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Structure-guided design enables development of a hyperpolarized molecular probe for the detection of aminopeptidase N activity in vivo

Dynamic nuclear polarization (DNP) is a cutting-edge technique that markedly enhances the detection sensitivity of molecules using nuclear magnetic resonance (NMR)/magnetic resonance imaging (MRI). This methodology enables real-time imaging of dynamic metabolic status in vivo using MRI. To expand the targetable metabolic reactions, there is a demand for developing exogenous, i.e., artificially designed, DNP-NMR molecular probes; however, complying with the requirements of practical DNP-NMR molecular probes is challenging because of the lack of established design guidelines. Here, we report Ala-[1-13C]Gly-d2-NMe2 as a DNP-NMR molecular probe for in vivo detection of aminopeptidase N activity. We developed this probe rationally through precise structural investigation, calculation, biochemical assessment, and advanced molecular design to achieve rapid and detectable responses to enzyme activity in vivo. With the fabricated probe, we successfully detected enzymatic activity in vivo. This report presents a comprehensive approach for the development of artificially derived, practical DNP-NMR molecular probes through structure-guided molecular design.


Binding energy calculations
As stated in Fig. 2, the calculated binding energies are similar for the substrates (Ala-NH2, Ala-Gly, and Ala-Gly-NMe2) when Glu384 is deprotonated and the hydrogen-bond is absent ( fig. S1).  (34) Theoretically, T1 relaxation is decomposed to each relaxation mechanism according to the following eq. 1. Here, inversion of each T1 contribution is expressed as R1 X , as shown in eq. 2

T1 relaxation analysis
where R1 tot is total of T1 relaxation components; R1 DD is T1 relaxation by dipole-dipole interaction, here we especially focus on dipole-dipole interaction between 13 C and 1 H nuclei that mainly contributes to the relaxation; R1 CSA is T1 relaxation of chemical shift anisotropy; R1 O2 is T1 relaxation by dipole-dipole interaction between 13 C and O2 molecules existing in the solvent; R1 res is residual factors including spin rotation and scalar coupling (35). In this paper, R1 DD and R1 O2 for each and R1 CSA for Ala-[1-13 C]Gly-d2-NMe2 at 9.4 T were determined according to the reported procedure (34). The result is shown in fig. S2. The further information for R1 CSA is shown in fig.  S13.

Fig. S3. Synthetic route for Ala-[1-13 C]Gly-d2-NMe2.
In an early stage of this study, different protocol was employed for deuteration and Cbzprotection steps of glycine ( fig. S4). Through this scheme, Ala-[1-13 C]Gly-d2-NMe2 was obtained in 33% total yield with 83% deuterated efficiency at a-position of Gly-moiety. This sample was used for the experiments shown in Fig. 5D.

In vitro experiment under thermal equilibrium state
Detection of APN-activity using Ala-[1-13 C]Gly-d2-NMe2 under thermal equilibrium state Figs. S5 and S6 are the whole spectra (x-offset: -25 ppm to 225 ppm) of the thermally equilibrated 13 C NMR spectra shown in Fig. 5B. No peak except ones of Ala-[1-13 C]Gly-d2-NMe2, [1-13 C]Gly-d2-NMe2, and DMSO (an internal standard) was observed. The detailed procedure is described in Materials and Methods section.  Figs. S7 and S8 show the thermally equilibrated 13 C NMR spectra of Ala-[1-13 C]Gly-d2-NMe2 incubated in homogenate of mouse tumor xenograft (MIA PaCa-2) in the presence or the absence of phebestin, an APN inhibitor. In these experiments, it was confirmed that the same reaction with that in kidney homogenate (shown in Fig. 5B and figs. S5 and S6) occurred although the conversion of Ala-[1-13 C]Gly-d2-NMe2 was slower than that in Fig. 5B and fig. S5.  Stability of Ala-[1-13 C]Gly-d2-NMe2 in mouse serum We assessed the stability of Ala-[1-13 C]Gly-d2-NMe2 (3) in mouse serum. There is a concern that Ala-[1-13 C]Gly-d2-NMe2 may be metabolized in blood before reaching at the region of interest because APN is known to also exist in blood as a soluble form (43). In this experiment, no conversion of Ala-[1-13 C]Gly-d2-NMe2 was observed within 21 min after incubation in mouse serum. After 28 min incubation, the peak of the metabolite [1-13 C]Gly-d2 (4) was gradually appeared. The detailed procedure is described in Materials and Methods section.

In vivo experiment under hyperpolarized state
Recovery experiment in the hyperpolarized study for in vivo detection of APN activity In the study for detection of APN-activity in vivo shown in Fig. 6, we confirmed the recovery of APN-activity one day after administration of phebestin ( fig. S10). This result indicates that the DNP-MRS with the current APN probe enables repetitive measurements without affecting APNactivity. (right) Expanded spectra in representative voxels a-d. The voxel size is 2.8 mm × 2.8 mm × 10 mm. The left peak is Ala-[1-13 C]Gly-d2-NMe2 (3, red circle), and the right peak is [1-13 C]Gly-d2-NMe2 (4, orange circle). The image was acquired at 18 s to 26 s after injection of the hyperpolarized probe. 13 C spectra are with denoising process. The voxel size is 2.8 mm × 2.8 mm × 10 mm. Inset figure: expanded spectra in representative voxels a-c. The left peak is Ala-[1-13 C]Gly-d2-NMe2 (3, red circle), and the right peak is [1-13 C]Gly-d2-NMe2 (4, orange circle). figs. S12A and S12C correspond to Fig. 6G; figs S12B and S12D correspond to fig. S11, respectively. 13 C spectra of figs. S12A and S12B are with denoising process; 13 C spectra of figs. S12C and S12D are without denoising process.

Magnetic field dependency and R1 CSA
T1 and R1 of Ala-[1-13 C]Gly-d2-NMe2 were measured at 9.4, 11.7, and 14.1 T. The results are summarized in fig. S13. R1 values were proportional to the square of the magnetic field (B0 2 ). This trend indicates that R1 CSA is much larger than the other B0-dependent R1 factors. From this plot, the R1 CSA of Ala-[1-13 C]Gly-d2-NMe2 can be determined as the product of the coefficient of the approximate line and the square of magnetic field strength (44,45). The R1 CSA at 9.4 T was 14.9 × 10 -3 s -1 . The T1 at 3.0 T was predicted to be 62.5 s.  Table S1. T1 values measured at 3.0 T under hyperpolarized state [a] [a] The corrected T1 values are shown. The corrected T1 values were calculated from apparent T1 values considering the magnetization loss due to successive flip angles. Dissolved hyperpolarized solution (DPBS containing 0.68 mM EDTA, 1.0 mL) was added into 2.0 mL of DPBS, human serum, D2O or phosphate buffer (PB, 100 mM, pH = 7.4), and then subjected to T1 measurement (3.0 T, ca. 37 °C).

8-1. General Synthesis
Unless otherwise noted, all materials including dry solvents were obtained from commercial suppliers and used without further purification. Unless otherwise noted, work-up and purification procedures were performed with reagent-grade solvents under air. Analytical thin-layer chromatography (TLC) was performed using E. Merk silica gel 60 F254 precoated plates (0.25 mm). The developed chromatogram was analyzed with UV lamp (254 nm) and ethanolic ninhydrin. Flash column chromatography was performed with silica gel 60N (Kanto Chemical Co., spherical, neutral, 40-50 µm) in manual operation, or with an Isolera Spektra instrument equipped with a Biotage ® Sfär Silica HC D 10 g or Sfär Silica HC D 25 g cartridge in automatic operation.
How to treat the current probe (Ala-[1-13 C]Gly-d2-NMe2) Because Ala-[1-13 C]Gly-d2-NMe2 is air-sensitive as a free-amine form, this compound should be treated quickly under air and stored in inert gas. After neutralization in aqueous solution, this compound is relatively stable but long storage in acidic condition causes decomposition. This compound is highly hygroscopic, and does not become solid easily, so that lyophilization may be needed to be repeated a few times. Although the solution turned brown or red sometimes during lyophilization (probably, due to oxidation), the samples could be used for hyperpolarization without any problems.
Hyperpolarized studies were performed by a Hypersense DNP polarizer (Oxford Instruments, UK) using microwave irradiation at 94.069 GHz and 100 mW under 2.8 mbar at 1.45 K, and a 3 T MR Solutions animal scanner (MR SOLUTIONS Ltd., USA). The signal processing of MRS data was performed using the low rank denoising technique as described previously (41).

8-2. Binding energy calculations
The substrate-APN structures for Ala-NH2-APN, Ala-Gly-APN, and Ala-Gly-NMe2-APN were modeled, using the E384Q mutant crystal structure with polyalanine (PDB ID: 4NAQ) and replacing the Gln384 side chain with glutamate (32). Hydrogen atoms and the Glu384 side-chain were energetically optimized with CHARMM version 40b (46), while the positions of all nonhydrogen atoms were fixed, and all titratable atoms groups were maintained in their standard protonation states at pH 7 (i.e., acidic groups were ionized, and basic groups were protonated) if not otherwise specified. The protonation pattern of the titratable residues was calculated, solving the linear Poisson-Boltzmann equation with the MEAD program (47). The difference in electrostatic energy between the two protonation states, protonated and deprotonated, in a reference model system was calculated using a known experimentally measured pKa value (e.g., 4.0 for Asp (48)). The difference in the pKa value of the protein relative to the reference system was added to the known reference pKa value. The experimentally measured pKa values employed as references were 12.0 for Arg, 4.0 for Asp, 9.5 for Cys, 4.4 for Glu, 10.4 for Lys, 9.6 for Tyr (48), and 7.0 and 6.6 for the Ne and Nd atoms of His, respectively (49)(50)(51). All other titratable sites were fully equilibrated to the protonation state of the target site during titration. The dielectric constants were set to 4 for the protein interior and 80 for water. All other water molecules, whose hydrogen bond orientations were not specifically reported, were considered implicitly. All computations were performed at 300 K, pH 7.0, and with an ionic strength of 100 mM. The linear Poisson-Boltzmann equation was solved using a three-step grid-focusing procedure at resolutions of 2.5, 1.0, and 0.3 Å. The ensemble of the protonation patterns was sampled by the Monte Carlo (MC) method with the Karlsberg program (52). The MC sampling yielded the probabilities [protonated] and [deprotonated] of the two protonation states of the molecule. Using the calculated protonation pattern, hydrogen atoms of the protonated residues were generated explicitly. The geometry was optimized using a QM/MM approach. The restricted density functional theory (DFT) method was employed with the B3LYP functional and LACVP* basis sets using the QSite program (53). The QM region was defined as substrate, Zn 2+ , water molecules (HOH-104, 1218, and 1776), the side chains of Gln208, Glu350, Glu384, His383, His387, Glu406, and Tyr472, and the side chain and backbone of Ala348. All atomic coordinates were fully relaxed in the QM region. In the MM region, the positions of hydrogen atoms were optimized using the OPLS2005 force field (54), while the positions of the heavy atoms were fixed. The binding energy between the substrate and APN was calculated based on the QM/MM-optimized structure, using the MOE program (55). See Data S1 for the QM/MM-optimized atomic coordinates.

8-3. Synthesis
Synthesis of S2 (36) To a 50-mL round-bottom flask containing a magnetic stirring bar were added [1-13 C]Gly (S1) (304 mg, 4.00 mmol), ruthenium on activated carbon (121 mg, 1.5 mol% for Ru, 5%wt), and D2O (7.0 mL). The vessel was equipped with a condenser and a H2 balloon. After evacuation of air and filling back H2 (repeated three times), the mixture was stirred at 85 °C for 21 h. After cooling to room temperature, the resulting mixture was filtrated through Celite ® . The filtrate was concentrated under reduced pressure. The deuterated product (S2) was obtained (313 mg, quant.) as a yellowish solid. This product was used for the next step without further purification and characterization.

Synthesis of S3
To a 25-mL round-bottom flask containing a magnetic stirring bar were added [1-13 C]Gly-d2 (S2) (313 mg, 4.00 mmol) and H2O (3.0 mL). After addition of Et3N (1.12 mL, 2.0 equiv), N-(benzyloxycarbonyloxy)succinimide (CbzOSu, 1.05 g, 1.1 equiv) in MeCN (1.2 mL) was added into the vessel. The mixture was stirred at room temperature for 13 h. The resulting mixture was evaporated to remove MeCN and Et3N. To the remained aqueous solution was added saturated NaHCO3 aq. (10 mL), and then the solution was washed with Et2O (5.0 mL). The aqueous phase was acidified with 1 M HCl aq. to adjust pH into ca. 3. When white precipitation appeared, the resulting mixture was extracted with EtOAc (15 mL × 6). The organic phase was dried over anhydrous Na2SO4, filtrated, and concentrated under reduced pressure. The residue was confirmed to contain the desired product S3 and hydroxysuccinimide with 1 H NMR. This crude was used for the next step without further purification.

Synthesis of 4
To a 50-mL round-bottom flask containing a magnetic stirring bar were added Cbz-[1-13 C]Gly-d2-NMe2 (S4) (636 mg, 2.66 mmol) and palladium on activated carbon (28.3 mg, 1.0 mol% for Pd, 10%wt). The vessel was equipped with a three-way cock and a N2 balloon, and then purged with N2 through evacuation and filling back N2 three times. After addition of MeOH (13 mL), the N2 balloon was replaced with a H2 balloon, and the vessel was purged with H2. The mixture was stirred for 1.5 h and filtered through Celite ® . The filtrate was concentrated under reduced pressure. The desired compound [1-13 C]Gly-d2-NMe2 (4) was obtained (276 mg, 99% yield) as a colorless oil

8-4. Biochemical experiments
Expression and purification of recombinant human APN The cDNA coding human aminopeptidase N (D66-K967, Uniprot ID: P15144) was cloned by PCR from cDNA library of A204 cells (primers are shown below) and subcloned into the expression vector pEFs (42). The leader sequence of the immunoglobulin kappa chain and hexahistidine tag were fused at the N-terminus and C-terminus, respectively. A proline residue was inserted between the leader sequence and APN-coding sequence. The expression and purification of the proteins were conducted as previously described (56). Expi293F cells were transfected with the expression vector and cultured according to the manufacturer's protocol. After 4 days of culture, the cells were centrifuged at 3000 × g for 30 min at 4 °C, and the supernatant was dialyzed at 4 °C against nickel-nitrilotriacetic acid (Ni-NTA)-binding buffer (20 mM Tris-HCl (pH = 7.6 at r.t.), 500 mM NaCl, and 5 mM imidazole). The dialyzed samples were purified using Ni-NTA agarose beads (#30210, QIAGEN) and Econo-Pac Chromatography Columns (Bio-Rad). The eluted samples were dialyzed against size exclusion chromatography (SEC) buffer (10 mM HEPES (pH = 7.4 at r.t.), and 50 mM NaCl) and purified using a Superdex 26/60 column (GE Healthcare). The concentration of the protein was determined by measuring the absorbance at 280 nm using a NanoDrop ND-1000 (Thermo Fisher Scientific). The purity of the final preparation was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie Brilliant Blue (CBB) staining.

8-5. Chemical shift and T1 measurements under thermal equilibrium state
Chemical shift change in Fig. 3B was measured with a JEOL ECS400 (9.4 T). Measurement conditions: 100 mM each of the probe candidate and the product in phosphate buffer containing 10% D2O (90 mM, pH = 7.4), 37 °C, pH was adjusted with 1 M NaOH aq.
The T1 values under thermal equilibrium state in Figs. 3B and 3D and fig. S13 were measured with a JEOL ECS400 (9.4 T), JEOL ECA 500 (11.7 T), and JEOL ECA 600 (14.1 T) by the inversion recovery method. Representative measurement conditions (for Fig. 3B): 500 mM of the compound in phosphate buffer containing 10% D2O (90 mM, pH = 7.4), 37 °C, 9.4 T, pH was adjusted with 35 wt% DCl in D2O and 40 wt% NaOD in D2O. In vitro detection of APN activity under hyperpolarized state for 13 C MR spectroscopy in vitro (Fig. 5C) Probe solution A (25.0 µL) prepared above was hyperpolarized (dissolution buffer: 4.0 mL of DPBS containing 0.68 mM EDTA). To 2.0 mL of DPBS was added 1.0 mL of the hyperpolarized solution. Dynamic 13 C MR spectrum was acquired using a 3 T MRI scanner (repetition time = 1 s, flip angle = 10°). The apparent T1 was obtained by curve fitting using the following eq. 3 where I(t) is the signal intensity at an arbitrary time t; T1 app is the apparent T1; y0 is the signal offset. The fitting algorithm was implemented in ImageJ. The corrected T1 was obtained using the following eq. 4 where T1 corr is the corrected T1; θ is the flip angle; TR is the repetition time.
In vivo detection of APN activity in human pancreatic tumor xenograft bearing mouse (Fig. 6, S10-S12) Probe solution B (40 or 80 µL for Figs. 6B, 6C, and fig. S10; 120 µL for Fig. 6G and figs S12A, and S12C; 80 µL for figs. S11, S12B, and S12D) prepared above was hyperpolarized (dissolution buffer: 4.0 mL of DPBS containing 0.68 mM EDTA). The hyperpolarized solution (ca. 400 µL) was injected into the tail vein of athymic nude mice bearing a tumor xenograft (MIA PaCa-2) in the hind leg. Dynamic 13 C MR spectrum was acquired using a 3 T MRI scanner using a 17 mm homebuilt 13 C solenoid coil placed inside of a 1 H saddle coil, which site-specifically detects a tumor region embedded in the right leg. The non-localized 13 C MRS were obtained with a spectral width of 3333 Hz, repetition time of 1 s, a flip angle of 10°. In this homebuilt 13 C solenoid coil area, approximately 75% of the tissue volume is attributed to tumor regions based on the analysis of the FLASH anatomical MRI scans, which have been addressed in our previous studies (57,58). Anatomical images were acquired using a T2-weighted fast spin-echo imaging sequence with a field of view of 28 mm × 28 mm with a 256 × 256 matrix, a 2 mm section thickness with a 2 mm intersection gap, a repetition time of 2500 ms, an effective echo time of 68 ms, an echo-train length of 8 echoes. All of the animal studies were approved by the National Cancer Institute Animal Care and Use Committee (NCI-CCR-ACUC) in accordance with the Guide for the Care and Use of Laboratory Animal Resources. Note: When the hyperpolarized solution is at a low magnetic field region close to the earth's magnetic field, the hyperpolarized signal will be reduced because of scalar relaxation by nearby 14 N nuclei. To avoid this problem, it is recommended to handle hyperpolarized solutions at a magnetic field strength above a certain level or to use a magnet for sample transfer (40,59).

Administration of phebestin
The stock solution of phebestin (56.6 µL, 10 mM in DMSO) was dissolved into H2O (43.4 µL). The diluted solution was intravenously injected into mice (10 mg/kg) 24 h after the measurement of APN-activity of the day 1.