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.
Highlights
Fragment-based drug discovery (FBDD) is a validated technique to uncover inhibitors for many protein classes and has resulted in four FDA-approved drugs [1,2,3,4]
15 N-Plasmodium falciparum (Pf) GCN5 and 1 HPfGCN5, and those that bind to both proteins (Table 1)
An analysis of the range of Protein-observed 19F (PrOF) NMR ∆δ and 1 H CPMG NMR percent resonance decreases showed no major differences between the proteins, indicating a similar responsiveness to binding (Figure S6)
Summary
Fragment-based drug discovery (FBDD) is a validated technique to uncover inhibitors for many protein classes and has resulted in four FDA-approved drugs [1,2,3,4]. Compounds in FBDD libraries are typically of low complexity and low molecular weight (average MW ≤ 300 g/mol), allowing these libraries to more effectively sample chemical space, with smaller library sizes than traditional high throughput screening libraries (average MW ≤ 500 g/mol) [5]. Fragments often have weak affinity for their target protein, making it essential to use screening assays that can detect compounds with affinity values in the high μM to low mM range.
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