Nonmevalonate terpene biosynthesis enzymes as antiinfective drug targets: substrate synthesis and high-throughput screening methods.
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.
Illarionova
Victoria
V
Lehrstuhl für Organische Chemie und Biochemie, Department Chemie, Technische Universität München, Lichtenbergstrasse 4, D-85747 Garching, Germany.
Kaiser
Johannes
J
Ostrozhenkova
Elena
E
Bacher
Adelbert
A
Fischer
Markus
M
Eisenreich
Wolfgang
W
Rohdich
Felix
F
D019747:Aldose-Ketose Isomerases / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D000890:Anti-Infective Agents / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D008826:Microbial Sensitivity Tests; D015394:Molecular Structure; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D010088:Oxidoreductases / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D019761:Phosphorus-Oxygen Lyases / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013997:Time Factors
D000890:Anti-Infective Agents; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D010088:Oxidoreductases; C114231:1-deoxy-D-xylulose 5-phosphate reductoisomerase; C522017:IspD protein, E coli; D017853:Phosphotransferases (Alcohol Group Acceptor); C404502:IspE protein, E coli; D019761:Phosphorus-Oxygen Lyases; C400181:ISPF protein, E coli; D019747:Aldose-Ketose Isomerases