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Targeting interactions with these hotspots maximises the efficiency of ligand binding. Existing methods are capable of identifying hotspots but often lack assays to quantify ligand binding and direct elaboration at these sites. Herein, we describe a fragment\u2010based competitive\n                    <jats:sup>19<\/jats:sup>\n                    F Ligand Based\u2005NMR (LB\u2010NMR) screening platform that enables routine, quantitative ligand profiling focused at ligand\u2010binding hotspots. As a proof of concept, the method was applied to 4\u2032\u2010phosphopantetheine adenylyltransferase (PPAT) from\n                    <jats:italic>Mycobacterium abscessus<\/jats:italic>\n                    (\n                    <jats:italic>Mabs<\/jats:italic>\n                    ). X\u2010ray crystallographic characterisation of the hits from a 960\u2010member fragment screen identified three ligand\u2010binding hotspots across the PPAT active site. From the fragment hits a collection of\n                    <jats:sup>19<\/jats:sup>\n                    F reporter candidates were designed and synthesised. By rigorous prioritisation and use of optimisation workflows, a single\n                    <jats:sup>19<\/jats:sup>\n                    F reporter molecule was generated for each hotspot. Profiling the binding of a set of structurally characterised ligands by competitive\n                    <jats:sup>19<\/jats:sup>\n                    F\u2005LB\u2010NMR with this suite of\n                    <jats:sup>19<\/jats:sup>\n                    F reporters recapitulated the binding affinity and site ID assignments made by ITC and X\u2010ray crystallography. This quantitative mapping of ligand binding events at hotspot level resolution establishes the utility of the fragment\u2010based competitive\n                    <jats:sup>19<\/jats:sup>\n                    F\u2005LB\u2010NMR screening platform for hotspot\u2010directed ligand profiling\n                  <\/jats:p>","DOI":"10.1002\/anie.202406846","type":"journal-article","created":{"date-parts":[[2024,6,19]],"date-time":"2024-06-19T11:09:35Z","timestamp":1718795375000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["A Fragment\u2010Based Competitive\n                    <sup>19<\/sup>\n                    F\u2005LB\u2010NMR Platform For Hotspot\u2010Directed Ligand Profiling"],"prefix":"10.1002","volume":"63","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2858-076X","authenticated-orcid":false,"given":"William J.","family":"McCarthy","sequence":"first","affiliation":[{"name":"Yusuf Hamied Department of Chemistry University of Cambridge  Lensfield Road Cambridge UK CB2 1EW"},{"name":"Present Address: Molecular Structure of Cell Signalling Laboratory The Francis Crick Institute  1 Midland Road London UK NW1 1AT"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1152-4312","authenticated-orcid":false,"given":"Sherine E.","family":"Thomas","sequence":"additional","affiliation":[{"name":"Department of Biochemistry University of Cambridge  Tennis Court Road Cambridge UK CB2 1GA"},{"name":"Present Address: Department of Pathology University of Cambridge  Tennis Court Road Cambridge UK CB2 1PD"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tayo","family":"Olaleye","sequence":"additional","affiliation":[{"name":"Yusuf Hamied Department of Chemistry University of Cambridge  Lensfield Road Cambridge UK CB2 1EW"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jennifer A.","family":"Boland","sequence":"additional","affiliation":[{"name":"Yusuf Hamied Department of Chemistry University of Cambridge  Lensfield Road Cambridge UK CB2 1EW"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"R. Andres","family":"Floto","sequence":"additional","affiliation":[{"name":"University of Cambridge Molecular Immunity Unit MRC Laboratory of Molecular Biology  Cambridge UK CB2 0QH"},{"name":"VPD Heart Lung Research Institute, Department of Medicine University of Cambridge  Cambridge UK CB2 0BB"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Glyn","family":"Williams","sequence":"additional","affiliation":[{"name":"Yusuf Hamied Department of Chemistry University of Cambridge  Lensfield Road Cambridge UK CB2 1EW"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2708-8992","authenticated-orcid":false,"given":"Tom L.","family":"Blundell","sequence":"additional","affiliation":[{"name":"Department of Biochemistry University of Cambridge  Tennis Court Road Cambridge UK CB2 1GA"},{"name":"VPD Heart Lung Research Institute, Department of Medicine University of Cambridge  Cambridge UK CB2 0BB"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0205-5630","authenticated-orcid":false,"given":"Anthony G.","family":"Coyne","sequence":"additional","affiliation":[{"name":"Yusuf Hamied Department of Chemistry University of Cambridge  Lensfield Road Cambridge UK CB2 1EW"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chris","family":"Abell","sequence":"additional","affiliation":[{"name":"Yusuf Hamied Department of Chemistry University of Cambridge  Lensfield Road Cambridge UK CB2 1EW"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2024,8,12]]},"reference":[{"key":"e_1_2_8_1_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi3005126"},{"key":"e_1_2_8_2_1","doi-asserted-by":"publisher","DOI":"10.1038\/nchem.217"},{"key":"e_1_2_8_3_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrd.2016.109"},{"key":"e_1_2_8_4_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrd3847"},{"key":"e_1_2_8_5_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrd.2016.23"},{"key":"e_1_2_8_6_1","doi-asserted-by":"publisher","DOI":"10.2217\/fon-2020-0542"},{"key":"e_1_2_8_7_1","doi-asserted-by":"publisher","DOI":"10.1039\/C9MD90044F"},{"key":"e_1_2_8_8_1","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa2103695"},{"key":"e_1_2_8_9_1","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa1902328"},{"key":"e_1_2_8_10_1","unstructured":"FDA Approves Capivasertib with Fulvestrant for Breast Cancer.FDA2023. 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