphilin is likely to be constitutively expressed in many ectothermic animals. The results suggest that the saxiphilin gene is evolutionarily as old as an ancestral gene encoding bilobed transferrin, an Fe3–binding and transport protein which has been identified in several arthropods and all the vertebrates which have been studied.

An isoform of the paralytic shellfish poison (PSP)-specific receptor saxiphilin, from the tropical centipedeEthmostigmus rubripes,was used as the basis for a radiometric, high-throughput, microtiter plate assay for this group of toxins. Characterization of the assay revealed that it was able to detect several representatives from the various structural PSP subgroups and yet was insensitive toward tetrodotoxin. To test the utility of the assay as a seafood toxin-monitoring tool, the assay was subjected to a variety of marine organism extracts, some of which were known to contain PSPs, and whole extract toxicity expressed as STX equivalents (STXeq) was measured by two methods: First, by comparison of values from a screening assay with a standard STX inhibition curve and, second, for highly active extracts, by calculation using the IC50from a full inhibition curve of the extract. For extracts which could be quantified by both methods, there was almost 100% correlation between the derived values. STXeq derived by both methods from the bioassay highly correlated with absolute toxin quantities from HPLC analysis.

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