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.
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Research Article|
September 14 2010
Fragment-based screening by X-ray crystallography, MS and isothermal titration calorimetry to identify PNMT (phenylethanolamine N-methyltransferase) inhibitors
Nyssa Drinkwater;
Nyssa Drinkwater
*University of Queensland, Institute for Molecular Bioscience, Division of Chemistry and Structural Biology, Brisbane, Queensland 4072, Australia
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Hoan Vu;
Hoan Vu
†Griffith University, Eskitis Institute, Brisbane, Queensland 4111, Australia
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Kimberly M. Lovell;
Kimberly M. Lovell
‡Department of Medicinal Chemistry, University of Kansas, Lawrence, KS 66045-7582, U.S.A.
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Kevin R. Criscione;
Kevin R. Criscione
‡Department of Medicinal Chemistry, University of Kansas, Lawrence, KS 66045-7582, U.S.A.
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Brett M. Collins;
Brett M. Collins
*University of Queensland, Institute for Molecular Bioscience, Division of Chemistry and Structural Biology, Brisbane, Queensland 4072, Australia
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Thomas E. Prisinzano;
Thomas E. Prisinzano
‡Department of Medicinal Chemistry, University of Kansas, Lawrence, KS 66045-7582, U.S.A.
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Sally-Ann Poulsen;
Sally-Ann Poulsen
†Griffith University, Eskitis Institute, Brisbane, Queensland 4111, Australia
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Michael J. McLeish;
Michael J. McLeish
§Department of Chemistry and Chemical Biology, Indiana University–Purdue University Indianapolis (IUPUI), 402 N. Blackford St, LD 326D, IN 46202, U.S.A.
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Gary L. Grunewald;
Gary L. Grunewald
‡Department of Medicinal Chemistry, University of Kansas, Lawrence, KS 66045-7582, U.S.A.
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Jennifer L. Martin
Jennifer L. Martin
1
*University of Queensland, Institute for Molecular Bioscience, Division of Chemistry and Structural Biology, Brisbane, Queensland 4072, Australia
1To whom correspondence should be addressed (email j.martin@imb.uq.edu.au).
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Publisher: Portland Press Ltd
Received:
April 27 2010
Revision Received:
June 29 2010
Accepted:
July 20 2010
Accepted Manuscript online:
July 20 2010
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2010 Biochemical Society
2010
Biochem J (2010) 431 (1): 51–61.
Article history
Received:
April 27 2010
Revision Received:
June 29 2010
Accepted:
July 20 2010
Accepted Manuscript online:
July 20 2010
Citation
Nyssa Drinkwater, Hoan Vu, Kimberly M. Lovell, Kevin R. Criscione, Brett M. Collins, Thomas E. Prisinzano, Sally-Ann Poulsen, Michael J. McLeish, Gary L. Grunewald, Jennifer L. Martin; Fragment-based screening by X-ray crystallography, MS and isothermal titration calorimetry to identify PNMT (phenylethanolamine N-methyltransferase) inhibitors. Biochem J 1 October 2010; 431 (1): 51–61. doi: https://doi.org/10.1042/BJ20100651
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