Screening for inhibitors of the terpenoid biosynthetic pathway that may be used as herbicides

摘要

A method (I) of screening for inhibitors of enzymes in the biosynthetic pathway that produces terpenoids via 1-deoxy-D-xylulose 5-phosphate in plants, comprises incubating plastids in the presence of a labeled precursor, ATP, CTP (or a source of CTP) and a candidate inhibitor and detecting the formation of labeled products, is new. Independent claims are included for the following: (1) a method (I) for screening for the presence of inhibition of at least one enzyme in the biosynthetic pathway to terpenoids via 1-deoxy-D-xylulose 5-phosphate in plants, comprising: (a) preparing a suspension of cells or plastids of a plastid-bearing organism in a culture medium for supporting the metabolism of the cells or plastids at least to the extent of the biosynthetic pathway; (b) adding to the suspension a predetermined amount of a carbon-13-, carbon-14-, deuterium-, or tritium-labeled biochemical precursor for generating terpenoids via the pathway; (c) incubating the mixture obtained in step (b) for a predetermined period of time at a predetermined temperature; (d) separating from the incubated mixture obtained in step (c), a fraction comprising a product or intermediate downstream from 1-deoxy-D-xylulose-5-phosphate in the pathway; (e) detecting the concentration of labeled product(s) in the fraction obtained in step (d); (f) repeating step (b) with the addition of a predetermined amount of a chemical test sample under otherwise identical conditions; (g) repeating steps (c) to (e) with the mixture obtained in step (f) under the same conditions as in steps (c) to (e); and (h) determining the presence of inhibition of at least one enzyme in the pathway by observation of whether the concentration of labeled product(s) detected in step (e) is higher than that detected when step (e) is repeated in step (g); (2) a method (II) for inhibiting the growth of a plant by treatment with a herbicide selected from a class of compounds exhibiting inhibition of the biosynthetic pathway to terpenoids via 1-deoxy-D-xylulose 5 phosphate in the test (I); (3) a method (III) of inhibiting, in the chloroplasts of plants, an enzyme in the biosynthetic pathway to terpenoids via 1-deoxy-D-xylulose 5 phosphate by treatment with a compound that inhibits that biosynthetic pathway identified in the test (I); (4) an inhibitor (IV) exhibiting inhibition in (I); (5) a method (V) for increasing the titer of an intermediate in the biosynthetic pathway via 1-deoxy-D-xylulose 5 phosphate upstream from IPP (undefined) in plants, plant cells or plastids by treatment with a synthetic inhibitor exhibiting inhibition in (I); (6) plant cells or plasmids containing a synthetic inhibitor and exhibiting a titer of an intermediate in the biosynthetic terpenoid pathway via 1-deoxy-D-xylulose 5 phosphate upstream from IPP which is increased over the titer absent in the inhibitor; (7) 14-carbon-, 13-carbon-, deuterium- or tritium-labelled 1-deoxy-D-xylulose 5 phosphate or 2C-methyl-D-erythritol 4-phosphate; (8) tritiated 2C-methyl-D-erythritol 4-phosphate or tritiated 1-deoxy-xylulose 5-phosphate; (9) 2C--methyl-D-erythritol 4-phosphate tritiated at positions 1 and/or 3 or in the methyl group; and (10) 1-deoxy-D-xylulose 5-phosphate, tritiated in positions 1, 3 and/or 4.