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

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.

Fragment-to-Lead Medicinal Chemistry Publications in 2020

“… 67 Another study combines protein- and ligand-observed experiments in a multiplexed screen that contains mixtures of fragments and bromodomain proteins. 68 Clearly, the NMR community remains very active in providing increasingly efficient FBDD screening technologies.…”

Fragment-Based Discovery of AF9 YEATS Domain Inhibitors

“…Using our fragment-based lead discovery platform [ 32 , 33 ], we screened 89 cocktails with 10 compounds each against the AF9 YEATS domain using NMR ligand-observed spectra, i.e., Saturation Transfer Difference (STD), WaterLOGSY, and Carr–Purcell–Meiboom–Gill (CPMG) [ 34 , 35 , 36 ] ( Figure 1 A). In principle, only compounds that bind will show signals in the STD spectrum.…”

19F NMR viewed through two different lenses: ligand-observed and protein-observed19F NMR applications for fragment-based drug discovery

“…10a and b). 35,[127][128][129][130][131][132] In a cross-validation study of PrOF NMR, a 930-member fragment library was screened against the first bromodomain of 5FW-labeled BRD4 (BRD4 D1) in mixtures of five using both PrOF NMR and ligand-observed 1 H CPMG NMR (with and without competitor validation). 41 Comparing the individual fragments, there was 85% overlap in hit detection when using competitors.…”

“…10c). 131 This work on selective bromodomain inhibitor development aided by PrOF NMR highlights 19 F NMR as a useful tool from screening through chemical probe development.…”