Fragment-based screening by X-ray crystallography, MS and isothermal titration calorimetry to identify PNMT (phenylethanolamine N-methyltransferase) inhibitors

Abstract

CNS (central nervous system) adrenaline (epinephrine) is implicated in a wide range of physiol. and pathol. conditions. PNMT (phenylethanolamine N-methyltransferase) catalyzes the final step in the biosynthesis of adrenaline, the conversion of noradrenaline (norepinephrine) to adrenaline by methylation. To help elucidate the role of CNS adrenaline, and to develop potential drug leads, potent, selective and CNS-active inhibitors are required. The fragment screening approach has advantages over other lead discovery methods including high hit rates, more efficient hits and the ability to sample chem. diversity more easily. In the present study we applied fragment-based screening approaches to the enzyme PNMT. We used crystallog. as the primary screen and identified 12 hits from a small com. library of 384 drug-like fragments. The hits include nine chems. with two fused rings and three single-ring chem. systems. Eight of the hits come from three chem. classes: benzimidazoles (a known class of PNMT inhibitor), purines and quinolines. Nine of the hits have measurable binding affinities (.apprx.5-700 .mu.M) as detd. by isothermal titrn. calorimetry and all nine have ligand efficiencies of 0.39 kcal/mol per heavy atom or better (1 kcal4.184 kJ). We synthesized five elaborated benzimidazole compds. and characterized their binding to PNMT, showing for the first time how this class of inhibitors interact with the noradrenaline-binding site. Finally, we performed a pilot study with PNMT for fragment-based screening by MS showing that this approach could be used as a fast and efficient first-pass screening method prior to characterization of binding mode and affinity of hits.