A fragment-based screen for inhibitors of Escherichia coli N5-CAIR mutase

Abstract

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 N⁵-carboxyaminoimidazole ribonucleotide (N⁵-CAIR) by the enzyme N⁵-CAIR mutase. The difference in pathways between humans and microbes indicate that N⁵-CAIR mutase is a potential antimicrobial drug target. To identify inhibitors of E. coli N⁵-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 N⁵-CAIR mutase with K_d 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 N⁵-CAIR mutase. The five fragments were assessed for their ability to inhibit enzyme activity. Four out of the 5 showed inhibition with K_i 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 B. anthracis N⁵-CAIR mutase and human AIR carboxylase suggesting a common pharmacophore.