Combined Protein- And Ligand-Observed NMR Workflow to Screen Fragment Cocktails against Multiple Proteins: A Case Study Using Bromodomains

Jorden A. Johnson, Noelle M. Olson, Madison J. Tooker, Scott K. Bur, William C.K. Pomerantz

Research output: Contribution to journalArticlepeer-review

6 Scopus citations

Abstract

As fragment-based drug discovery has become mainstream, there has been an increase in various screening methodologies. Protein-observed 19F (PrOF) NMR and 1H CPMG NMR are two fragment screening assays that have complementary advantages. Here, we sought to combine these two NMR-based assays into a new screening workflow. This combination of protein- and ligand-observed experiments allows for a time- and resource-efficient multiplexed screen of mixtures of fragments and proteins. PrOF NMR is first used to screen mixtures against two proteins. Hit mixtures for each protein are identified then deconvoluted using 1H CPMG NMR. We demonstrate the benefit of this fragment screening method by conducting the first reported fragment screens against the bromodomains of BPTF and Plasmodium falciparum (Pf) GCN5 using 467 3D-enriched fragments. The hit rates were 6%, 5% and 4% for fragments binding BPTF, PfGCN5, and fragments binding both proteins, respectively. Select hits were characterized, revealing a broad range of affinities from low µM to mM dissociation constants. Follow-up experiments supported a low-affinity second binding site on PfGCN5. This approach can be used to bias fragment screens towards more selective hits at the onset of inhibitor development in a resource- and time-efficient manner.

Original languageEnglish (US)
Article number3949
JournalMolecules
Volume25
Issue number17
DOIs
StatePublished - Sep 2020

Bibliographical note

Funding Information:
Funding: Research reported in this publication was supported by National Institute of General Medical Sciences Grant R01GM121414, National Science Foundation RUI1806228, and CHE-1904071. J.A.J. and N.M.O. were supported by National Institutes of Health Biotechnology Training Grants 5T32GM008347-27 and 5T32GM008347-29, respectively.

Publisher Copyright:
© 2020 by the authors.

Keywords

  • 3D fragments
  • BPTF inhibitor
  • H CPMG NMR
  • PfGCN5 inhibitor
  • PrOF NMR

PubMed: MeSH publication types

  • Journal Article

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