Extremely potent triterpenoid inducers of the phase 2 response: correlations of protection against oxidant and inflammatory stress

Abstract

A series of synthetic triterpenoid (TP) analogues of oleanolic acid are powerful inhibitors of cellular inflammatory processes such as the induction by IFN-gamma of inducible nitric oxide synthase (iNOS) and of cyclooxygenase 2 in mouse macrophages. Here, we show that these analogues are also extremely potent inducers of the phase 2 response [e.g., elevation of NAD(P)H-quinone oxidoreductase and heme oxygenase 1], which is a major protector of cells against oxidative and electrophile stress. Moreover, like previously identified phase 2 inducers, the TP analogues use the antioxidant response element-Nrf2-Keap1 signaling pathway. Thus, induction of the phase 2 response and suppression of the iNOS induction was abrogated in nrf2(-/-) and keap1(-/-) mouse embryonic fibroblasts. The high potency of TP analogues in inducing the phase 2 response and blocking inflammation depends on the presence of activated Michael reaction (enone) functions at critical positions in rings A and C. The most potent TP doubles NAD(P)H-quinone oxidoreductase in murine hepatoma cells at 0.28 nM and has an IC(50) for suppression of iNOS induction in primary mouse macrophages of 0.0035 nM. The direct interaction of this TP with thiol groups of the Keap1 sensor for inducers is demonstrated spectroscopically. The antiinflammatory and phase 2 inducer potencies of 18 TP are closely linearly correlated (r(2) = 0.91) over 6 orders of magnitude of concentration. Thus, in addition to blocking inflammation and promoting differentiation, these TP exhibit another very important protective property: the induction of the phase 2 response.

Fig. 3.

Correlation of potencies of 18 TP as inducers of NQO1 in Hepa1c1c7 murine hepatoma cells, expressed as CD values, and for suppression of iNOS induction by IFN-γ in primary mouse macrophages, expressed as IC₅₀ values. The linear correlation coefficient is r² = 0.91. TP-230 is the butyl ester of CDDO, TP-241 is the piperidyl amide derivative of CDDO, and TP-218 is the ethyl ester of CDDO. Other structures are shown in Fig. 1.

Fig. 1.

Structures and NQO1 inducer potencies (CD values) of TP.

Fig. 2.

SAR of suppression of induction of iNOS and COX-2 mRNA (A) and protein (B) by TP. RAW cells were incubated with 10 ng/ml IFN-γ and various TP (Northern, 100 nM for 16 h; Western, 300 nM for 24 h). Total RNA and protein were isolated, separated electrophoretically, and transferred to membranes, and COX-2 and iNOS expression was analyzed by Northern and Western blotting.

Fig. 4.

Nrf2 and Keap1 are essential for induction of phase 2 response and for suppression of iNOS induction. (A) Induction of NQO1 as a function of concentration by TP-225 in MEF cells of WT (•), Nrf2^–/– (○), and Keap1^–/– (▴) mice. Cells (10⁴ per well) were plated in 96-well plates and 24 h later were exposed to serial dilutions of TP-225 for a further 48 h. NQO1 activity was determined in cell lysates. (B) CDDO imidazolide (TP-235) suppresses production of NO in WT Nrf2, but not in knockout, fibroblasts. Nrf2^+/+ and Nrf2^–/– fibroblasts (10⁵ cells per well) were plated in 96-well plates and incubated with 10 ng/ml IFN-γ, 10 ng/ml TNF-α, and TP-235 for 24 h. NO was measured as nitrite accumulation in the medium by the Griess reaction.

Fig. 5.

Inhibition of binding of [³H]Dex-mes to Keap1 by TP-225 (•), TP-155 (○), and TP-156 (▴). Keap1 (100 μl, 35–40 pmol) was incubated with 10 μlof each competitor in a range of concentrations for 20 min at 25°C in 20 mM Tris·HCl/0.005% Tween 20/0.1 mM EDTA (pH 8.0). Then, 10 μl of [³H]Dex-mes (25 pmol) was added, and incubation was continued for an additional 60 min. [³H]Dex-mes bound to protein was separated from unbound steroid by gel filtration, and the radioactivity in the protein fraction was determined by scintillation counting. Only partial structures (rings C and D) are shown.

Fig. 6.

Reaction of TP-225 with sulfhydryl groups in 20 mM Tris·HCl/0.005% Tween 20/1% acetonitrile (vol/vol) (pH 8.0) at 25°C. (A) UV spectrum of 12.5 μM TP-225 before (solid line) or immediately after (dotted line) it was allowed to react with 125 μM DTT. (B) UV spectrum of 10 μM TP-225 before (solid line) or immediately after (dotted line) it was allowed to react with 8.75 μM Keap1. The protein was present at the same concentration in the reference cuvette. (C) Time course of the reaction of 3.5 μM TP-225 with 0.5 μM Keap1 measured by the decrease in absorbance at 250 nm. (D) Titration of TP-225 (from a 150 μM stock solution) in 2-μl aliquots into a solution of 8 μM Keap1 in a final volume of 200 μl.

Fig. 7.

TP protect cells against oxidative stress. (A) SAR of suppression of ROS formation by various TP. U937 cells were exposed to 10 nM TP for 24 h, incubated with 10 μM2′,7′-dichlorodihydrofluorescein diacetate for 30 min, and then challenged with 250 μM tert-butyl hydroperoxide for 15 min. Mean fluorescence intensity of 10⁴ cells was determined by flow cytometry. (B) TP-225 protects against photooxidative damage. Human retinal pigment epithelial cells (ARPE-19) were incubated with TP-225 for 24 h. They then were loaded with 25 μM all-trans-retinaldehyde for 2 h, exposed to UVA (340–400 nm) for 15 min, and finally incubated for an additional 18 h. Cell survival was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay.