Parallelized Ligand Screening Using Dissolution Dynamic Nuclear Polarization

Abstract

Protein−ligand interactions are frequently screened using nuclear magnetic resonance (NMR) spectroscopy. The dissociation constant (KD) of a ligand of interest can be determined via a spin-spin relaxation measurement of a reporter ligand, in a single scan when using hyperpolarization by means of dissolution dynamic nuclear polarization (D-DNP). Despite nearly instantaneous signal acquisition, a limitation of D-DNP for the screening of protein−ligand interactions is the required polarization time on the order of tens of minutes. Here, we introduce a multiplexed NMR experiment, where a single hyperpolarized ligand sample is rapidly mixed with protein injected into two flow cells. NMR detection is achieved simultaneously on both channels, resulting in a chemical shift resolved spin relaxation measurement. Spectral resolution allows the use of reference compounds for accurate quantification of concentrations. Simultaneous use of two concentration ratios between protein and ligand broadens the range of KD that is accurately measurable in a single experiment to at least an order of magnitude. In a comparison of inhibitors for the protein trypsin, the average KD values of benzamidine and benzylamine were found to be 12.6±1.4 μM and 207±22.3 μM from three measurements, based on KD = 142 μM assumed known for the reporter ligand 4-(trifluoromethyl)benzene-1-carboximidamide. Typical confidence ranges at 95% evaluated for single experiments were (8.3 μM, 20 μM) and (151 μM, 328 μM). The multiplexed detection of two or more hyperpolarized samples increases throughput of D-DNP by the same factor, improving the applicability to most multi-point measurements that would traditionally be achieved using titrations.

The Effect of Gadolinium Doping in [13C6,2H7]Glucose Formulations on 13C Dynamic Nuclear Polarization at 3.35 T

“…Chemicals [1][2][3][4][5][6][7][8][9][10][11][12][13] For the [ 13 C 6 , 2 H 7 ]glucose formulations, three stock solutions were prepared by dissolving OX063 to a concentration of 20 mM in D 2 O with and without addition of 0.5 M solution of Gd-DOTA to the following Gd-DOTA concentrations: 0 mM, 2 mM and 6 mM.…”

“…The dissolution dynamic nuclear polarization (dDNP) technology, that can enhance the liquid-state nuclear magnetic resonance (NMR) signal of 13 C nuclei by 3-4 orders of magnitude compared to ambient conditions, has improved the abilities of magnetic resonance in applications from analytical NMR, [1,2] reaction monitoring, [3,4] ligand screening [5,6] to real-time measurements of metabolism ex vivo and in vivo in preclinical models [7] and in vivo in human subjects. [8][9][10] In this method, the compound of interest is mixed with a free radical in a solution that vitrifies upon rapid freezing to cryogenic temperatures.…”

Characterization of Chemical Exchange Using Relaxation Dispersion of Hyperpolarized Nuclear Spins

“…These aliquots were hyperpolarized on 19 F in a HyperSense DNP polarizer (Oxford Instruments, Abingdon, U.K.), by irradiation with 100 mW of 94.055 GHz microwaves for 20 min, at a temperature of 1.4 K. 300 μL of trypsin (AMERSCO, Road Solon, OH) solutions of 2 and 4 μM in 50 mM tris(hydroxy-methyl)aminomethane buffer at pH 8, or of 4 and 10 μM in 50 mM acetate buffer at pH 5 were pre-loaded into a sample loop of a stopped-flow injection device. 24,25 Subsequently, hyperpolarized ligand was dissolved in 4 mL buffer that had been pre-heated until reaching a vapor pressure of 5 bar. This dissolution buffer was of the same composition as that used for preparation of trypsin samples, at pH 5 or pH 8.…”

Hyperpolarized NMR Spectroscopy: d‐DNP, PHIP, and SABRE Techniques

“…The polarization build-up starts with the formation of hyperpolarized species C* (i.e.,t he metal complex and parahydrogen), as shown in Equation (5). The polarizationo fs peciesC * decays with the relaxation rate constant, R C ¼ 1=T C 1 ,a ccording to Equation (6). This species takes part in an exchange process with the substrate that is governed by the substrate-dissociation rate constant (k d S ), accordingt oE quation (7), and by the substrate-associationr ate constant (k a s ), accordingt oE quation (8).…”

“…1 H), and the latter group for the polarization of nuclei with small gyromagnetic ratios (e.g. 13 C, 15 N, 31 P, 6 Li, and 129 Xe). Ah igh degree of hyperpolarization that is uniformly distributed over the entire sample is typicallya chieved for efficiently vitrifieds amples by capturing the spatial distribution of the PAsa tt he glass-transition temperature (T g ).…”