A high-throughput screening platform for inhibitors of the riboflavin biosynthesis pathway

3,4-Dihydroxy-2-butanone 4-phosphate synthase, 6,7-dimethyl-8-ribityllumazine synthase, and riboflavin synthase of the riboflavin biosynthetic pathway are potential targets for novel antiinfective drugs. This article describes a platform for high-throughput screening for inhibitors of these enzymes. The assays can be monitored photometrically and have been shown to be robust, as indicated by Z factors ⩾0.87. A 13C NMR assay for hit verification of 3,4-dihydroxy-2-butanone 4-phosphate synthase inhibitors is also reported.