Structural and Functional Studies of S-(2-Carboxyethyl)-L-Cysteine and S-(2-Carboxyethyl)-l-Cysteine Sulfoxide

Abstract

Insecticidal non-proteinogenic amino acid S-(2-carboxyethyl)-L-cysteine (β-CEC) and its assumed metabolite, S-(2-carboxyethyl)-l-cysteine sulfoxide (β-CECO), are present abundantly in a number of plants of the legume family. In humans, these amino acids may occur as a result of exposure to environmental acrylonitrile or acrylamide, and due to consumption of the legumes. The β-CEC molecule is a homolog of S-carboxymethyl-l-cysteine (carbocisteine, CMC), a clinically employed antioxidant and mucolytic drug. We report here detailed structural data for β-CEC and β-CECO, as well as results of in vitro studies evaluating cytotoxicity and the protective potential of the amino acids in renal tubular epithelial cells (RTECs) equipped with reporters for activity of seven stress-responsive transcription factors. In RTECs, β-CEC and the sulfoxide were not acutely cytotoxic, but activated the antioxidant Nrf2 pathway. β-CEC, but not the sulfoxide, induced the amino acid stress signaling, which could be moderated by cysteine, methionine, histidine, and tryptophan. β-CEC enhanced the cytotoxic effects of arsenic, cadmium, lead, and mercury, but inhibited the cytotoxic stress induced by cisplatin, oxaliplatin, and CuO nanoparticles and acted as an antioxidant in a copper-dependent oxidative DNA degradation assay. In these experiments, the structure and activities of β-CEC closely resembled those of CMC. Our data suggest that β-CEC may act as a mild activator of the cytoprotective pathways and as a protector from platinum drugs and environmental copper cytotoxicity.

Keywords: NRK-52E cell line; X-ray diffraction crystallography; amino acid stress signaling; antioxidants; carbocisteine; green fluorescent protein; heavy metal cytotoxicity; luciferase assay; transcriptional activation reporters.

Scheme 1

Preparation of S-(2-carboxyethyl)-L-cysteine (β-CEC, 1) and its consequent oxidation to S-(2-carboxyethyl)cysteine sulfoxide, which was obtained as a mixture of the (2R,4R)-epimer [(4R)-β-CECO, 2] and the (2R,4S)-epimer [(4S)-β-CECO, 3], due to emergence of a new chiral center around the sulfur atom.

Figure 1

Atomic numbering and displacement ellipsoids at the 50% probability level for β-CEC. An intramolecular N-H···S interaction is shown as a dashed line.

Figure 2

Molecular structure of S-(2-carboxyethyl)-l-cysteine sulfoxide. (a) Atomic numbering and displacement ellipsoids at the 50% probability level for (4R)-β-CECO. Intramolecular N-H···O interactions are shown as dashed lines. (b) Atomic numbering and displacement ellipsoids at the 50% probability level for (4S)-β-CECO.

Figure 3

Interaction energies in crystal structures. (a) A view of interactions between a central molecule of β-CEC in crystalline 1, shown as its Hirshfeld surface, and 14 molecules that share the interaction surfaces with the central molecule. (b) Calculated energies (electrostatic, polarization, dispersion, repulsion, and total) of pairwise interactions in 1 between the central molecule and those indicated by respective colors. (c) Energy framework for total pairwise interaction energies in 1. The cylinders link molecular centroids, and the cylinder thickness is proportional to the magnitude of the energies, such as those shown in (b). For clarity, the cylinders corresponding to energies < 5 kJ mol⁻¹ are not shown. (d) The total pairwise interaction energy framework in 2. For interaction energies in 2 and 3, also see Supplementary Materials Figures S5 and S6. For energy frameworks depicting electrostatic and dispersion energies in crystals of 1, 2, and 3, see Supplementary Materials Figures S7–S9.

Figure 4

Testing antioxidant activities of β-CEC and β-CECO (1:1 diastereomeric mixture). (a) Scavenging 40 μM hydrogen peroxide in the Amplex Red assay. (b) Protection of dsDNA degradation in presence of 50 μM copper/2 mM ascorbate/2 mM H₂O₂. Abbreviations: Ctrl, no H₂O₂; DTPA, diethylenetriaminepentaacetic acid; NAC, N-acetyl-L-cysteine; GSH, reduced glutathione; Ahd, l-2-aminohexanedioic acid. Concentrations of inhibitors—1 mM; DMSO—0.5% v/v. For each treatment, n = 3. The differences in means were probed by the one-way ANOVA; ø indicates no statistically significant difference between a treatment and “no inhibitor” control; otherwise, p < 0.05.

Figure 5

Transcriptional activation assay in rat renal proximal tubular epithelial cell line NRK-52E. (a) A general scheme of the reporter construct. Four to eight specific transcription factor binding sequences (transcription factor response elements, TREs) and the mCMV promoter regulate reporter luciferase, while the EF1 promoter provides for constant production of destabilized GFP and puromycin resistance selector. The flanking core insulators protect from epigenetic silencing of the reporter, while the piggyBac transposon ITRs secure accurate and efficient insertion of the reporter into the genomic DNA. (b) Heatmap of the reporter activation, expressed as log₂ (induction fold), to common inducers and inhibitors used to validate specificity of the reporters. See extended Supplementary Materials Table S14 for viabilities and SDs. Concentrations: lipopolysaccharide (LPS)—200 ng/mL; interleukin-1β (IL-1β)—10 ng/mL; tumor necrosis factor (TNF)—10 ng/mL; bardoxolone (CDDO-Me)—500 nM; nutlin—25 μM; tert-butyl hydroquinoline (tBHQ)—20 μM; CdCl₂—10 μM; ZnSO₄—100 μM; CoCl₂—250 μM; thapsigargin—50 nM; hydrogen peroxide (H₂O₂)—240 μM; tanespimycin (17-AAG)—250 nM; pyocyanin—80 μM.

Figure 6

Transcriptional activation in NRK-52E cells treated with β-CEC, CMC, and the sulfoxides for 18 h. See extended Supplementary Materials Table S15 for viabilities and SDs, as well as data on activation of the transcription factors HIF-1, p53, and MTF-1.

Figure 7

Transcriptional activation of the amino acid response pathway in NRK-52E cells treated with 4 mM β-CEC or CMC and co-treated with no or 4 mM amino acids for 18 h. The abbreviations are conventional one-letter abbreviations for amino acids, except for C standing for N-acetyl-L-cysteine. Responses significantly different from “not co-treated” in each group (n = 3, p < 0.05) are marked with # (more than “not co-treated”) and * (less than “not co-treated”). See Supplementary Materials Table S16 for viabilities and extended data.

Figure 8

Activation of p53 in NRK-52E cells co-treated with (a) cisplatin, (b) oxaliplatin, and 1 mM S-carboxyalkylcysteines for 24 h. Codes for the amino acids: 0—no amino acid; 1—β-CEC; 2—(4R)-β-CECO; 3—(4S)-β-CECO; 4—CMC; 5—(4R)-CMCO; 6—(4S)-CMCO. Responses significantly different from “no amino acid” (n = 3, p < 0.05) are marked with asterisks.

Figure 9

Viabilities of NRK-52E cells treated with common environmental nephrotoxic pollutants (gray bars) and co-treated with 1 mM β-CEC (green bars) for 18 h. Concentrations of the pollutants: NaAsO₂—15 μM; CdCl₂—30 μM; CuCl₂—480 μM; HgCl₂—30 μM; Pb(OAc)₂—1.2 mM; atrazine, diquat, and paraquat—300 μM; ochratoxin A—30 μM. Statistical significances (n = 3, p < 0.001) are marked with asterisks. See Supplementary Materials Table S18 for extended data.

Figure 10

Activation of the antioxidant pathway in NRK-52E cells treated with (a) CuCl₂, (b) CuO nanoparticles (30–50 nm), and co-treated with 1 mM S-carboxyalkylcysteines for 18 h.