title: Novel inhibitors of glutamyl-tRNA(Glu) reductase identified through cell-based screening of the heme/chlorophyll biosynthetic pathway.
abstract: The metabolite 5-aminolevulinic acid (ALA) is an early committed intermediate in the biosynthetic pathway of heme and chlorophyll formation. In plants, 5-aminolevulinic acid is synthesized via a two-step pathway in which glutamyl-tRNA(Glu) is reduced by glutamyl-tRNA(Glu) reductase (GluTR) to glutamate 1-semialdehyde, followed by transformation to 5-aminolevulinic acid catalyzed by glutamate 1-semialdehyde aminotransferase. Using an Escherichia coli cell-based high-throughput assay to screen small molecule libraries, we identified several chemical classes that specifically inhibit heme/chlorophyll biosynthesis at this point by demonstrating that the observed cell growth inhibition is reversed by supplementing the medium with 5-aminolevulinic acid. These compounds were further tested in vitro for inhibition of the purified enzymes GluTR and glutamate 1-semialdehyde aminotransferase as confirmation of the specificity and site of action. Several promising compounds were identified from the high-throughput screen that inhibit GluTR with an I(0.5) of less than 10 microM. Our results demonstrate the efficacy of cell-based high-throughput screening for identifying inhibitors of 5-aminolevulinic acid biosynthesis, thus representing the first report of exogenous inhibitors of this enzyme.
authors.affiliation: Monsanto Company, 700 Chesterfield Parkway, Chesterfield, Missouri, 63198, USA. paul.j.loida@monsanto.com
authors.forename: P J
authors.initials: PJ
authors.lastname: Loida
authors.forename: R L
authors.initials: RL
authors.lastname: Thompson
authors.forename: D M
authors.initials: DM
authors.lastname: Walker
authors.forename: C A
authors.initials: CA
authors.lastname: CaJacob
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D000622:Aminolevulinic Acid / Q000378:metabolism / Q000494:pharmacology; D017360:Arabidopsis / Q000235:genetics; D002734:Chlorophyll / Q000096:biosynthesis*; D003509:Cyclohexanecarboxylic Acids / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000145:classification / Q000494:pharmacology*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D005971:Glutamates / Q000378:metabolism; D006418:Heme / Q000096:biosynthesis*; D001467:Hordeum / Q000201:enzymology / Q000235:genetics; D020128:Inhibitory Concentration 50; D019751:Intramolecular Transferases / Q000037:antagonists & inhibitors / Q000378:metabolism; D007700:Kinetics; D009154:Mutation / Q000235:genetics; D012351:RNA, Transfer, Gln / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D017386:Sequence Homology, Amino Acid; D018724:Spinacia oleracea / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D003509:Cyclohexanecarboxylic Acids; D004791:Enzyme Inhibitors; D005971:Glutamates; D012351:RNA, Transfer, Gln; D011993:Recombinant Fusion Proteins; D002734:Chlorophyll; C012987:gabaculine; D006418:Heme; C057794:glutamate-1-semialdehyde; D000622:Aminolevulinic Acid; D000445:Aldehyde Oxidoreductases; C067282:glutamyl tRNA reductase; D019751:Intramolecular Transferases; C061049:glutamate-1-semialdehyde 2,1-aminomutase