A Rapid Screening Method to Detect Specific Inhibitors of Pyruvate Orthophosphate Dikinase as Leads for C₄ Plant-Selective Herbicides

Abstract

Plants using the C₄ photosynthetic pathway are highly represented among the world’s worst weeds, with only 4 C₄ species being agriculturally productive (maize, sorghum, millet, and sugar cane). With the C₄ acid cycle operating as a biochemical appendage of C₃ photosynthesis, the additional enzymes involved in C₄ 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 C₄ 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. (Journal of Biomolecular Screening 2005:67-75)