Accelerated Screening of Protein-Ligand Interactions via Parallel T2-Weighted 19F-MRI

Abstract

In drug discovery, ligands are sought that modulate the (mal-)function of medicinally relevant target proteins. In order to develop new drugs, typically a multitude of potential ligands are initially screened for binding and subsequently characterized for their affinity. Nuclear magnetic resonance (NMR) is a well-established and highly sensitive technology for characterizing such interactions. However, it has limited throughput, because only one sample can be measured at a time. In contrast, magnetic resonance imaging (MRI) is inherently parallel and MR parameters can conveniently be encoded in its images, potentially offering increased sample throughput. We explore this application using a custom-built 9-fold sample holder and a ¹⁹F-MRI coil. With this setup, we show that ligand binding can be detected by T₂-weighted ¹⁹F-MRI using 4-(trifluoromethyl)benzamidine (TFBA) and trypsin as the reporter ligand and target protein, respectively. Furthermore, we demonstrate that the affinity of nonfluorinated ligands can be determined in a competition format by monitoring the dose-dependent displacement of TFBA. By comparing ¹⁹F-T₂-weighted MR images of TFBA in the presence of different benzamidine (BA) concentrations-all recorded in parallel-the affinity of BA could be derived. Therefore, this approach promises parallel characterization of protein-ligand interactions and increased throughput of biochemical assays, with potential for increased sensitivity when combined with hyperpolarization techniques.

Figure 1

(a) Alderman–Grant coil designed to operate at 612 MHz corresponding to the ¹⁹F NMR Larmor frequency at 15.2 T. A 3D-printed coil holder (black) and coaxial RF cable (blue) are also shown. The schematic insert above shows the geometry of the coil tracks. (b) S₁₁ reflection curve of the unloaded Alderman–Grant resonator.

Figure 2

Sample holder assembly, 3D-printed using PLA. Nine individual sample capillaries, each with an OD of 2.4 mm, and filled with a sample volume of 150–200 μL, were thus accommodated.

Figure 3

¹⁹F limit of detection of TFBA in a capillary system. Five different TFBA concentrations (white) and their calculated SNR (yellow) values are shown from the FLASH experiment in one acquisition experiment. The imaging parameters were as follows: FOV, 30 mm × 30 mm; MTX, 32 × 32; SL, 5 mm; TR/TE = 100/4 ms; NEX, 200; TA, 10 min, 40 s. SNR was calculated as described in the Experimental Methods section, taking a slice ROI area covering 3.55 voxels, with a volume of 4.4 mm³ per sample.

Figure 4

Transverse relaxation time dependence on binding between trypsin and its reporter and competitor ligands, TFBA and BA. Spin–echo intensities extracted from the chosen ROI of the MR-image averaged 20 scans, of 25 mM TFBA obtained in the absence (black) and presence of 150 μM trypsin (blue), and in the presence of both 150 μM trypsin and 10 mM benzamidine (red). T₂ relaxation times were obtained by fitting the mean of three replicate measurements to a single exponential (error bars are ± standard deviation); T_2,f = 952 ms, T_2,nc = 231 ms, and T_2,c = 738 ms, (ParaVision) with a fit quality (R²) of 0.98, 0.98, and 0.97 (originLab), respectively. The corresponding relaxation rates are R_2,f = 1.05 s^–1, R_2,nc = 4.33 s^–1, and R_2,c = 1.36 s^–1.

Figure 5

¹⁹F T₂ map of TFBA obtained using MSME. Samples for noncompetitive binding to trypsin and competitive binding in the presence of BA were measured in a single experiment. The MSME experiment was a collection of nine samples with 10 echo times, ranging from 200 ms to 1100 ms. Fixed amounts of TFBA (A, 25 mM) were added to trypsin in different concentrations (B, 25 μM; C, 50 μM; D, 75 μM; and E, 150 μM) for noncompetitive experiments. Different concentrations of BA (F, 1 mM; G, 10 mM; H, 25 mM; and I, 50 mM) was then added to the fixed TFBA and trypsin at 150 μM for the competition format. The imaging parameters were as follows: FOV, 20 mm × 20 mm; MTX, 32 × 32; SL, 10 mm; TR/TE = 5418/100 ms; NEX, 20. The full set of 10 echo experiments required 76 min. The image displayed was obtained at an echo time of 200 ms. The crosslike shapes are due to a partial volume effect resulting from pixel dimensions approaching the diameter of the sample capillary.

Figure 6

Dependence of the TFBA ¹⁹F signal intensity (black) and R₂ rates (red) on titrating trypsin ([TFBA] = 25 mM). ¹⁹F-signal intensities are normalized with the sample without trypsin.

Figure 7

TFBA ¹⁹F signal intensity (black) and R₂ rates (red) in the competitive assay. BA was titrated with [TFBA] = 25 mM and [trypsin] = 150 μM. Lines connecting the data points are intended to guide the eye.