Nrf2/ARE-mediated antioxidant actions of pro-electrophilic drugs

Abstract

Living cells maintain a balance between oxidation and reduction, and perturbations of this redox balance are thought to contribute to various diseases. Recent attempts to regulate redox state have focused on electrophiles (EPs), which activate potent cellular defense systems against oxidative stress. One example of this approach is exemplified by carnosic acid (CA) and carnosol (CS), compounds that are found in the herb rosemary (Rosmarinus officinalis). Importantly, CA and CS themselves are not electrophilic, but in response to oxidation, become electrophilic, and then activate the Keap1/Nrf2/ARE (antioxidant-response element) transcription pathway to synthesize endogenous antioxidant "phase 2" enzymes. As a result of our efforts to develop these compounds as therapeutics for brain health, we have formulated two innovative criteria for drug development: the first concept is the use of pro-electrophilic drugs (PEDs) that are innocuous in and of themselves; and the second concept involves the use of compounds that are pathologically activated therapeutics (PATs);i.e., these small molecules are chemically converted to their active form by the very oxidative stress that they are designed to then combat. The chemical basis for PED and PAT drugs is embodied in the ortho- and para-hydroquinone electrophilic cores of the molecules, which are oxidized by the Cu(2+)/Cu(+) cycling system (or potentially by other transition metals). Importantly, this cycling pathway is under stringent regulation by the cell redox state. We propose that redox-dependent quinone formation is the predominant mechanism for formation of PED and PAT drugs from their precursor compounds. In fact, redox-dependent generation of the active form of drug from the "pro-form" distinguishes this therapeutic approach from traditional EPs such as curcumin, and results in a decrease in clinical side effects at therapeutic concentrations, e.g., lack of reaction with other thiols such as glutathione (GSH), which can result in lowering GSH and inducing oxidative stress in normal cells. We consider this pro-drug quality of PED/PAT compoundsto be a key factor for generating drugs to be used to combat neurodegenerative diseases that will be clinically tolerated. Given the contribution of oxidative stress to the pathology of multiple neurodegenerative diseases, the Keap1/Nrf2/ARE pathway represents a promising drug target for these PED/PAT agents.

Keywords: ABCC; ARE; ATP-binding cassette, subfamily C; Antioxidant-response element; CA; CS; Carnosol; DA; DMF; DOX; Dimethyl fumarate (also known as BG-12); Dopamine; Doxorubicin (also known as adriamycin); EP; Electrophile; Electrophilic counterattack; GCLC; GCLM; GSH; GST; Glutamyl cysteine ligase catalytic subunit; Glutamyl cysteine ligase modifier subunit; Glutathione; Glutathione-S-transferase; H(2)O(2); HO-1; HSE; HSF-1; HSP; Heat-shock factor-1; Heat-shock protein; Hemeoxygenase-1; Hydrogen peroxide; Keap1; Kelch-like ECH-associated protein 1; MS; Multiplesclerosis; NADPH quinone oxidoreductase 1; NEPP; NGF; NQO1; Na(+)-independent cystine-glutamate exchanger; Nerve growth factor; Neurite outgrowth-promoting prostaglandin; Nrf2; Nuclear factor (erythroid-derived 2)-like 2; PAT; PD; PED; Parkinson's disease; Pathologically activated therapeutic; Pro-electrophilic drug; RNS; ROS; Reactive nitrogen species; Reactive oxygen species; STR; Strongylophorin; TBHQ; Tert-butylhydroquinone; carnosic acid; heat-responsive element; xCT.

Fig. 1. Electrophile-Cell Interactions

A. Dose-response curves. Because all EPs have two opposing actions on cells, toxic and protective, these compounds manifest an inverse U shaped dose-response curve. There are three distinctive groups of EPs in terms of the broadness of their therapeutic index: EP1, none; EP2, limited; EP3, broad. B. Three distinctive groups of EPs. The size of the arrows indicates the relative strength of the opposing effects of GSH depletion vs. Electrophilic counterattack in response to EP treatment.

Fig. 2. Chemical conversion of geometric isomers

Geometric isomers of hydroquinones donate an electron and proton to Cu²⁺ and are thus converted to a semiquinone. Cu⁺ is reoxidized by an oxygen molecule. Para- and ortho-hydroquinones (A and B) convert to quinones, but meta-semiquinone (C) is not converted to a quinone form. Apparently for this reason, para- and ortho-hydroquinones, but not meta-hydroquinone, can activate the Keap1/Nrf2/ARE transcriptional pathway.

Fig. 3. Effects of various geometric isomers of hydroquinones on biological systems

A and B. ARE and HSE activation. HT22 cells transfected with the reporter constructs, ARE(GST-Ya)- or ptK-hHSP70-luciferase vector, were treated with vehicle or 2 µM hydroquinone geometric isomers 1, 2 or 3 (as indicated in Fig. 2). After 24 h, activity was measured by reporter gene assay, as described previously [–11]. Values are mean ± SD; *p < 0.05, **p < 0.01. C. Protective effects of geometric isomers. HT22 cells were seeded onto 24-well plates at a density of 4 × 10⁴ cells/cm². After a 5-h incubation, the various isomers of the compounds were added. One hour later, the cells were exposed to glutamate for 24 h and then subjected to a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) viability assay, as described previously [–109]. High concentrations (5 mM) of glutamate induce oxidative stress in HT22 cells due to GSH depletion, a process termed “oxidative glutamate toxicity” [110]. The various geometric isomers of hydroquinone compounds offer varying degrees of protection from this oxidative stress. Values are mean ± S.D. (n = 4) and represent percentage of control (in the absence of glutamate exposure). D. Antioxidant actions of geometric isomers. Summary of biological activity of the three geometric isomers in activating the Nrf2 cellular antioxidant defense system. “Antioxidant activity by electron donation” represents reductive power originated from the chemical reactions described in Fig. 2. “Antioxidant activity by Nrf2 activation” represents phase 2 induction by S-alkylation of Keap1 and Nrf2 activation. Note that ‘Total antioxidant actions’ represents the sum actions of “Antioxidant activity by electron donation” and “Antioxidant activity by Nrf2 activation.”

Fig. 4. Conversion of DA to DA Quinone

In Scenarios 1 and 2, DA quinone can be a toxic or protective substance, respectively. Circles show pro-electrophilic ortho-hydroquinone (left) and electrophilic ortho-quinone (right).

Fig. 5. Conversion of CA to CA Quinone

In Scenarios 1 and 2, CA quinone can be a toxic or protective substance, respectively. Circles show pro-electrophilic ortho-hydroquinone (left) and electrophilic ortho-quinone (right).

Fig. 6. Two Distinctive Groups of Electrophiles

A. Linear EPs. Linear EPs are exemplified by NEPP11 [8] and bardoxolone methyl [48]. Circles highlight the linear electrophilic cores of the compounds. B. Keap1 Activation by Cyclic EPs. Catechol-type CA is oxidized to a quinone form, with the carbon at position 14 [C(14)] becoming electrophilic (*). This CA quinone is subject to nucleophilic attack by the cysteine thiol of Keap1 to form an adduct. The Keap1-CA adduct results in release of Nrf2 protein from the Keap1/Nrf2 complex. Nrf2 can then be translocated into the nucleus, where it activates transcription of phase 2 enzymes via ARE transcriptional elements of the cognate genes. These phase 2 enzymes improve the redox state of neurons, contributing to an endogenous anti-oxidant defense system. Note that quinone formation is enhanced under oxidative stress, as described in the text. (*) indicates electrophilic carbons in NEPP11 [8], bardoxolone methyl [52] and CA [10] (the electrophilic carbons are subject to nucleophilic attack by cysteine thiols such as Cys151 on Keap1).

Fig. 7. Candidate PEDs

PEDs, as proposed here, consist of two types, ortho- and para-hydroquinones. Circles highlight the electrophilic cores of the structures.

Fig. 8. Redox-regulation of Cu²⁺/Cu⁺ recycling

Because H₂O₂ is a much better acceptor of electrons than O₂, the Cu²⁺/Cu⁺ recycling system is enhanced in the pathological state.

Fig. 9. The Keap1/Nrf2 pathway participates in electrophilic counterattack

The Keap1/Nrf2 pathway constitutes an electrophilic counterattack triggered by EPs, including DMF and CA. Note that EPs activate the pathway by triggering S-alkylation on the regulatory thiol of Keap1 protein.

Fig. 10. Astrocyte- and neuron-mediated neurotrophic actions of CA

CA preferentially acts on astrocytes, leading to the release NGF and GSH from astrocytes, both of which exert trophic actions on neurons [–78, 64, 65]. CA is also thought to act directly on neurons to upregulate TrkA, ERK1/2 and p62/ZIP via Nrf2 activation [12, 75, 79]. CA-activated Nrf2 then induces p62/ZIP expression, which plays an important role in mediating neurotrophic actions [12, 75, 79].