A Fragment-Based Screen for Inhibitors of Escherichia coli N5-CAIR Mutase.

Although purine biosynthesis is a primary metabolic pathway, there are fundamental differences between how purines are synthesized in microbes versus humans. In humans, the purine intermediate, 4- carboxy-5-aminoimidazole ribonucleotide (CAIR) is directly synthesized from 5-aminoimidazole ribonucleotide (AIR) and carbon dioxide by the enzyme AIR carboxylase. In bacteria, yeast and fungi, CAIR is synthesized from AIR via an intermediate N-carboxyaminoimidazole ribonucleotide (N-CAIR) by the enzyme N-CAIR mutase. The difference in pathways between humans and microbes indicate that N-CAIR mutase is a potential antimicrobial drug target. To identify inhibitors of N-CAIR mutase, a fragment-based screening campaign was conducted using a thermal shift assay and a library of 4,500 fragments. Twenty-eight fragments were initially identified that displayed dose-dependent binding to N-CAIR mutase with K values ranging from 9-309 μM. Of the 28, 14 were obtained from commercial sources for retesting; however, only 5 showed dose-dependent binding to N-CAIR mutase. The five fragments were assessed for their ability to inhibit enzyme activity. Four out of the 5 showed inhibition with K values of 4.8 to 159 μM. All fragments contained nitrogen heterocycles with 3 out of the 4 containing 5-membered heterocycles like those found in the substrate of the enzyme. The identified fragments show similarities to compounds identified from studies on N-CAIR synthetase and human AIR carboxylase suggesting a common pharmacophore.