An exceptionally potent inducer of cytoprotective enzymes: elucidation of the structural features that determine inducer potency and reactivity with Keap1

Abstract

The Keap1/Nrf2/ARE pathway controls a network of cytoprotective genes that defend against the damaging effects of oxidative and electrophilic stress, and inflammation. Induction of this pathway is a highly effective strategy in combating the risk of cancer and chronic degenerative diseases, including atherosclerosis and neurodegeneration. An acetylenic tricyclic bis(cyano enone) bearing two highly electrophilic Michael acceptors is an extremely potent inducer in cells and in vivo. We demonstrate spectroscopically that both cyano enone functions of the tricyclic molecule react with cysteine residues of Keap1 and activate transcription of cytoprotective genes. Novel monocyclic cyano enones, representing fragments of rings A and C of the tricyclic compound, reveal that the contribution to inducer potency of the ring C Michael acceptor is much greater than that of ring A, and that potency is further enhanced by spatial proximity of an acetylenic function. Critically, the simultaneous presence of two cyano enone functions in rings A and C within a rigid three-ring system results in exceptionally high inducer potency. Detailed understanding of the structural elements that contribute to the reactivity with the protein sensor Keap1 and to high potency of induction is essential for the development of specific and selective lead compounds as clinically relevant chemoprotective agents.

Figures

FIGURE 1.

Chemical structures of cyano enone inducers of the Keap1/Nrf2/ARE pathway.

FIGURE 2.

Induction of cytoprotective enzymes in vivo by topical application of TBE-31. SKH-1 hairless mice (n = 3 per group) were topically treated three times at 24 h intervals on their backs with 0.03, 0.1, or 0.3 μmol of TBE-31 dissolved in 80% aqueous acetone (v/v, 100 μl) over ∼5 cm2 area. Control skin received 80% acetone. The animals were euthanized 24 h after the last application, and dorsal skin within the treated areas was harvested. NQO1 (menadione as a substrate) and GST (CDNB as a substrate) specific activities were measured in cytosol fractions of skin sections. Means ± S.D. are shown. p < 0.01 (*), p < 0.001 (**), p < 0.0001 (***).

FIGURE 3.

Induction of cytoprotective enzymes in vivo by dietary administration of TBE-31. Three groups of SKH-1 hairless mice (n = 4 per group) were placed on either control diet or diet containing TBE-31. Group 1 received the control diet, Group 2 was fed the low-dose (0.1 μmol/3 g diet), and Group 3, the high-dose (0.3 μmol/3 g diet) TBE-31 containing diet. After 11 days of feeding, the animals were euthanized, and the specific activities of NQO1 (A) and GST (B) were determined in cytosols from liver, skin, stomach, and cerebral cortex. Means ± S.D. are shown. p < 0.01 (*), p < 0.001 (**), p < 0.0001 (***). C, Western blots for GSTA1, GSTM1, and GSTP1 in which aliquots from liver or stomach cytosols (100,000 × g supernatant fractions) from each animal were resolved by SDS/PAGE and transferred to Immobilon-P before being probed with the specific antibodies. Equal loading was confirmed by probing the blots with an antibody against GAPDH.

FIGURE 4.

Induction of NQO1 by cyano enones. Hepa1c1c7 cells (10,000 cells per well) were grown in 96-well plates for 24 h and exposed to serial dilutions of each inducer (A) or combinations of inducers (B) for 48 h. NQO1 activity and total protein concentrations were determined in cell lysates. Results are derived from average values of 8 replicate wells and are plotted as Median Effect. The standard deviations in each case were between 5–7% of the values.

FIGURE 5.

Induction of ARE-dependent gene expression by TBE-31 and its monocyclic derivatives: Requirement for Nrf2. A, AREc32 cells expressing luciferase gene under the transcriptional control of 8 tandemly arrayed copies of the ARE were grown in 96-well plates. After 24 h, cells were exposed to increasing concentrations of compounds. The ARE-driven luciferase reporter activity was determined in cell lysates 24 h later. The value of luciferase activity of cells treated with acetonitrile (control) was arbitrarily set at 1. Values are means ± S.D. B, mouse embryonic fibroblasts isolated from wild-type (closed symbols) or nrf2-knockout (open symbols) mice were grown in 96-well plates (seeding density of 20,000 cells per well) for 24 h, and exposed to serial dilutions of each inducer for further 24 h. NQO1 activity and total protein concentrations were determined in cell lysates. Results are shown as average values of 8 replicate wells. The standard deviations in each case were between 5 and 10% of the observed values.

FIGURE 6.

TBE-31 reacts with cysteine residues of Keap1. A, absorption spectra of 50 μm TBE-31, the reaction mixture of 50 μm TBE-31 and 10 μm Keap1 (TBE-31+Keap1) in 20 mm Tris-HCl/0.005% Tween 20 (pH 8.0) at 25 °C against Keap1 blank, and their difference spectrum. B, titration of TBE-31 delivering stoichiometric amounts of the compound into a solution of 8 μm Keap1 in 20 mm Tris-HCl/0.005% Tween 20 (pH 8.0) at 25 °C in the absence (closed symbols) or the presence (open symbols) of 250 μm sulforaphane. Absorption spectra of 100 μm MCE-1 (C) and MCE-5 (D) (dashed lines), their corresponding reaction mixtures with 10 μm Keap1 against Keap1 blank in 20 mm Tris-HCl/0.005% Tween 20 (pH 8.0) at 25 °C (gray lines), and their difference spectra (black lines).

FIGURE 7.

Formation of the reaction product of ring-C cyano enones with DTT is accompanied by enolization. A, 50 μm TBE-31 was added to a solution of 50 μm DTT in 20 mm Tris-HCl/0.005% Tween 20 (pH 8.0) at 25 °C and the absorption spectrum was recorded against a DTT blank. The pH of the solution was lowered by addition of small aliquots of HCl. Once the pH reached pH 2, NaOH was added to raise the pH to 9. Absorption spectra of 100 μm dihydro-MCE-1 (B) and 100 μm dihydro-MCE-5 (C) before (black lines) and after (gray lines) addition of 10 mm NaOH in phosphate-buffered saline (pH 7.4), containing 1% EtOH (v/v), at 25 °C.

SCHEME 1.

Proposal for the reaction of TBE-31 with Keap1.