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 physiological and pathological conditions. PNMT (phenylethanolamine N-methyltransferase) catalyses 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 chemical diversity more easily. In the present study we applied fragment-based screening approaches to the enzyme PNMT. We used crystallography as the primary screen and identified 12 hits from a small commercial library of 384 drug-like fragments. The hits include nine chemicals with two fused rings and three single-ring chemical systems. Eight of the hits come from three chemical classes: benzimidazoles (a known class of PNMT inhibitor), purines and quinolines. Nine of the hits have measurable binding affinities (~5-700 μM) as determined by isothermal titration calorimetry and all nine have ligand efficiencies of 0.39 kcal/mol per heavy atom or better (1 kcal≈4.184 kJ). We synthesized five elaborated benzimidazole compounds 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.

Synthesis of n-acetylglucosaminides dimers

“…Додаткову інформацію про структуру фрагмен-тів, які зв'язуються одночасно в сусідніх сайтах, можна отримати при спостереженні міжліганд-них ядерних ефектів Оверхаузера (ILOEs) [47]. На сьогодні розроблені практичні методи отриман-ня кількісних даних афінності, як наприклад, ізо-термічна титраційна калориметрія, що базуєть-ся на точному вимірюванні швидкості поглинан-ня або виділення тепла в процесі взаємодії дос-ліджуваної біомолекули з лігандом [48]. Цей метод є легким у виконанні, високочутливим, вичерп-но характеризує термодинаміку процесу і може використовуватися для вимірюванні широкого спектра значень KD.…”

Missing Fragments: Detecting Cooperative Binding in Fragment-Based Drug Design

“…edu.au/atb). 13 All of the 12 fragments identified by Drinkwater et al 9 contain at least one 5-or one 6-membered aromatic ring. In the structures of the complexes deposited in the PDB, this ring lies sandwiched between the side chains of Phe182 and Asn39 forming a π−π stacking interaction with the side chain of Phe182.…”

“…Drinkwater et al 9 also examined each member of three cocktails using electron spray ionization Fourier transform mass spectrometry (ESI-FTMS). These three cocktails collectively contained four fragments identified by X-ray crystallography.…”

“…Fragment F2:4 is imidazole. One of the 12 fragments identified by Drinkwater et al 9 was 4-bromoimidazole (F2:5; PDB 3KQM) with a binding affinity of measured by ITC to be 170 μM. Thus, F2:4 might also bind to PNMT.…”

“…As the binding of F1:4 and F2:4 is highly cooperative, it is likely that cocktail 4 would have appeared to be a false positive if deconvoluted. More than 25% of the hits obtained by Drinkwater et al 9 in the initial crystallographic screens were considered to be false positives. That is, it was not possible to identify an individual fragment that could account for the density observed.…”