Abstract
Numerous small molecules (termed inducers), many of which are electrophiles, upregulate cytoprotective responses and inhibit pro-inflammatory pathways by activating nuclear factor-erythroid 2 p45-related factor 2 (NRF2). Key to NRF2 activation is the ability to chemically modifying critical sensor cysteines in the main negative regulator of NRF2, Kelch-like ECH-associated protein 1 (KEAP1), of which C151, C273 and C288 are best characterized. This study aimed to establish the requirement for these cysteine sensor(s) for the biological activities of the most potent NRF2 activators known to date, the cyclic cyanoenones, some of which are in clinical trials. It was found that C151 in KEAP1 is the main cysteine sensor for this class of inducers, irrespective of molecular size or shape. Furthermore, in primary macrophage cells expressing C151S mutant KEAP1, at low concentrations, the tricyclic cyanoenone TBE-31 is inactive as an activator of NRF2 as well as an inhibitor of lipopolysaccharide-stimulated gene expression of the pro-inflammatory cytokines IL6 and IL1β. However, at high inducer concentrations, NRF2 activation proceeds in the absence of C151, albeit at a lower magnitude. Our findings highlight the intrinsic flexibility of KEAP1 and emphasize the critical importance of establishing the precise dose of NRF2 activators for maintaining on-target selectivity.
Highlights
The Kelch-like ECH-associated protein 1 (KEAP1)/nuclear factor-erythroid 2 p45-related factor 2 (NRF2)/antioxidant response elements (ARE) pathway is at the forefront of cellular defense
It was found that the semisynthetic triterpenoid CDDO-imidazolide [CDDO-Im, 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole)] (Fig. 1)[17] and the closely related compound, RTA-408, required C151 for their ability to cause NRF2 stabilization[39,40], implicating C151 as the sensor cysteine for these pentacyclic cyanoenones
We show that C151 in KEAP1 is the main cysteine sensor for the cyanoenone class of inducers, irrespective of their molecular size or shape
Summary
The KEAP1/NRF2/ARE pathway is at the forefront of cellular defense. Induction of this pathway is protective against various conditions of stress. To allow testing for new inducers, a highly quantitative and robust bioassay was developed which measures changes in the NQO1 enzyme activity in murine Hepa1c1c7 cells; the Concentration that Doubles the specific activity of NQO1 (CD value) provides a measure of inducer potency[13] Using this assay, extremely high NQO1 inducer potency, with CD values in the low nanomolar concentration range, was reported for the semisynthetic triterpenoids, such as CDDO (2-cyano3,12-dioxooleana-1,9(11)-dien-28-oic acid, Fig. 1) and its analogs with cyanoenone functionalities as Michael acceptors[14]. Many tricyclic and monocyclic compounds containing cyanoenone functionalities were designed and synthesized, and their potencies were evaluated using the quantitative NQO1 bioassay in Hepa1c1c7 cells Among these derivatives, the acetylenic tricyclic bis(cyanoenone), (±)-(4bS,8aR,10aS)-10a-ethynyl-4b,8,8trimethyl-3,7-dioxo-3,4b,7,8,8a,9,10,10a-octahydrophenanthrene-2,6-dicarbonitrile (TBE-31, Fig. 1) is the most potent inducer, active at low and even sub-nanomolar concentrations (CD = 0.9 nM)[20,21,22]. It was shown that oral administration of TBE-31 to wild type, but not NRF2-knockout mice that had been rendered obese and insulin resistant by consumption of high-fat and high-fructose diet, reversed insulin resistance, and attenuated obesity and liver disease[27]
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