Fragment-Based Approach Identifies a Novel Inhibitory Site on DHPS

Abstract

Dihydropteroate synthase (DHPS) is an essential enzyme in the bacterial folate biosynthetic pathway. It catalyzes the condensation of 6-hydroxymethyl-7,8-dihydropterin pyrophosphate (DHPP) with p-aminobenzoic acid (pABA) to form the folate intermediate, 7,8-dihydropteroate. DHPS is the target of the sulfonamide class of antibiotics. Widespread resistance to sulfonamides has decreased their clinical use. The active site of DHPS is comprised of three sub-sites: the structured “pterin” site, the flexible pABA site, and the anion binding pocket. Most of the drug resistant mutations have been mapped to the pABA sub-site of DHPS. using an NMR ligand-based screening approach, a number of structurally unrelated fragment-like small molecules have been identified that inhibit the enzymatic activity of DHPS from Bacillus anthracis (Ba), Yersinia pestis (Yp), and Staphylococcus aureus (Sa). Fragment hits were shown to target the three sub-pockets of the active site and a novel site distinct from the active site. The latter site potentially inhibits via an allosteric mechanism and has been characterized by high resolution X-ray crystallography.We screened the Maybridge® Fragment library of 1,100 fragments using water ligand observed gradient spectroscopy (waterLOGSY) as a primary screen which resulted in a hit rate of 6.7 %. Of the 74 hits, 25 were shown to inhibit DHPS activity using two independent enzyme activity assays. A total of eight compounds inhibited the activity of DHPS from three different species (Ba, Yp, and Sa). In addition to screening for inhibition, the fragment hits were validated using a number of biophysical techniques including 2D NMR, SPR, competition waterLOGSY, and X-ray crystallography. Herein, we focus on two fragment hits for which high-resolution x-ray crystal structures are available