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Nonmevalonate terpene biosynthesis enzymes as antiinfective drug targets: substrate synthesis and high-throughput screening methods.
- Source :
-
The Journal of organic chemistry [J Org Chem] 2006 Nov 10; Vol. 71 (23), pp. 8824-34. - Publication Year :
- 2006
-
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.
- Subjects :
- Aldose-Ketose Isomerases biosynthesis
Anti-Infective Agents chemistry
Enzyme Inhibitors chemical synthesis
Enzyme Inhibitors chemistry
Enzyme Inhibitors pharmacology
Escherichia coli Proteins biosynthesis
Microbial Sensitivity Tests
Molecular Structure
Multienzyme Complexes biosynthesis
Oxidoreductases biosynthesis
Phosphorus-Oxygen Lyases biosynthesis
Phosphotransferases (Alcohol Group Acceptor) biosynthesis
Stereoisomerism
Structure-Activity Relationship
Time Factors
Aldose-Ketose Isomerases antagonists & inhibitors
Anti-Infective Agents chemical synthesis
Anti-Infective Agents pharmacology
Drug Evaluation, Preclinical methods
Escherichia coli Proteins antagonists & inhibitors
Multienzyme Complexes antagonists & inhibitors
Oxidoreductases antagonists & inhibitors
Phosphorus-Oxygen Lyases antagonists & inhibitors
Phosphotransferases (Alcohol Group Acceptor) antagonists & inhibitors
Subjects
Details
- Language :
- English
- ISSN :
- 0022-3263
- Volume :
- 71
- Issue :
- 23
- Database :
- MEDLINE
- Journal :
- The Journal of organic chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 17081012
- Full Text :
- https://doi.org/10.1021/jo061466o