Combined Protein- and Ligand-Observed NMR Workflow to Screen Fragment Cocktails against Multiple Proteins: A Case Study Using Bromodomains
As fragment-based drug discovery has become mainstream, there has been an increase in various screening methodologies. Protein-observed <sup>19</sup>F (PrOF) NMR and <sup>1</sup>H CPMG NMR are two fragment screening assays that have complementary advantages. Here, we sought t...
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| Format: | Article |
| Language: | English |
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MDPI AG
2020-08-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/25/17/3949 |
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| author | Jorden A. Johnson Noelle M. Olson Madison J. Tooker Scott K. Bur William C.K. Pomerantz |
| author_facet | Jorden A. Johnson Noelle M. Olson Madison J. Tooker Scott K. Bur William C.K. Pomerantz |
| author_sort | Jorden A. Johnson |
| collection | DOAJ |
| description | As fragment-based drug discovery has become mainstream, there has been an increase in various screening methodologies. Protein-observed <sup>19</sup>F (PrOF) NMR and <sup>1</sup>H 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 <sup>1</sup>H CPMG NMR. We demonstrate the benefit of this fragment screening method by conducting the first reported fragment screens against the bromodomains of BPTF and <i>Plasmodium falciparum</i> (<i>Pf</i>) GCN5 using 467 3D-enriched fragments. The hit rates were 6%, 5% and 4% for fragments binding BPTF, <i>Pf</i>GCN5, 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 <i>Pf</i>GCN5. 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. |
| first_indexed | 2024-03-10T16:42:57Z |
| format | Article |
| id | doaj.art-7a3dc3b4272f40e8a2cdabf9472d7965 |
| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-03-10T16:42:57Z |
| publishDate | 2020-08-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Molecules |
| spelling | doaj.art-7a3dc3b4272f40e8a2cdabf9472d79652023-11-20T11:50:47ZengMDPI AGMolecules1420-30492020-08-012517394910.3390/molecules25173949Combined Protein- and Ligand-Observed NMR Workflow to Screen Fragment Cocktails against Multiple Proteins: A Case Study Using BromodomainsJorden A. Johnson0Noelle M. Olson1Madison J. Tooker2Scott K. Bur3William C.K. Pomerantz4Department of Chemistry, University of Minnesota, Minneapolis, MN 55445, USADepartment of Chemistry, University of Minnesota, Minneapolis, MN 55445, USADepartment of Chemistry, Gustavus Adolphus College, St. Peter, MN 56028, USADepartment of Chemistry, Gustavus Adolphus College, St. Peter, MN 56028, USADepartment of Chemistry, University of Minnesota, Minneapolis, MN 55445, USAAs fragment-based drug discovery has become mainstream, there has been an increase in various screening methodologies. Protein-observed <sup>19</sup>F (PrOF) NMR and <sup>1</sup>H 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 <sup>1</sup>H CPMG NMR. We demonstrate the benefit of this fragment screening method by conducting the first reported fragment screens against the bromodomains of BPTF and <i>Plasmodium falciparum</i> (<i>Pf</i>) GCN5 using 467 3D-enriched fragments. The hit rates were 6%, 5% and 4% for fragments binding BPTF, <i>Pf</i>GCN5, 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 <i>Pf</i>GCN5. 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.https://www.mdpi.com/1420-3049/25/17/3949PrOF NMR<sup>1</sup>H CPMG NMRBPTF inhibitor<i>Pf</i>GCN5 inhibitor3D fragments |
| spellingShingle | Jorden A. Johnson Noelle M. Olson Madison J. Tooker Scott K. Bur William C.K. Pomerantz Combined Protein- and Ligand-Observed NMR Workflow to Screen Fragment Cocktails against Multiple Proteins: A Case Study Using Bromodomains Molecules PrOF NMR <sup>1</sup>H CPMG NMR BPTF inhibitor <i>Pf</i>GCN5 inhibitor 3D fragments |
| title | Combined Protein- and Ligand-Observed NMR Workflow to Screen Fragment Cocktails against Multiple Proteins: A Case Study Using Bromodomains |
| title_full | Combined Protein- and Ligand-Observed NMR Workflow to Screen Fragment Cocktails against Multiple Proteins: A Case Study Using Bromodomains |
| title_fullStr | Combined Protein- and Ligand-Observed NMR Workflow to Screen Fragment Cocktails against Multiple Proteins: A Case Study Using Bromodomains |
| title_full_unstemmed | Combined Protein- and Ligand-Observed NMR Workflow to Screen Fragment Cocktails against Multiple Proteins: A Case Study Using Bromodomains |
| title_short | Combined Protein- and Ligand-Observed NMR Workflow to Screen Fragment Cocktails against Multiple Proteins: A Case Study Using Bromodomains |
| title_sort | combined protein and ligand observed nmr workflow to screen fragment cocktails against multiple proteins a case study using bromodomains |
| topic | PrOF NMR <sup>1</sup>H CPMG NMR BPTF inhibitor <i>Pf</i>GCN5 inhibitor 3D fragments |
| url | https://www.mdpi.com/1420-3049/25/17/3949 |
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