The prioritized molecules were evaluated for synthesizability and, in some cases, adapted based on feasibility. To increase redundancy and likelihood of success, precursors and synthetic intermediates were also submitted for testing (Supplementary Table ). Synthesis routes and data for all 41 molecules generated in this study are described in the Supplementary Information. All synthesized compounds were submitted to a F NMR displacement assay, a highly sensitive technique to confirm that the compounds bind to the protein pocket of interest. Here, a well characterized F-containing reporter molecule was used at 50 µM concentration in presence of 2 µM VCB complex . Addition of the compounds to be tested at 500 µM concentration led to different degrees of displacement of the reporter probe from the binding site, and therefore to reappearance of the F NMR signal in the spectrum (Figure ). Only compound 1 showed a dose-dependent displacement of the F NMR probe and was submitted to protein-observed NMR experiments. Protein labeled selectively with C methyl groups in the residues Ile, Val, Leu, and Met was used to obtain a KD = 258 ± 8 µM, by fitting the dose-dependent shifts (Figure ). The competitive behavior was confirmed in a time-resolved fluorescence resonance energy transfer (TR-FRET) assay using a Cy5-labeled VHL Tracer analog , revealing displacement of IC50 = 264 ± 30 µM. Based on these results, the diastereomeric mixture of compound 1 was co-crystallized in complex with VCB.