Identification of inhibitors of N5-carboxyaminoimidazole ribonucleotide synthetase by high-throughput screening

The increasing risk of drug-resistant bacterial infections indicates that there is a growing need for new and effective antimicrobial agents. One promising, but unexplored area in antimicrobial drug design is de novo purine biosynthesis. Recent research has shown that de novo purine biosynthesis in microbes is different from that in humans. The differences in the pathways are centered around the synthesis of 4-carboxyaminoimidazole ribonucleotide (CAIR) which requires the enzyme N⁵-carboxyaminoimidazole ribonucleotide (N⁵-CAIR) synthetase. Humans do not require and have no homologs of this enzyme. Unfortunately, no studies aimed at identifying small-molecule inhibitors of N⁵-CAIR synthetase have been published. To remedy this problem, we have conducted high-throughput screening (HTS) against Escherichia coli N⁵-CAIR synthetase using a highly reproducible phosphate assay. HTS of 48,000 compounds identified 14 compounds that inhibited the enzyme. The hits identified could be classified into three classes based on chemical structure. Class I contains compounds with an indenedione core. Class II contains an indolinedione group, and Class III contains compounds that are structurally unrelated to other inhibitors in the group. We determined the Michaelis–Menten kinetics for five compounds representing each of the classes. Examination of compounds belonging to Class I indicates that these compounds do not follow normal Michaelis–Menten kinetics. Instead, these compounds inhibit N⁵-CAIR synthetase by reacting with the substrate AIR. Kinetic analysis indicates that the Class II family of compounds are non-competitive with both AIR and ATP. One compound in Class III is competitive with AIR but uncompetitive with ATP, whereas the other is non-competitive with both substrates. Finally, these compounds display no inhibition of human AIR carboxylase:SAICAR synthetase indicating that these agents are selective inhibitors of N⁵-CAIR synthetase.