Screening of Protein–Ligand Binding Using a SABRE Hyperpolarized Reporter

Abstract

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 (KD) 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 KD 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.