An Exceptionally Potent Inducer of Cytoprotective Enzymes

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.

Covalent Modifiers: A Chemical Perspective on the Reactivity of α,β-Unsaturated Carbonyls with Thiols via Hetero-Michael Addition Reactions

“…33c Variable temperature NMR also demonstrated the reversibility of the reaction, as 411 was regenerated upon heating of the DTT- 411 adduct. 33c Other cyanoenones, such as tricyclic TBE-31 ( 434 ) 149 and monocyclic compounds 435-437 , 150 have shown similar reactivity towards thiols using the same UV and 1 H NMR methods (Figure 35). Surprisingly, compounds 439-440 showed no reactivity with DTT and 441-442 showed greatly reduced reactivity requiring 10 equiv of DTT for adduct formation, thereby demonstrating the importance of the surrounding structure on electrophilicity.…”

An Improved Synthesis of a Hydroxymethyl Tricyclic Ketone from Cyclohexanone, the Key Processes for the Synthesis of a Highly Potent Anti-inflammatory and Cytoprotective Agent

“…1 The oral administration of 1 indicated excellent oral bioavailability, and resulted in a dose-dependent induction of cytoprotective enzymes, NAD(P)H:quinone oxidoreductase 1 (NQO1), and glutathione S-transferase (GST) in the stomach, skin, and liver. 2 Furthermore, longterm (five days per week for four weeks) daily topical applications in small quantities (200 nmol) of 1 caused a robust systemic induction of the Keap1/Nrf2/ARE pathway and decreased 6-thioguanine incorporation into DNA of skin, blood, and liver of azathioprine-treated mice, indicating extraordinary bioavailability and efficacy. 3 Tricyclic compound 1 is orally highly active against aflatoxininduced liver cancer in rats.…”

Electron affinity of tricyclic, bicyclic, and monocyclic compounds containing cyanoenones correlates with their potency as inducers of a cytoprotective enzyme

“…We have previously shown that cyanoenones react with cysteine residues in Keap1, the main negative regulator of Nrf2. 6,7,14 In a variety of cell lines and animal tissues, we have demonstrated that Nrf2 activation by cyanoenones leads to the coordinate transcriptional upregulation of NQO1 together with other Nrf2-target genes, such as multiple isoforms of glutathione S-transferase (GST), heme oxygenase 1, -glutamyl cysteine ligase catalytic (GCLC) subunit, as well as an ARE-luciferase reporter, a direct readout of Nrf2-mediated transcription. 6,7,14 Furthermore, Nrf2 is required for the NQO1 inducer activity of cyanoenones as NQO1 induction is not observed in Nrf2-deficient cells.…”

“…6,7,14 In a variety of cell lines and animal tissues, we have demonstrated that Nrf2 activation by cyanoenones leads to the coordinate transcriptional upregulation of NQO1 together with other Nrf2-target genes, such as multiple isoforms of glutathione S-transferase (GST), heme oxygenase 1, -glutamyl cysteine ligase catalytic (GCLC) subunit, as well as an ARE-luciferase reporter, a direct readout of Nrf2-mediated transcription. 6,7,14 Furthermore, Nrf2 is required for the NQO1 inducer activity of cyanoenones as NQO1 induction is not observed in Nrf2-deficient cells. 6,7 The electron affinity of these new compounds can be calculated via the energy of their lowest unoccupied molecular orbital, E (LUMO) in eV, and was quantified at two quantum mechanical levels: the semiempirical AM1, 16 and the density functional theory (DFT) 17 (see Table 1).…”

“…6,7,14 Furthermore, Nrf2 is required for the NQO1 inducer activity of cyanoenones as NQO1 induction is not observed in Nrf2-deficient cells. 6,7 The electron affinity of these new compounds can be calculated via the energy of their lowest unoccupied molecular orbital, E (LUMO) in eV, and was quantified at two quantum mechanical levels: the semiempirical AM1, 16 and the density functional theory (DFT) 17 (see Table 1). Briefly, no solvent model was used for the calculation of the LUMO energies.…”