Novel inhibitors of glutamyl-tRNA(Glu) reductase identified through cell-based screening of the heme/chlorophyll biosynthetic pathway

The metabolite 5-aminolevulinic acid (ALA) is an early committed intermedia te in the biosynthetic pathway of heme and chlorophyll formation. In plants , 5-aminolevulinic acid is synthesized via a two-step pathway in which glut amyl-tRNA(Glu) is reduced by 1-semitamyl-tRNA(Glu) reductase (GluTR) to glu tamate1-semialdehyde, followed by transformation to 5-aminolevulinic acid c atalyzed by glutamate 1-semialdehyde aminotransferase. Using an Escherichia coil cell-based high-throughput assay to screen small molecule libraries, we identified several chemical classes that specifically inhibit heme/chlor ophyll biosynthesis at this point by demonstrating that the observed cell g rowth inhibition is reversed by supplementing the medium with 5-aminolevuli nic acid. These compounds were further tested in vitro for inhibition of th e purified enzymes GLuTR and glutamate 1-semialdehyde aminotransferase as c onfirmation of the specificity and site of action. Several promising compou nds were identified from the high-throughput screen that inhibit GluTR with an I-0.5 of less than 10 mu M. Our results demonstrate the efficacy of cel l-based high-throughput screening for identifying inhibitors of 5-aminolevu linic acid biosynthesis, thus representing the first report of exogenous in hibitors of this enzyme. (C) 1999 Academic Press.