Determination of Ligand-Binding Affinity (<i>K</i>_d) Using Transverse Relaxation Rate (<i>R</i>₂) in the Ligand-Observed ¹H NMR Experiment and Applications to Fragment-Based Drug Discovery

High hit rates from initial ligand-observed NMR screening can make it challenging to prioritize which hits to follow up, especially in cases where there are no available crystal structures of these hits bound to the target proteins or other strategies to provide affinity ranking. Here, we report a reproducible, accurate, and versatile quantitative ligand-observed NMR assay, which can determine Kd values of fragments in the affinity range of low μM to low mM using transverse relaxation rate R2 as the observable parameter. In this study, we examined the theory and proposed a mathematical formulation to obtain Kd values using non-linear regression analysis. We designed an assay format with automated sample preparation and simplified data analysis. Using tool compounds, we explored the assay reproducibility, accuracy, and detection limits. Finally, we used this assay to triage fragment hits, yielded from fragment screening against the CRBN/DDB1 complex

Demonstrating In-Cell Target Engagement Using a Pirin Protein Degradation Probe (CCT367766)

Demonstrating intracellular protein target engagement is an essential step in the development and progression of new chemical probes and potential small molecule therapeutics. However, this can be particularly challenging for poorly studied and noncatalytic proteins, as robust proximal biomarkers are rarely known. To confirm that our recently discovered chemical probe <b>1</b> (CCT251236) binds the putative transcription factor regulator pirin in living cells, we developed a heterobifunctional protein degradation probe. Focusing on linker design and physicochemical properties, we generated a highly active probe <b>16</b> (CCT367766) in only three iterations, validating our efficient strategy for degradation probe design against nonvalidated protein targets

Discovery of Potent, Orally Bioavailable, Small-Molecule Inhibitors of WNT Signaling from a Cell-Based Pathway Screen

WNT signaling is frequently deregulated in malignancy, particularly in colon cancer, and plays a key role in the generation and maintenance of cancer stem cells. We report the discovery and optimization of a 3,4,5-trisubstituted pyridine <b>9</b> using a high-throughput cell-based reporter assay of WNT pathway activity. We demonstrate a twisted conformation about the pyridine–piperidine bond of <b>9</b> by small-molecule X-ray crystallography. Medicinal chemistry optimization to maintain this twisted conformation, cognisant of physicochemical properties likely to maintain good cell permeability, led to <b>74</b> (CCT251545), a potent small-molecule inhibitor of WNT signaling with good oral pharmacokinetics. We demonstrate inhibition of WNT pathway activity in a solid human tumor xenograft model with evidence for tumor growth inhibition following oral dosing. This work provides a successful example of hypothesis-driven medicinal chemistry optimization from a singleton hit against a cell-based pathway assay without knowledge of the biochemical target