The role of natural products in revealing NRF2 function

Abstract

Covering: up to 2020The transcription factor NRF2 is one of the body's major defense mechanisms, driving transcription of >300 antioxidant response element (ARE)-regulated genes that are involved in many critical cellular processes including redox regulation, proteostasis, xenobiotic detoxification, and primary metabolism. The transcription factor NRF2 and natural products have an intimately entwined history, as the discovery of NRF2 and much of its rich biology were revealed using natural products both intentionally and unintentionally. In addition, in the last decade a more sinister aspect of NRF2 biology has been revealed. NRF2 is normally present at very low cellular levels and only activated when needed, however, it has been recently revealed that chronic, high levels of NRF2 can lead to diseases such as diabetes and cancer, and may play a role in other diseases. Again, this "dark side" of NRF2 was revealed and studied largely using a natural product, the quassinoid, brusatol. In the present review, we provide an overview of NRF2 structure and function to orient the general reader, we will discuss the history of NRF2 and NRF2-activating compounds and the biology these have revealed, and we will delve into the dark side of NRF2 and contemporary issues related to the dark side biology and the role of natural products in dissecting this biology.

Figure 1.. The NRF2 field is growing rapidly.

The graph shown lists the number of publications in pubmed by year.

Figure 2.. The canonical NRF2 pathway.

NRF2 is sequestered by the CUL3-RBX1 E3 ubiquitin ligase complex through the KEAP1 adapter protein, which binds the ETGE and DLG motifs of NRF2 in a 2:1 KEAP1:NRF2 ratio. A) Under basal, unstressed conditions, the CUL3 complex ubiquitylates NRF2 at one of the seven lysines residing between the ETGE and DLG motifs. B) Ubiquitylated NRF2 is then extracted from the CUL3 complex through the action of p97-UFD1-NPL4 mediated by UBXN7. C) Ubiquitylated NRF2 is transferred to the 26S proteasome, where it is destroyed. D) When cells are challenged with an oxidative or xenobiotic insult, one of the sensor cysteines of KEAP1 can become modified, which causes a structural rearrangement, releasing the DLG motif, and stopping subsequent ubiquitylation. Please see the text for alternate explanations and other models. E) The inhibited CUL3 complex blocks further NRF2 degradation, allowing NRF2 levels to rise in the cytosol. F) They then translocate to the nucleus, where they can bind to sMAF proteins and initiate ARE-regulated transcriptional programs.

Figure 3.. NRF2 domain architecture and structure.

A) NRF2 is comprised of seven domains termed Neh1–7 that have been defined according to biological function and homology to other protein domains. The numbering shown in the figure is for the human protein. Defined biological function of each of the domains is shown above the given domain and explained in greater detail in the text. B) The only structure of an unliganded domain of NRF2 is the NMR structure of the Neh1 domain shown (PDB ID 2LZ1). C) A crystal structure of the CNC bZip transcription factor MAFA as a potential model for the NRF2 Neh1 domain when bound to an obligate DNA-binding partner (PDB ID 4EOT). The high homology between bZip transcription factors argue this is a likely model for NRF2 in the active form, but to date, no structure has been solved.

Figure 4.. The KEAP1 Kelch domain bound to ETGE and DLG containing peptides.

A) and B) The ETGE motif binds to a series of positively charged amino acids in the KEAP1 Kelch domain. The ETGE forms a loop in the binding pose. Two views are shown: from the top A) and the side B). (PDB ID 5WFV). C) and D) The DLG containing peptide shows a pose like the ETGE, but shows fewer contacts, explaining the decreased affinity. The rest of the peptide forms an alpha-helical structure, but this is not known to be physiological or significant due to lack of larger structural data. Two views are shown: from the top C) and the side D). (PDB ID 3WN7).

Figure 5.. KEAP1 domain architecture and structure.

A) KEAP1 is comprised of three structural domains the BTB domain, the IVR domain, and the Kelch domain. The numbering shown is for the human protein. The assigned functions of each of the domains is shown above each domain. Human KEAP1 has 27 cysteines that can work as sensors. The most important cysteine sensors are also shown. For a detailed discussion of domain and cysteine function, see the text. B) The BTB domain of KEAP1 forms a functional dimer to bind to a single NRF2 protein. This dual binding mode is essential for physiologic function. (PDB ID 4CXI). C) The BTB domain bound to the N-terminus of CUL3. (PDB ID 5NLB). D) The unliganded Kelch domain of KEAP1. (PDB ID 5WFV).

Figure 6.. The KEAP1 BTB domain.

A) The apo BTB domain of KEAP1 showing Cys151 in green. B) The BTB domain of KEAP1 bound to the A ring of bardoxolone. This structure has been used to argue for dissociation of KEAP1 from the CUL3 complex upon activation by electrophiles. (PDB ID 4CXI). C) The KEAP1 BTB domain bound to CUL 3 with Cys150 highlighted in green. (PDB ID 4CXT). (PDB ID 5NLB).

Figure 7.. The conversion of glucoraphanin to sulforaphane by the plant enzyme Myrosinase.

Normally, in cruciferous vegetables, sulforaphane is in the glycosylated form. It is thought that when plants are attacked by herbivores, the level of Myrosinase increases, releasing sulforaphane and deterring the herbivore. This has important implications in the use of sulforaphane as a drug, since glucoraphanin is poorly bio-available. In the liver of mammals, glucoraphanin is reduced to glucoerucin. Both of these forms are substrates for Myrosinase. Once the carbohydrate is hydrolyzed, the resulting product undergoes a spontaneous Lossen rearrangement to the final, NRF2 activating isothiocyante.

Figure 8.. The NRF2 activators discussed in the text.

A) Curcumin from Curcuma longa. B) A curcumin derivative with more potent NRF2 activation and better pharmacological properties. C) A curcumin derivative with more potent NRF2 activation and better pharmacological properties. D) Cinnamaldehyde from Cinnamomum verum. E) Bixin from Bixa orellana. The red box is to differentiate natural product derived compounds from natural products.

Figure 9.. Withaferin A and a semi-synthetic derivative.

A) Withaferin A (from Withania somnifera)has been assigned many modes of action, but it is a known NRF2 activator as verified by our lab, however the precise mechanism by which it activates NRF2 is more complex than simple Cys151 adduction (See text for further discussion). B) A semi-synthetic withaferin A derivative that does not inhibit the proteasome but inhibits p97 and activates NRF2. The red box is to differentiate natural product derived compounds from natural products.

Figure 10.. NRF2 activating compounds that have been used in neuroprotective studies.

In addition to these, other compounds discussed in other sections shown in other figures have been used in neuroprotective studies. A) Carnosic acid from Rosmarinus officinalis. B) Sulfuretin from Rhus verniciflua. C) Methysticin from Piper methysticum. D) Resveratrol. E) Thymol from Thymus vulgaris. F) 6-Dehydrogingerdione from Zingiber officinale. G) Xanthohumol from Humulus lupulus. H) Hydroxytyrosol from Olea europaea. I) 6-Shogaol from Zingiber officinale. J) Cardamonin from Alpinia katsumadae. K) Honokiol from Magnolia virginiana. L) Costunolide from Saussurea costus. M) Mangiferin from Mangifera indica. N) Chlorogenic acid from coffee. O) Lipoamide. Please see the text for details.

Figure 11.. Natural product derived compounds.

A) Oleanolic acid is isolated in large quantity from olive (Olea europaea) waste and has been shown to have modest anti-inflammatory action but does not show NRF2 activation activity. B) Addition of a Michael acceptor to the A ring produced a μM NRF2 activating compound. C) Addition of a second Michael acceptor to the C ring led to an approximately order of magnitude increase in NRF2 activation activity, but it is not understood why. D) Electronic modulation of the A ring Michael acceptor gave another order of magnitude increase and the compound bardoxolone (CDDO), one of the most potent NRF2 activators known. Me Bardoxolone (more commonly CDDO-Me) only showed a modest increase in potency, but became orally bio-available, whereas bardoxolone must be injected. E) The imidazole variant has been in many studies but does not seem to be more efficacious. However, as discussed in the text, there are subtle differences between the activities of the varios CDDO derivatives for yet undescribed reasons. F) Omaveloxolone is a recent iteration from Reata Pharmaceuticals that is in clinical trials for several indications (see text). G) Dimethyl fumarate is a synthetic derivative of a primary metabolite but is included since it is the only compound to be used in humans that uses NRF2 activation as its primary proposed mode of action.

Figure 12.. The geopyxins offer insight into the advantages of non-covalent NRF2 activation.

A) A series of ent-kaurane diterpenoids were shown to activate NRF2. Geopyxin C from Geopyxis aff. majalis, a fungus occurring in the lichen Pseudevernia intensa, was shown to potently activate NRF2 in a KEAP1-Cys151 manner. B) Geopyxin F from Geopyxis sp. AZ0066 inhabiting the lichen Pseudevernia intensa was shown to be a modest activator of the NRF2 pathway. However, geopyxin F was shown to activate NRF2 in a KEAP1-dependent, but Cys151-independent manner. Moreover, geopyxin F showed greater protection of cells against toxicants and that this protection was NRF2-dependent.

Figure 13.. The GSK-3β/NRF2/β-TrCP regulatory axis.

GSK-3β can phosphorylate the Neh6 domain of NRF2 making it an enhanced substrate for the CUL1/β-TrCP/RBX1 complex. GSK-3β is inhibited by Ser9 phosphorylation mediated by PKC or AKT/PKB, which are both activated by PDK1. AKT/PKB can also be activated by AMPK or inhibited by PHLPP2. PI3K converts PIP2 to PIP3, which activates PDK1. The action of PI3K can be reversed by PTEN. The letters A-D in the figure refer to sites of modulation by the compounds in Figure 14.

Figure 14.. NRF2 modulating compounds that modulate the GSK-3β/NRF2/β-TrCP regulatory axis.

A) PI3K inhibitors that inhibit NRF2 by blocking PI3K. Wortmannin from Penicillium funiculosum. Despxo-narchinol A and narchinol B from Nardostachys jatamansi. Shikonin from Lithospermum erythrorhizon. Kaempferol from Brassica oleracea var. viridis. B) PKC modulators. Chelerythrine (Chelidonium majus) inhibits PKC and sauchinone (Saururus chinensis) activates PKC leading to inhibition of NRF2 and activation of NRF2, respectively. C) Compounds that activate AKT/PKB by increasing the activity of AMPK. Nectandrin B from Myristica fragrans. Emodin from Rheum hybridum. Esculentoside A from Phytolacca esculenta. Amentoflavone from Ginkgo biloba. Butin from Vernonia anthelmintica. Pterostilbene from blueberries. Apelin 13 from humans. D) Miscellaneous compounds that activate AKT/PKB by unknown mechanisms or through routes described in the text. 2-(penta-1,3-diynyl)-5-(3,4-dihydroxybut-1-ynyl)thiophene (PDDYT) from Echinops grijsii. Rosmarinic acid from Rosmarinus officinalis. Igalan from Inula helenium L. Oxymatrine from Sophora flavescens. Morin from Maclura pomifera. Totarol from Podocarpus totara. Melittin from honeybee (Apis mellifera) venom.

Figure 15.. The dark side of NRF2 has led to a search for NRF2 inhibitors.

A) The first NRF2 pathway inhibitor to be revealed was all-trans retinoic acid (ATRA). However, this was not without controversy as some groups reported ATRA to be an NRF2 activator. In any case, ATRA revealed RXR-𝛼 as a negative regulator of NRF2 transcription and defined the Neh7 domain as the site of RXR-𝛼 binding. B) Brusatol is a quassinoid that inhibits the synthesis of NRF2 and is the most potent NRF2 pathway inhibitor known. Despite potential off-target effects, brusatol (Brucia javanica) has been used extensively to probe the NRF2 pathway and reveal the intricacies of the dark-side of NRF2. C) Brucein C (Brucia javanica) was found to be inactive in NRF2 pathway assays. D) Bruceantin (Brucea antidysenterica) has been shown to be more potent than brusatol at inhibiting NRF2 function. These three molecules, and others of the class, show interesting SAR related to the lipid ester. E) and F) Febrifugine (Dichroa febrifuga) and halofuginone, a semi-synthetic derivative of febrifuginone, were shown to block prolyl-tRNA synthetase, thus blocking NRF2 synthesis and confirmed some of the studies conducted by brusatol, cementing the importance of the discovery and development of an NRF2 inhibitor. G) Wogonin (Scutellaria baicalensis) has been shown to decrease NRF2 mRNA levels and to reverse chemoresistance. However, conflicting studies have shown this to be an NRF2 activating compound.