Identification of Antioxidant Methyl Derivatives of Ortho-Carbonyl Hydroquinones That Reduce Caco-2 Cell Energetic Metabolism and Alpha-Glucosidase Activity

Abstract

α-glucosidase, a pharmacological target for type 2 diabetes mellitus (T2DM), is present in the intestinal brush border membrane and catalyzes the hydrolysis of sugar linkages during carbohydrate digestion. Since α-glucosidase inhibitors (AGIs) modulate intestinal metabolism, they may influence oxidative stress and glycolysis inhibition, potentially addressing intestinal dysfunction associated with T2DM. Herein, we report on a study of an ortho-carbonyl substituted hydroquinone series, whose members differ only in the number and position of methyl groups on a common scaffold, on radical-scavenging activities (ORAC assay) and correlate them with some parameters obtained by density functional theory (DFT) analysis. These compounds' effect on enzymatic activity, their molecular modeling on α-glucosidase, and their impact on the mitochondrial respiration and glycolysis of the intestinal Caco-2 cell line were evaluated. Three groups of compounds, according their effects on the Caco-2 cells metabolism, were characterized: group A (compounds 2, 3, 5, 8, 9, and 10) reduces the glycolysis, group B (compounds 1 and 6) reduces the basal mitochondrial oxygen consumption rate (OCR) and increases the extracellular acidification rate (ECAR), suggesting that it induces a metabolic remodeling toward glycolysis, and group C (compounds 4 and 7) increases the glycolysis lacking effect on OCR. Compounds 5 and 10 were more potent as α-glucosidase inhibitors (AGIs) than acarbose, a well-known AGI with clinical use. Moreover, compound 5 was an OCR/ECAR inhibitor, and compound 10 was a dual agent, increasing the proton leak-driven OCR and inhibiting the maximal electron transport flux. Additionally, menadione-induced ROS production was prevented by compound 5 in Caco-2 cells. These results reveal that slight structural variations in a hydroquinone scaffold led to diverse antioxidant capability, α-glucosidase inhibition, and the regulation of mitochondrial bioenergetics in Caco-2 cells, which may be useful in the design of new drugs for T2DM and metabolic syndrome.

Keywords: DFT; antioxidants; diabetes; docking; hydroquinones; methyl derivatives.

Scheme 1

Synthesis of methyl derivatives of ortho-carbonyl hydroquinones. R corresponds to those shown in Table 1.

Figure 1

Correlation among calculated thermodynamic parameters and experimental ORAC values. (A) BDE of OH1 versus ORAC. (B) BDE of OH2 versus ORAC. (C) IP versus ORAC. (D) PA of OH2 versus ORAC. (E) BDE of OH1 for the anions deprotonated at OH2 versus ORAC.

Figure 2

In silico molecular docking for ortho-carbonyl hydroquinone series against the α-glucosidase enzyme. (A) Binding free energy components for the α-glucosidase–hydroquinone (3 and 10) complexes calculated by MM-GBSA analysis; all energies are in kcal/mol and pIC50 for ortho-carbonyl hydroquinones. (B) Correlations of calculated MMGBSAΔG values with experimental IC₅₀ inhibitory values, expressed as pIC50 = −log [IC50]. The r square value from linear regression and p-value for Spearman’s correlation (n = 10) are shown. (C–E) 3D maps binding interactions of compound 3 and 10 against the α-glucosidase enzyme. Ligand compound 3 (A,C) and compound 10 (B,D) exposure points are indicated with gray coloring, while the binding interactions of ligands with α-glucosidase are indicated with red lines. (C) Binding free energy components for the α-glucosidase–hydroquinone (3 and 10) complexes calculated by MM-GBSA analysis; all energies are in kcal/mol.

Figure 3

Effect of ortho-carbonyl methyl-substituted hydroquinones on proliferation, mitochondrial respiration, and glycolysis in Caco-2 cells. (A) Effect of compounds on the MTT reduction in Caco-2. Cells were treated with compounds (1, 5, 10, 30, 50, and 100 µM) for 24 h. (B) Dot-plot for basal mitochondrial OCR (basal mitoOCR) vs. glycolysis. The red square represents to control condition. Three groups of compounds are highlighted in blue, yellow, and green. (C) Dendrogram with five and two parameters of mitochondrial respiration and glycolysis, respectively, affected by compounds (100 µM) for 24 h of treatment. Data are shown as means ± SD of three independent experiments. ** p < 0.01, *** p < 0.001 control, and n.s.: not significant.

Figure 4

(A–D) Effect of selected compounds (100 µM, compounds 5 and 10) on mitochondrial respiration and (E–H) glycolysis at 24 h of treatment. (I,J) Evaluation of antioxidant effects in the in vitro model of menadione-dependent ROS production. Effects of compounds 5 and 10 on (I) mitochondrial ROS and (J) ROS in Caco-2 cells treated with menadione. The mitoROS and cytosolic ROS were evaluated with mitoSOX red and DHE, respectively. Data are shown as means ± SD of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001, control. n.s.: not significant. #: ** p < 0.01 for compound vs. compound + Menadione.