Structure-activity relationship of capsaicin analogs and transient receptor potential vanilloid 1-mediated human lung epithelial cell toxicity

Abstract

Activation of intracellular transient receptor potential vanilloid-1 (TRPV1) in human lung cells causes endoplasmic reticulum (ER) stress, increased expression of proapoptotic GADD153 (growth arrest- and DNA damage-inducible transcript 3), and cytotoxicity. However, in cells with low TRPV1 expression, cell death is not inhibited by TRPV1 antagonists, despite preventing GADD153 induction. In this study, chemical variants of the capsaicin analog nonivamide were synthesized and used to probe the relationship between TRPV1 receptor binding, ER calcium release, GADD153 expression, and cell death in TRPV1-overexpressing BEAS-2B, normal BEAS-2B, and primary normal human bronchial epithelial lung cells. Modification of the 3-methoxy-4-hydroxybenzylamide vanilloid ring pharmacophore of nonivamide reduced the potency of the analogs and rendered several analogs mildly inhibitory. Correlation analysis of analog-induced calcium flux, GADD153 induction, and cytotoxicity revealed a direct relationship for all three endpoints in all three lung cell types for nonivamide and N-(3,4-dihydroxybenzyl)nonanamide. However, the N-(3,4-dihydroxybenzyl)nonanamide analog also produced cytotoxicity through redox cycling/reactive oxygen species formation, shown by inhibition of cell death by N-acetylcysteine. Molecular modeling of binding interactions between the analogs and TRPV1 agreed with data for reduced potency of the analogs, and only nonivamide was predicted to form a "productive" ligand-receptor complex. This study provides vital information on the molecular interactions of capsaicinoids with TRPV1 and substantiates TRPV1-mediated ER stress as a conserved mechanism of lung cell death by prototypical TRPV1 agonists.

Fig. 1.

Annotated structure of nonivamide.

Fig. 2.

Quantification of TRPV1 mRNA in TRPV1-OE, BEAS-2B, and NHBE cells by RT-qPCR (A) and correlation of copy number with the LD₅₀ of nonivamide for each cell type (B). Copy numbers values were compared using Student's t test. Correlation values are in the order of NHBE, BEAS-2B, and TRPV1-OE cells and were analyzed using the correlation plot analysis feature provided by GraphPad Prism 5.0 software.

Fig. 3.

Dose response curves for calcium flux in TRPV1-OE cells treated with capsaicinoid analogs: nonivamide (▵), N-(3,4-dihydroxybenzyl)nonanamide (□), N-(3-hydroxy-4-methoxybenzyl)nonanamide (○), 3-methoxy-4-(nonamidomethyl)phenyl sulfate (▿), N-benzylnonanamide (▴), N-(3-methoxybenzyl)nonanamide (▾), N-(3,4-dimethoxybenzyl)nonanamide (◊), N-(4-trifluoromethylbenzyl)nonanamide (●), and N-(4-hydroxybenzyl)nonanamide (♦). Statistically significant calcium flux, p < 0.05 by one-way ANOVA, was observed for nonivamide and the 3,4-DiOH analog at concentrations equal to and greater than as indicated by *. Data are represented as percentage of response relative to ionomycin ± S.E.M. (n ≥3).

Fig. 4.

Comparison of TRPV1-OE cell viability at 2.5 μM (A), 25 μM (B), and 100 μM (C) and analog-induced calcium flux at 2 μM (A), 20 μM (B), and 100 μM (C) analog concentration. Cell viability was assayed after 24-h treatment, and calcium flux was measured in real time over 1 min using a BMG Labtech NOVOstar plate reader as described under Materials and Methods. Data (mean ± S.E.M.) (left axis) are percentage of maximum attainable calcium response (▴) and percentage of cell viability relative to untreated controls (■). Analogs are arranged on the x-axis in order of lowest to highest cytotoxicity (left to right). Correlations between these two indices of TRPV1 activation are reported as the Pearson R value and two-tailed p value under Results and in the supplemental data.

Fig. 5.

A to E, comparison of calcium flux (▴) and GADD153 induction (■) (A, C, and E) in TRPV1-OE cells at 2 μM (A), 20 μM (B), and 100 μM (C) analog concentration and GADD153 (▴) and cell viability (■) (B, D, and E) using 2 and 2.5 μM (B), 20 and 25 μM (D), and 100 μM (F) analog. G and H, comparison of GADD153 expression (▴) and cytotoxicity (■) for BEAS-2B (G) and NHBE (H) cells treated with 200 μM analog. Percentage of maximum calcium flux was measured in real time for 1 min using a NOVOStar plate reader, GADD153 mRNA expression was measured using RT-qPCR (4-h treatment), and cell viability (24-h treatment) was measured using the Dojindo assay, as described under Materials and Methods. Analogs are arranged on the x-axis in all graphs from least to most potent (left to right) in calcium flux and cytotoxicity assays. Correlations between the indices of TRPV1 activation are reported as the Pearson R value and two-tailed p value under Results.

Fig. 6.

Assessment of TRPV1 dependence and non-TRPV1-mediated/redox stress-mediated toxicity for nonivamide and N-(3,4-dihydroxybenzyl)nonanamide in TRPV1-OE cells. *, statistically significant difference (p < 0.05) from untreated controls. #, statistical difference between treatment with analog alone or with LJO-328 or NAC (p < 0.05) using one-way ANOVA. Data are represented as the mean ± S.D. (n = 3).

Fig. 7.

Dose response curves for inhibition of nonivamide- (2.5 μM) induced calcium flux in TRPV1-OE cells treated by capsaicinoid analogs: N-(3-hydroxy-4-methoxybenzyl)nonanamide (○), 3-methoxy-4-(nonamidomethyl)phenyl sulfate (▿), N-benzylnonanamide (▴), N-(3-methoxybenzyl)nonanamide (▾), N-(3,4-dimethoxybenzyl)nonanamide (◊), N-(4-trifluoromethylbenzyl)nonanamide (■), and N-(4-hydroxybenzyl)nonanamide (♦). Data are normalized to nonivamide only and were fit using the sigmoidal dose response (variable slope) equation to estimate the IC₅₀ value. *, concentration at which statistically significant inhibition, p < 0.05 by one-way ANOVA, was observed. Data are the mean and S.E.M. (n = 3).

Fig. 8.

Images depicting predicted binding interactions between TRPV1 transmembrane helices 3 and 4 (residues Ser510–Ile569) (Gavva et al., 2004) and capsaicinoid analogs. Lowest energy (green) and most highly populated (purple) poses for nonivamide (A), N-(3-hydroxy-4-methoxybenzyl)nonanamide (B), N-(3,4-dihydroxybenzyl)nonanamide (C), N-(3-methoxybenzyl)nonanamide (D), N-(4-hydroxybenzyl)nonanamide (E), N-benzylnonanamide (F), N-(3,4-dimethoxybenzyl)nonanamide (G), and N-(4-trifluoromethybenzyl)nonanamide (H) are shown. For nonivamide, the structure represents both the lowest energy and highest populated structure.