PEPTIDE IDR-1002 REGULATES THE ANTIOXIDANT AND ANTI-INFLAMMATORY RESPONSES BY ACTIVATING THE KEAP1-NRF2 SIGNALING PATHWAY

Peptide synthesis

The immunomodulatory peptides IDR-1018 (VRLIVAVRIWRR-NH2), IDR-HH2 (VQLRIRVAVIRA-NH2), IDR-1 (KSRIVPAI-PVSLL-NH2), and IDR-1002, (VQRWLIVWRIRK-NH2) with the C-terminal amidated were synthesized by solid-phase Fmoc chemistry (CPC Scientific, USA) and obtained at a purity greater than 95%.

Antibodies and reagents

Primary and secondary antibodies used: mouse β-actin (sc-47778), laminin (sc-7293), GAPDH-IgG (sc-32233), and anti-mouse IgG-BP-HRP (sc-525409) (Santa Cruz Biotechnology, USA); Histone H3 Rabbit (9717), rabbit NQO1 (62262S), and anti-rabbit IgG-HRP (7074S) (Cell Signaling Technology, USA); rabbit Nrf2 (ADI-KAP-TF125) and HO-1 (ADI-OSA-150-F) (Enzo Life Sciences, USA). Additional reagents included non-fat dry milk (Bio-Rad, USA), Luminol (Millipore, USA), protease and phosphatase inhibitor cocktail (cOmplete™, Roche, Switzerland), tert-butyl hydroperoxide (TBHP; Sigma-Aldrich, cat. 458139), ferrous sulfate (FeSO₄; cat. 7782-63-0), D, L-sulforaphane (SFN; cat. S4441), Tris-HCl, NaCl, 1-chloro-2,4-dinitrobenzene (CDNB), hydrogen peroxide (H₂O₂; cat. 7722-84-1), DCFDA (cat. 4091-99-0), Igepal CA-930, Na-pyrophosphate, NaF, Na-orthovanadate, RIPA buffer (cat. R0278), and chemiluminescent substrate (Millipore, USA). TNFα was purchased from (ACROBiosystem-Switzerland; cat. TNA-H4211). Cell culture media and supplements were obtained from BPS Bioscience (USA), including MEM, Thaw Medium 1, 1K nutrient medium, fetal bovine serum (FBS), non-essential amino acids, sodium pyruvate, penicillin/streptomycin, geneticin (cat. 79533), and Luciferase ONE-Step reagent (cat. 60690). Trypsin-EDTA 1X (cat. T2601), L-glutathione reduced (gsh, cat. G4251), Bradford Reagent (cat. B6916) and MTT (3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (cat. 475989) were purchased from Sigma-Aldrich.

Cell culture

ARE-Hep-G2 cells (Hep-G2 cells) (BPS Bioscience, cat. 60513) which express luciferase under control of ARE sequences were cultured in 1K medium containing DMEM supplemented with 10% FBS, 1% non-essential amino acids, 1 mM sodium pyruvate, 1% penicillin/streptomycin, and 600 μg/mL geneticin at 37°C in a humidified atmosphere with 5% CO₂. Cells between passages 8–23 were used. BEC cells (bovine umbilical vein endothelial cells) were kindly provided by Dr. Carmen Clapp from the Institute of Neurobiology, National Autonomous University of Mexico (UNAM). These cells were immortalized by transfection with HPV16 E6E7 oncogenes, that extend their replicative lifespan of primary BEC from 40 to more than 120 passages without signs of senescence. Importantly, BEC cells retain key endothelial characteristics, including the uptake of acetylated low-density lipoproteins, von Willebrand factor expression, specific lectin binding, and proliferative responses to vascular endothelial growth factor.²² HEK-293 cells were purchased from the American Type Culture Collection (ATCC; cat. CRL-1573). HEK-293 and BEC cells were cultured in DMEM supplemented with 10% FBS, 10,000 U/mL penicillin, and 1 mg/mL streptomycin, at 37°C in 5% CO₂. Cells between passages 8 and 20 were used.

Luciferase reporter assay

Hep-G2 cells were plated at 40,000 cells/well in 96-well plates with 45 μL of 1K medium without geneticin. Cells were treated with 5 μl of IDR-1002 (final concentration: 0.5–300 μM) or FeSO₄ (positive control; final concentration: 100 μM). After 8–10 h incubation at 37°C, 100 μL of Luciferase ONE-Step reagent was added and the plate was shaken for 15 min. Luminescence was measured using a Varioskan^TM LUX plate reader (Thermo Fisher Scientific, USA). Fold induction was calculated by normalizing to untreated controls. TBHP, FeSO₄, or SFN were used as Nrf2-activators.

MTT cell viability assay

Hep-G2 and BEC cells were seeded in 96-well plates at 5 x 10⁴ cells/mL and incubated for 24 h at 37 ^°C. Cells were treated with IDR-1002 (1–100 μM) or TBHP (10 μM) for 8 h. Then, 50 μL of 10% MTT solution was added, and incubation continued for 4 h. Medium was removed and formazan crystals were solubilized in 100% DMSO. Absorbance was read at 570 nm using a Varioskan^TM LUX reader.

Intracellular ROS quantification

ROS levels were measured in BEC cells using the DCFDA (2’,7’-dichlorofluorescein diacetate) fluorescence assay.²³ Cells were pretreated with IDR-1002 (1-50 μM) for 4 h, followed by incubation with 30 μM DCFDA at 37 °C for 30 min. Cells were then washed with PBS and stimulated with 50 μM H₂O₂ for 20 min. Fluorescence was measured at excitation/emission wavelengths of 485/530 nm using a Varioskan^TM LUX reader.

Protein extraction and Western blotting

BEC cells were grown in 6-well plates to ~90% confluence, serum-starved for ≥ 4 h, and then treated with 1, 10, 25, or 50 μM IDR-1002 for 1 h or 4 h before lysis for subsequent Western blots of Nrf2, HO-1, and NQO1. Total protein (cytosolic plus nuclear from control and treated cells) was extracted by washing cells with cold PBS and lysing them with 80 μL of a cold buffer containing 20 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% Igepal CA-930, 10 mM Na-pyrophosphate, and 50 mM NaF supplemented with 1 mM Na-orthovanadate and 1x protease inhibitors. Lysates were centrifuged at 13,000 × g (20 min, 4°C), and supernatants collected and transferred to ice-cold Eppendorf tubes. Fractions (Cytoplasmic and Nuclear) were prepared using 80 μL of RIPA buffer and sonicated with a Qsonica Q125 sonicator (Newtown, USA) adjusted to 25% amplitude for a total of 15 s (3 × 5 s, with 7 s rest in between). Protein concentration was measured by the Bradford method using BSA as standard. Samples (50-60 μg) were separated by 10% SDS-PAGE and transferred in a wet chamber to 0.22 μm nitrocellulose membranes for 1 h at 250–300 mA. Detection was performed using appropriate antibodies and Immobilon HRP chemiluminescent kit. Imaging was done with the LI-COR Odyssey system.

Nrf2 and antioxidant enzymes quantification

Levels of Nrf2, HO-1, NQO1 and GCLM were measured using competitive ELISA kits (MyBioSource, USA), according to the manufacturer’s instructions.

Glutathione S-transferase activity

BEC cells were treated with 50 μM IDR-1002 for 6, 12, 18, or 24 h. Cell lysates were prepared in Tris-HCl buffer (50 mM, pH 7.4) containing 150 mM NaCl and protease inhibitors. GST activity was determined by measuring the absorbance at 340 nm of the 1-chloro-2,4-dinitrobenzene assay (CDNB)-glutathione (GSH) conjugation assay.

TNFα quantification

TNFα levels in BEC cells supernatants were measured by ELISA per the manufacturer’s protocol (MyBioSource, USA).

Statistical analysis

All data are presented as mean ± standard deviation (SD). Analyses were conducted using GraphPad Prism 8 (GraphPad Software, USA). Unpaired Student’s t test was used for two-group comparisons while one-way ANOVA with Tukey’s post hoc test was applied for multiple comparisons. Statistical significance is indicated by *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns = not significant and ND = not detected.

IDR peptides promote Nrf2 nuclear translocation in HEK293 cells

Several synthetic protein-protein interaction (PPI) inhibitors of Keap1-Nrf2 have been reported to activate the Nrf2 pathway; however, their use is limited by low selectivity and cytotoxicity effects.^24,25 In this context, IDR peptides have emerged as promising alternatives due to their immunomodulatory properties and low cytotoxic potential.^17,26–28 Notably, among the IDR peptides group, IDR-1002 exhibits a potent inhibitory NF-κB activity.^1,21 Thus, we initially explored the ability of IDR-1002 and three other IDR peptides, namely IDR-1018, IDR-HH2, and IDR-1, to activate Nrf2 nuclear translocation. HEK293 cells were treated with 10 μM of each peptide and Nrf2 abundance was assessed in whole-cell, cytoplasmic, and nuclear fractions by Western blot ( Figure 1A, B). Among all peptides tested, IDR-1002 induced the strongest nuclear translocation of Nrf2. IDR-HH2 also promoted Nrf2 nuclear accumulation, albeit to a lesser extent, while IDR-1 had minimal effect. To confirm the dose-dependency of IDR-1002, increasing concentrations (up to 50 μM) were tested ( Figure 1C, D). A 15-fold increase in nuclear Nrf2 abundance was observed at 50 μM compared to a 6-fold increase at 10 μM, supporting IDR-1002 as the most potent Nrf2 activator among the tested peptides. These data suggest that sequence variations between IDR peptides influence their ability to modulate the Nrf2 signaling pathway. Based on these results and those we previously reported on NF-κB inhibition,²¹ IDR-1002 was selected for subsequent experiments.

Figure 1. Nrf2 nuclear translocation induced by IDR peptides in HEK293 cells and by IDR-1002 in bovine endothelial cells (BEC cells).

(A, C) Western blot detection showing the relative abundance of the Nrf2 protein (MW ~100 kDa) in whole (W), cytoplasmic (C), and nuclear (N) protein enriched extracts from HEK293 cells. Cells were treated for 1 h with 10 μM IDR-1002, IDR-1018, IDR-1, and IDR-HH2 or with 50 μM IDR-1002. β-Actin (MW ~42 kDa) and laminin (MW ~70 kDa) were used as loading controls for the cytoplasmic and nuclear extracts, respectively. (B, D) Nrf2 nuclear accumulation is expressed as fold changes normalized to the untreated control (CTRL). (E) Representative Western blot showing nuclear Nrf2 levels in BEC cells treated for 1 h with 1, 10, 25, and 50 μM IDR-1002 and a positive control, tert-butyl hydroperoxide (TBHP, 10 μM). The graph above the blot shows the densitometric analysis of the relative fold change in nuclear Nrf2 levels, normalized to the untreated control (CTRL). Histone H3 (H3, 17 kDa) was used as a nuclear loading control. (F) Quantification of nuclear Nrf2 levels by competitive ELISA in BEC cells treated under the same conditions as in (E). Mean values for IDR-1002 at 10, 25, and 50 μM, as well as TBHP, were significantly increased compared to CTRL. Comparisons not indicated by asterisks were not statistically significant (ns) or not detect (ND). These results are representative of three independent experiments (n = 3). Bars indicate the mean ± standard deviation (SD). Asterisks indicate a statistically significant difference, determined by two-way ANOVA, followed by Tukey's post hoc test with the following significance levels: *p < 0.05, **p < 0.01 and ***p < 0.001.

IDR-1002 activates Nrf2 nuclear translocation in BEC cells

Next, to determine whether IDR-1002 activates Nrf2 in other cell types, BEC cells were treated with increasing concentrations of the peptide. Given the central role of endothelial cells in redox signaling and inflammation, and the antagonistic interplay between NF-κB and Nrf2 in vascular homeostasis,^19,29–32 BEC cells are considered a relevant model. IDR-1002 induced a concentration-dependent increase in Nrf2 nuclear translocation ( Figure 1E). Also, the amount of Nrf2 measured in BEC cells lysates by ELISA increased with IDR-1002 treatment from 10 to 50 μM ( Figure 1F). These data indicate that IDR-1002 activated Nrf2 nuclear translocation in BEC cells.

IDR-1002 induces Nrf2 transcriptional activity

Under basal conditions, Nrf2 is retained in the cytoplasm by the kelch domain of Keap1. Upon activation, Nrf2 dissociates from Keap1, translocates to the nucleus and binds to ARE in target genes promoters, driving the expression of antioxidant genes.^33–35 To evaluate the Nrf2 transcriptional activity Hep-G2 cells stably expressing the ARE-luciferase reporter, were treated with IDR-1002. A concentration-dependent increase in ARE-luciferase activity was observed with an EC₅₀ of 18.57 μM ( Figure 2A). Importantly, cell viability assays confirmed that IDR-1002 did not induce cytotoxicity neither in Hep-G2 or BEC cells at 1, 10, 25, 50, or 100 μM (Extended data Figure E1).

Figure 2. IDR-1002 activates the Nrf2 transcriptional pathway and downstream phase II antioxidant enzymes in BEC cells.

(A) Measurement of Nrf2-mediated transcriptional activity in HepG2-ARE-luciferase reporter cells treated with IDR-1002 (0.5 to 300 μM) for 8 h. Activity is measured as a fold change in luminescence relative to the untreated control, resulting in an EC₅₀ of 18.57 μM. (B, C) Western blot analysis of the protein levels of the antioxidant enzymes HO-1 (28 kDa) and NQO1 (29 kDa) in BEC cells treated for 4 h with IDR-1002 1, 10, 25, and 50 μM. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 37 kDa) served as a loading control for both blots. Iron (II) sulfate (FeSO₄, 150 μM) was used as a positive control. The graph above the blot represents the densitometric analysis of the relative fold change in HO-1 expression, normalized to the unstimulated control. Mean values for HO-1 at 10, 25, and 50 μM IDR-1002, and those for NQO1 at 25 and 50 μM IDR-1002 were significantly different from the control group. (D, E, F) ELISA quantification of the protein levels of the antioxidant enzymes, HO-1 (D), NQO1 (E), and GCLM (F) in BEC cells treated with 50 μM of IDR-1002 for 2 or 4 h. Protein expression was normalized to total protein content and compared to the respective time-matched untreated control (CTRL 2 h or CTRL 4 h). Treatment with IDR-1002 significantly increased the expression of these antioxidant enzymes in a time-dependent manner. Comparisons not indicated by asterisks were not statistically significant (ns) or not detect (ND). These results are representative of three independent experiments (n = 3). Bars indicate the mean ± standard deviation (SD). Asterisks indicate a statistically significant difference, determined by two-way ANOVA, followed by Tukey's post hoc test with the following significance levels: *p < 0.05, **p < 0.01 and ***p < 0.001.

IDR-1002 induces the expression of antioxidant enzymes

The Nrf2-dependent phase II antioxidant enzymes HO-1, NQO-1 and GCLM, mitigate oxidative stress and can suppress NF-κB activity.^7,36,37 Western blot analysis confirmed the increased expression of HO-1 and NQO1 following IDR-1002 treatment ( Figure 2B, C) supporting its role in Nrf2 activation and downstream antioxidant gene induction. This was further corroborated by ELISA-based quantification of HO-1, NQO1 and GCLM, at 2- and 4-h post-treatment with 50 μM IDR-1002, demonstrating time-dependent upregulation of these enzymes ( Figure 2D, E, F).

IDR-1002 induces glutathione S-transferase activity

Glutathione S-transferases (GSTs) are a family of phase II detoxifying enzymes that catalyze the conjugation of glutathione (GSH) to reactive intermediates, thus protecting against oxidative stress.³⁸ To determine whether IDR-1002 activates GSTs, BEC cells were treated with 50 μM IDR-1002 for 18, and 24 h. GST activity increased significantly at 18 h ( Figure 3A), with a no significant slight reduction at 24 h ( Figure 3B), compared with the untreated control for each time point. No GST activity was detected at 6 and 12 h (data not shown). These results suggest that IDR-1002 contributes to cellular protection by inducing GST-mediated detoxification responses.

Figure 3. Induction of GST activity by IDR-1002 in BEC cells.

BEC cells were treated with 50 μM IDR-1002 for 18 (A) and 24 h (B). Sulforaphane (SFN, 10 μM) was used as a positive control. Glutathione S-transferase (GST) activity was quantified after each treatment using the 1-chloro-2,4-dinitrobenzene (CDNB) colorimetric assay. IDR-1002-stimulated cells showed a significant increase in GST activity compared to the untreated control. Data are representative of three independent experiments (n = 3) and are presented as the mean ± standard deviation (SD). Statistical significance was determined by the two-way ANOVA with Tukey’s post hoc test. Asterisks indicate significance levels as follows: *p < 0.05, **p < 0.01, and ***p < 0.001.

IDR-1002 reduces intracellular ROS production

To assess the antioxidant effect of IDR-1002, intracellular ROS levels were measured in BEC cells treated with H₂O₂ following IDR-1002 pre-treatment. BEC cells were pre-incubated with the indicated concentrations of the peptide for 4 h and subsequently incubated with 50 μM H₂O₂ for 15 min. Basal ROS levels remained unchanged in both control and in the presence of 50 μM IDR-1002-treated cells, indicating that IDR-1002 did not exert pro-oxidant effects ( Figure 4). In H₂O₂-stimulated cells, treatment with IDR-1002 resulted in a dose-dependent reduction in ROS levels. At 25 and 50 μM, IDR-1002 decreased ROS levels by approximately 2.4- and 5.4-fold respectively, compared to the H₂O₂-treated control. These findings are consistent with previous studies reporting that 18 μM IDR-1002 reduces H₂O₂-induced ROS production by 1.6-fold.¹² Altogether, these results indicate that IDR-1002 exerts its antioxidant effects through the activation of the Nrf2 pathway.

Figure 4. IDR-1002 reduces ROS production in H₂O₂-stimulated BEC cells.

BEC cells were pretreated with 1, 10, 25, and 50 μM IDR-1002 for 4 h and subsequently incubated with 50 μM H₂O₂ for 15 min to induce oxidative stress. Intracellular ROS production was then quantified using the 2',7'-dichlorofluorescin diacetate (DCFH-DA) fluorescence assay. Data are representative of three independent experiments (n = 3) and are presented as the mean ± standard deviation (SD). Asterisks indicate a statistical difference compared to the H₂O₂-treated control, with a significance level of ***p < 0.001.

IDR-1002 reduces TNFα expression

Chronic oxidative stress is closely linked to inflammation, and TNFα is a key cytokine involved in various redox-related pathologies, including cancer, Alzheimer’s, Parkinson’s, diabetes, Crohn’s, and cardiovascular disorders.^39–44 ROS can induce TNFα production, and elevated TNFα can further amplify ROS levels.

To investigate the effect of IDR-1002 on TNFα expression, BEC cells were pre-incubated with 1, 10, 25 and 50 μM IDR-1002 for 1 hour and subsequently incubated with 10 ng/mL TNFα for an additional 1 h. Then, supernatants were collected and TNFα quantification was performed by ELISA. IDR-1002 significantly reduced TNFα levels at concentrations as low as 10 μM ( Figure 5), confirming its anti-inflammatory activity and supporting our previous results as NF-κB inhibitor in macrophage cells.²¹

Figure 5. IDR-1002 decreases TNFα expression in BEC cells stimulated with TNFα.

BEC cells were pretreated with 1, 10, 25, and 50 μM IDR-1002 for 1 h and subsequently incubated with 10 ng/mL TNFα for an additional 1 h. Supernatants were collected and TNFα levels were quantified by ELISA. These results are representative of three independent experiments (n = 3). Bars indicate the mean ± SD, and asterisks indicate statistical difference, ** p <0.01; ***P ˂0.001, n = 3, by the 2-way ANOVA method post hoc Tukey.