Nanoparticle-Catalyzed Transamination under Tumor Microenvironment Conditions: A Novel Tool to Disrupt the Pool of Amino Acids and GSSG in Cancer Cells

Abstract

Catalytic cancer therapy targets cancer cells by exploiting the specific characteristics of the tumor microenvironment (TME). TME-based catalytic strategies rely on the use of molecules already present in the TME. Amino groups seem to be a suitable target, given the abundance of proteins and peptides in biological environments. Here we show that catalytic CuFe₂O₄ nanoparticles are able to foster transaminations with different amino acids and pyruvate, another key molecule present in the TME. We observed a significant in cellulo decrease in glutamine and alanine levels up to 48 h after treatment. In addition, we found that di- and tripeptides also undergo catalytic transamination, thereby extending the range of the effects to other molecules such as glutathione disulfide (GSSG). Mechanistic calculations for GSSG transamination revealed the formation of an imine between the oxo group of pyruvate and the free -NH₂ group of GSSG. Our results highlight transamination as alternative to the existing toolbox of catalytic therapies.

Keywords: Alanine; Amino acids; Cancer therapy; Copper; Glutamine; Glutathione; Nanocatalysis; Pyruvate; Transamination.

Figure 1

Different nanoparticle-catalyzed strategies developed for cancer therapy. (a) Bioorthogonal catalysis based on pro-drug activation strategies typically require a transition-metal catalyst including Pd, Pt, Au, Rh, or Cu to form a cytotoxic compound by either removing a chemical group from a pro-drug or binding two low-toxicity molecules. (b) Reactions employed in the context of nanocatalytic therapy in the TME: (i) GSH oxidation, (ii) ^•OH or (iii) O₂ generation using endogenous H₂O₂, and (iv) glucose oxidation. (c) New scenario potentially enabled by the internalization of transition-metal leaching nanoparticles. In particular, Cu²⁺ catalyzes the transamination reaction between the −NH₃⁺ group attached to α-C of an amino acid/peptide and the keto group of pyruvate to yield d/l-alanine and the corresponding keto-acid derived from the amino acid/peptide. Reactions target key biomolecules in the cell: glutamine, glutamic acid, aspartic acid, and GSSG.

Figure 2

¹H NMR analysis of the transamination reaction in the presence of CuFe₂O₄ nanoparticles: (a) Schematic display of the transamination reaction between selected amino acids (glutamine, glutamic acid, and aspartic acid acting as amino donors) and pyruvate to yield α-ketoacid acid and alanine. (b) ¹H NMR analysis of the glutamine–pyruvate transamination reaction at different times. (c) UPLC-MS chromatogram of the produced alanine from the glutamine–pyruvate transamination (m/z = 90, [M + H]⁺). (d) Control experiment using CuFe₂O₄ as catalyst in the absence of GSH using glutamine as the amino acid substrate. (e) ¹H NMR spectra of the glutamic acid–pyruvate transamination at different reaction time intervals. (f) Evolution of alanine concentration with reaction time in a system containing 1 mM GSH using glutamic acid as the amino acid substrate (additional ¹H NMR spectra and UPLC-MS chromatograms can be found in Figure S5 and Figure S6). Alanine derived from aspartic acid–pyruvate transamination reaction was also found in (g), the ¹H NMR spectra corresponding to the aspartic acid–pyruvate transamination at different reaction time intervals. (h) UPLC-MS analysis of alanine derived from aspartic acid–pyruvate transamination (additional ¹H NMR spectra/UPLC-MS chromatograms are depicted in Figure S6 and Figure S7). Reaction conditions for all experiments: [Cu] = 6 mM, [pyruvate] = 30 mM, [amino acid] = 45 mM, [GSH] = 5 mM, pH = 7.4 (Na₂HPO₄/NaH₂PO₄ 1M), T = 37 °C.

Figure 3

Cu-catalyzed transamination of GSH-GSSG in the presence of CuFe₂O₄ nanoparticles. (a) Cu²⁺ released from CuFe₂O₄ nanoparticles first catalyzes GSH oxidation with dissolved O₂, giving GSSG; then it furthers catalyzes its transamination with pyruvate. (b) ¹H NMR and (c) UPLC-MS analysis of the generation of alanine from transamination of GSSG at different reaction times. (d, e) MS analysis of the formation of α-ketoGSSG and the depletion of GSH at various reaction times. Reaction conditions: [Cu] = 6 mM, [pyruvate] = 30 mM, [GSH] = 5 mM, pH = 7.4 (Na₂HPO₄/NaH₂PO₄ 1 M), T = 37 °C.

Figure 4

(a) ΔG values for the 1,3-H shift with HPO₄^2– acting as the H-transferring agent. (b) Depiction of the most stable conformers of TS-II A and TS-II B. Dotted black lines indicate Cu–ligand bonds, thin yellow lines represent TS bonds, and distances are displayed in Å. (c) Experimental reaction rates at different pH values after 7 h. (d) Thermodynamic aspects of the transamination process. Computational protocols: DFT calculations⁻ were carried out with ωB97X-D/Def2-QZVPP//ωB97X-D/6-31+G(d,p),⁻ SMD⁻ (solvent = water) was included in all the calculations, standard state = 1 M, T = 37 °C.

Figure 5

Tracking the intracellular transamination induced by CuFe₂O₄ nanoparticles. (a) Intracellular glutamine concentrations decrease for both control and treated U251-MG cells. Glutamine is a key metabolite for cells as one of the major nitrogen sources and is used both for the TCA cycle or for fatty acid/nucleotide biosynthesis. Treatment with CuFe₂O₄ nanoparticles significantly decreased glutamine levels especially after 72 h. (b) Monitoring intracellular alanine concentration revealed different profiles in control/treated U251-MG cells. After 24 and 48 h, the alanine concentration was significantly larger for treated U251, suggesting that artificial transamination had been successfully induced. (c) Pyruvate concentration present in cell media at different incubation times with 0.05 mg·mL^–1 CuFe₂O₄ nanoparticles. As a result of the Cu-catalyzed transamination reaction, the concentration of pyruvate was lower after 48 and 72 h in comparison to the control experiment. Intracellular pyruvate could not be determined with sufficient accuracy due to low concentration levels. (d) Confocal microscopy analysis of U251-MG cells revealed the internalization of CuFe₂O₄ nanoparticles in the form of aggregates (highlighted in yellow). (e) Intracellular copper levels of U251-MG cells treated with 50 μg·mL^–1 of CuFe₂O₄ showed a strong increase of copper concentration up to 48 h, followed by a decrease at 72 h. (f) Schematic illustration of some possible catalytic pathways of the intracellular amino acid pool: glutamine (or other amino acids) can enter different metabolic routes to enable ATP or lipid biosynthesis. However, internalization of CuFe₂O₄ nanoparticles increases the intracellular concentration of Cu²⁺, a catalyst that promotes artificial amino acid/pyruvate transamination, as well as that of other species with suitable chemical structure such as GSH and GSSG. For GSSG this reaction competes with the reduction of GSSG to GSH by glutathione reductase. Statistically significant differences were expressed as follows: *p < 0.05, **p < 0.005, ***p < 0.0005, and ****p < 0.00005.