An Inhibitor-in-Pieces Approach to DAHP Synthase Inhibition: Potent Enzyme and Bacterial Growth Inhibition

Abstract

3-Deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase catalyzes the first step in the shikimate biosynthetic pathway and is an antimicrobial target. We used an inhibitor-in-pieces approach, based on the previously reported inhibitor DAHP oxime, to screen inhibitor fragments in the presence and absence of glycerol 3-phosphate to occupy the distal end of the active site. This led to DAHP hydrazone, the most potent inhibitor to date, K i = 10 ± 1 nM. Three trifluoropyruvate (TFP)-based inhibitor fragments were efficient inhibitors with ligand efficiencies of up to 0.7 kcal mol −1 /atom compared with 0.2 kcal mol −1 /atom for a typical good inhibitor. The crystal structures showed the TFPbased inhibitors binding upside down in the active site relative to DAHP oxime, providing new avenues for inhibitor development. The ethyl esters of TFP oxime and TFP semicarbazone prevented E. coli growth in culture with IC 50 = 0.21 ± 0.01 and 0.77 ± 0.08 mg mL −1 , respectively. Overexpressing DAHP synthase relieved growth inhibition, demonstrating that DAHP synthase was the target. Growth inhibition occurred in media containing aromatic amino acids, suggesting that growth inhibition was due to depletion of some other product(s) of the shikimate pathway, possibly folate.

Role of Half-of-Sites Reactivity and Inter-Subunit Communications in DAHP Synthase Catalysis and Regulation

“…Subunits’ conformations are indicated by shape, along with the ligands. Trifluoropyruvate oxime (TO) and trifluoropyruvate semicarbazone (TS) are non-TS mimic inhibitor fragments that do not extend into the E4P binding site . Literature structures are 5CKS, 7RUD, 7RUE, 1GG1, 1QR7, 1N8F, and 1KFL …”

“…DAHPS­(Phe) was crystallized and the structures solved largely as described previously. , For most structures, the basic crystallization conditions were the same, with only ligands and additives changing. DAHPS­(Phe) (5 mg/mL) was cocrystallized with different ligands using hanging drops in 0.1 M trilithium citrate tetrahydrate and 10% (w/v) PEG 3350 over 2 M ammonium sulfate.…”

“…Among the proposals for how inter-subunit communications contribute to catalysis, energy from ligand binding and/or the chemical step in one subunit promoting product release from its partner is consistent with product release being rate-limiting for DAHPS. Although not all DAHPS inhibitors demonstrate half-of-sites binding, 3 intersubunit interactions must be considered when attempting to design potent inhibitors of the αCKS superfamily.…”

“…α-Carboxyketose synthases (αCKSs) produce 7–9 carbon monosaccharides and are antimicrobial targets that have been the focus of numerous inhibition studies. − The main superfamily members are 3-deoxy- d - arabino heptulosonate 7-phosphate synthase (DAHPS), , 3-deoxy- d - manno -2-octulosonate-8-phosphate synthase (KDO8PS), , and NeuB, a sialic acid synthase. , DAHPS catalyzes the first step of the shikimate biosynthetic pathway in all bacteria, plants, and fungi. Escherichia coli produces three DAHPSs, one each feedback-inhibited by phenylalanine (DAHPS­(Phe)), tyrosine, and tryptophan .…”