Abstract
The nonmevalonate isoprenoid pathway is an established target for antiinfective drug development. This paper describes high-throughput methods for the screening of 2C-methyl-D-erythritol synthase (IspC protein), 4-diphosphocytidyl-2C-methyl-D-erythritol synthase (IspD protein), 4-diphosphocytidyl-2C-methyl-D-erythritol kinase (IspE protein), and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF protein) against large compound libraries. The assays use up to three auxiliary enzymes. They are all monitored photometrically at 340 nm and are robust as documented by Z-factors of >or=0.86. 13C NMR assays designed for hit verification via direct detection of the primary reaction product are also described. Enzyme-assisted methods for the preparation, on a multigram scale, of isoprenoid biosynthesis intermediates required as substrates for these assays are reported. Notably, these methods enable the introduction of single or multiple 13C labels as required for NMR-monitored assays. The preparation of 4-diphosphosphocytidyl-2C-methyl-D-erythritol 2-phosphate in multigram quantities is described for the first time.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Aldose-Ketose Isomerases / antagonists & inhibitors*
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Aldose-Ketose Isomerases / biosynthesis
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Anti-Infective Agents / chemical synthesis*
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Anti-Infective Agents / chemistry
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Anti-Infective Agents / pharmacology*
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Drug Evaluation, Preclinical / methods*
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Enzyme Inhibitors / chemical synthesis
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Enzyme Inhibitors / chemistry
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Enzyme Inhibitors / pharmacology
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Escherichia coli Proteins / antagonists & inhibitors*
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Escherichia coli Proteins / biosynthesis
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Microbial Sensitivity Tests
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Molecular Structure
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Multienzyme Complexes / antagonists & inhibitors*
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Multienzyme Complexes / biosynthesis
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Oxidoreductases / antagonists & inhibitors*
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Oxidoreductases / biosynthesis
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Phosphorus-Oxygen Lyases / antagonists & inhibitors*
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Phosphorus-Oxygen Lyases / biosynthesis
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Phosphotransferases (Alcohol Group Acceptor) / antagonists & inhibitors*
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Phosphotransferases (Alcohol Group Acceptor) / biosynthesis
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Stereoisomerism
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Structure-Activity Relationship
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Time Factors
Substances
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Anti-Infective Agents
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Enzyme Inhibitors
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Escherichia coli Proteins
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Multienzyme Complexes
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Oxidoreductases
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1-deoxy-D-xylulose 5-phosphate reductoisomerase
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IspD protein, E coli
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Phosphotransferases (Alcohol Group Acceptor)
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IspE protein, E coli
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Phosphorus-Oxygen Lyases
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ISPF protein, E coli
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Aldose-Ketose Isomerases