Screening of Protein-Ligand Binding Using a SABRE Hyperpolarized Reporter

Hyperpolarization through signal amplification by reversible exchange (SABRE) provides a facile means to enhance nuclear magnetic resonance (NMR) signals of small molecules containing an N-heterocycle or other binding site for a polarization transfer catalyst. A purpose-designed reporter ligand, which is capable of binding both to a target protein and to the catalyst, makes the sensitivity enhancement by this technique compatible with the measurement of a range of biomolecular interactions. The ¹H polarization of the reporter ligand 4-amidinopyridine, which is targeting trypsin, is used to screen ligands that are not themselves hyperpolarizable by SABRE. The respective protein-ligand dissociation constants (K_D) are determined by an observed change in the R₂ relaxation rate of the reporter. A calculation of expected signal changes indicates that the accessible ligand K_D values extend over several orders of magnitude, while the concentrations of target proteins and ligands can be reduced considering the sensitivity gains from hyperpolarization. In general, the design of a single, weakly binding ligand for a target protein enables the use of SABRE hyperpolarization for ligand screening or other biophysical studies involving macromolecular interactions.