Previous reporters of Nrf2 activation have utilized the ARE fused to coding regions of firefly luciferase or human alkaline phosphatase in vitro or in vivo. The ARE-GFP construct was used to screen the Spectrum library, and 45 hits were identified (Shaw et al., 2010
). The ARE-based reporters allow monitoring effects of antioxidant response induced by Nrf2 stabilization only after 24 hr or longer. We have constructed a reporter system that allows immediate monitoring of drug-induced Nrf2 stabilization in the form of Neh2-luciferase fusion protein. The reporter appears to be a physiological surrogate for Nrf2 based on several observations: (1) Keap1 overexpression inhibits the reporter activity, whereas Keap1 depletion stabilizes the reporter (); (2) canonical activators of Nrf2, which have been shown to act by alkylating Keap1, lead to expected increases in the Neh2-luciferase activity and protein (Figure S1B
; ); (3) representatives of all previously known classes of Nrf2 activators as well as the majority of ARE-GFP screen hits (Shaw et al., 2010
) were identified in the Spectrum library using the Neh2-luc reporter, further validating the assay (); and (4) activators of Nrf2 discovered in this screen protect neurons from oxidative death via an Nrf2-dependent mechanism in astrocytes ().
The power of the Neh2-luc reporter allowed us to discriminate between direct and indirect effects on reporter stabilization induced by compounds tested in HTS, and, to our knowledge, for the first time identify gedunin as a direct activator of Nrf2. Recent studies suggest that gedunins are potent Hsp90 inhibitors (Brandt et al., 2008
). Celastrol, a quinone methide triterpenoid, is a known Hsp90 inhibitor (Zhang et al., 2008
) as well, and its derivative, dihydrocelastrol, was also found as a modest hit in the screen. Based only on structural similarities between gedunin and celastrol, one could speculate that gedunin utilizes a similar mechanism of action via disrupting the interaction between Hsp90 and Cdc37, the cochaperone providing a bridge between Hsp90 and client tyrosine kinases (Zhang et al., 2008
), which being detached from the Hsp90 complex undergo fast inactivation (usually within 40–45 min). Of note, triterpenoids have been described as Nrf2 activators using ARE-reporter mice and NQO1 induction levels (Yates et al., 2007
), and induce neuroprotection in a transgenic model of Huntingtons disease (Stack et al., 2010
). Withanolides, closer analogs of gedunins, have been long known as inducers of NQO1 (Dinkova-Kostova et al., 2004
), and are also known to disrupt Hsp90-Cdc37 interaction (Yu et al., 2010
If gedunin works via the same mechanism as the aforementioned compounds, we should observe the delayed effect of Hsp90 downregulation with all three compounds, e.g., gedunin, geldanamycin, and TSA. However, the latter two show 3 hr lag period in reporter activation, in contrast to the immediate effect induced by gedunin (). We may speculate that the direct effect of gedunin originates from its competition with Nrf2 for Keap1 based on the comparatively modest activation amplitude and observed plateau in the time course of reporter activation (). This is in contrast to alkylating agents that drive the system to the maximum activation linearly (see quercetin and catechol in ). The plateau is a characteristic of re-equilibration of the system with reversible binding, or in other words, gedunins may bind Keap1 reversibly. It is tempting to speculate that gedunins compete with Nrf2 for Keap1 binding: the possibility to design mild peptide-type inhibitors displacing Nrf2 from Keap1 like p62 does in vivo (Komatsu et al., 2010
) has been discussed in the paper with the resolved crystal structure of Neh2-Keap1 DGR (Tong et al., 2007
). This speculation is supported by computer modeling: gedunins fit perfectly into the same Keap1-binding pocket as Nrf2 (), closely following the bending of the 83FEGTE79 portion of the Nrf2 peptide ().
Schematic Representation of Different Mechanisms of Nrf2 Level Regulation and Plausible Mechanism of Gedunin Action
An important unanswered question is the mechanism of the “switch” effect demonstrated for our best hits, fisetin and NDGA. The time course of NDGA and fisetin clearly shows that they exert an immediate effect upon addition to the reporter cell line; therefore, they act “as is” without prior chemical modification. Both NDGA and fisetin have adjacent hydroxy groups on a freely rotating phenyl ring. This might suggest that these adjacent hydroxy groups lead to reduction of a critical disulfide bond. However, there is some doubt that fisetin and NDGA work via this mechanism because the flavones are strong reducing agents capable of immediate reduction of dithionitrobenzoate, a model disulfide, whereas NDGA is not. In addition, luteolin, a flavone with potent reducing properties, with 3,4-dihydroxy-phenyl group present in fisetin, but hydroxyl group in position 5, not 3, is a very poor Nrf2 activator. Moreover, catechol, being a very potent reducing agent, does show a 20 min lag period, which may reflect initial “priming,” most likely oxidation that results in formation of its form capable of alkylating Keap1. The fact that luteolin and catechol do not behave the same way argues against this potential mechanism and points to the special structural requirements for a “switch” mechanism of Nrf2 activation.
A common and intriguing feature of our most promising hits, fisetin and NDGA, is their steep concentration response, reminiscent of a ligand binding to a receptor. Of note, a common feature of these hits is that they all have been reported to act as inhibitors of protein tyrosine kinases, and NDGA in particular was reported to target IGF1-R kinase. We also identified genistein (100% reporter activation), which is well known for targeting this class of enzymes. Phosphorylation of Tyr141 in Keap1 is catalyzed by an unknown protein tyrosine kinase and is critical for Keap1 stability (Jain et al., 2008
). Protein tyrosine kinases are also known to be stabilized by Hsp90, inhibitors of which also came out in our screen as hits.
The analysis of kinetics of individual hits leads to the model scheme of Nrf2 regulation shown in . A key role is played by Keap1 Cys151, 273, 288, for which modification with alkylating agents causes a dramatic change in Keap1 conformation leading to Nrf2 stabilization. If Keap1 in vivo has a zinc atom in the structure, we may hypothesize that the small planar Zn2+
chelators identified in HTS may target and destabilize the thiol pair in Keap1 as well. The delayed effect of cadmium may reflect the inhibition of thioredoxin reductase/thioredoxin system, eventually compromising the redox status of key cysteines in Keap1. Regulation of Keap1 stability via Hsp90-Cdc37-tyrosine kinase interaction is upstream of immediate activation pathways. Hsp90 is a target for TSA and geldanamycin, whereas NDGA and fisetin inhibit tyrosine kinase activity. Gedunin, in addition to intercalation into the Hsp90-Cdc37 interface, exerts an immediate effect on Nrf2 stabilization, possibly by disrupting Nrf2-Keap1 interaction. With respect to fisetin and NDGA, we also cannot rule out a possibility of targeting an unknown site at the interface of Keap1 subunits () resulting in an immediate change in Keap1 conformation and stabilization of Nrf2 because the scaffold of fisetin closely resembles those of the hits generated by the virtual screen in Wu et al. (2010)
Canonical activators of Nrf2 such as TBHQ, isothiocyanates, and the recently identified AL-I (Hur et al., 2010
) appear to act by modifying key cysteines in Keap1, the negative regulator of Nrf2 stability. A major potential problem with electrophile activators of Nrf2 is their ability to induce toxicity, particularly in cells vulnerable to redox stress such as neurons afflicted by ischemia or neurodegeneration. The challenge is to find Nrf2 activators that do not add to the overall oxidative load, and the reporter provides a valuable resource for future developments toward such medications. Here, we discover a number of Nrf2 activators that are nontoxic to neurons over the range of concentrations optimal for reporter activation (Figures S6E–S6I
Activation of Nrf2 plays a key role in the antioxidant defense of the central nervous system and has been shown to be important for neuroprotection in several acute and chronic neuropathological conditions such as stroke, intracerebral hemorrhage, Parkinsons disease, Huntingtons disease, and amyotrophic lateral sclerosis. Yet, Nrf2 activators such as TBHQ, sulforaphane, or CDDO-triterpenoid are only now making their way into the clinic (Shih et al., 2005
; Chen et al., 2009
; Vargas et al., 2008
). These findings highlight the biological and clinical importance of a real-time assay for screening and design of Nrf2 activators. The developed Neh2-luc reporter is perfectly suited for HTS purposes, for studying the mechanistic details of drug action, and by analogy with HIF ODD-luc system (Safran et al., 2006
), we are confident that the reporter may be successfully used for in vivo imaging of Nrf2 activators in animals.