Bütikofer, Matthias, Gabriela R. Stadler, Harindranath Kadavath, Riccardo Cadalbert, Felix Torres, and Roland Riek. “Rapid Protein–Ligand Affinity Determination by Photoinduced Hyperpolarized NMR.” Journal of the American Chemical Society 146, no. 26 (July 3, 2024): 17974–85.
https://doi.org/10.1021/jacs.4c04000.
The binding affinity determination of protein−ligand complexes is a cornerstone of drug design. State-of-the-art techniques are limited by lengthy and expensive processes. Building upon our recently introduced novel screening method utilizing photochemically induced dynamic nuclear polarization (photo-CIDNP) NMR, we provide the methodological framework to determine binding affinities within 5−15 min using 0.1 mg of protein. The accuracy of our method is demonstrated for the affinity constants of peptides binding to a PDZ domain and fragment ligands binding to the protein PIN1. The method can also be extended to measure the affinity of nonphoto-CIDNPpolarizable ligands in competition binding experiments. Finally, we demonstrate a strong correlation between the ligand-reduced signals in photo-CIDNP-based NMR fragment screening and the well-established saturation transfer difference (STD) NMR. Thus, our methodology measures protein−ligand affinities in the micro- to millimolar range in only a few minutes and informs on the binding epitope in a single-scan experiment, opening new avenues for early stage drug discovery approaches.