Based on the structure of the activators and the type of reaction with cysteine, indirect inhibitors of the Keap1-Nrf2 interaction can be classified into several categories such as Michael acceptors, oxidizable diphenols, isothiocyanates, dithiolethiones and diallyl sulfides, polyenes, and others.

"Recap of Part I: Advances in Research on Nrf2 Activators Based on Natural Products and Their Derivatives (Part I)"

Dithiolethiones and Diallyl Sulfides

Dithiolethiones are sulfur-containing five-membered cyclic compounds that exhibit cytoprotective effects against carcinogenic substances. 3H-1,2-dithiole-3-thione (D3T) is the simplest dithiole compound and can be extracted from cruciferous vegetables such as cabbage and kale. Diallyl sulfides, on the other hand, are a class of organosulfur compounds found in the Allium genus of vegetables (e.g., garlic, onion), which effectively inhibit various cancer cells. Research has shown that the disulfide bonds in dithiolethiones and diallyl sulfides can undergo thiol-disulfide exchange reactions with the sulfhydryl groups of cysteines, modifying Keap1 and inhibiting the ubiquitination of Nrf2, thereby upregulating the expression of phase II detoxifying enzymes. Common Nrf2 activators in this category include 3H-1,2-dithiole-3-thione and diallyl sulfide. Furthermore, oltipraz, the earliest drug approved by the FDA for the treatment of schistosomiasis, has also been found to increase the expression of phase II detoxifying enzymes such as GSH, helping to eliminate reactive oxygen species (ROS) in the body.

Vicinal Dithiols

Vicinal dithiols can be converted into electrophilic disulfide bonds within the body. Studies have shown that 1,2-ethanedithiol and 2,3-dimercaptosuccinic acid can activate Nrf2. Another variant of dithiol, (R)-lipoic acid, or alpha-lipoic acid, is found in nearly all foods, with slightly higher concentrations in spinach and broccoli compared to other foods, and can also be generated endogenously. (R)-Lipoic acid is a molecular nutrient and antioxidant that has been shown to induce the expression of various antioxidant enzymes through the activation of Nrf2.

Trivalent Arsenic Compounds

The high affinity of arsenic(III) towards thiols is considered one of the causes of acute and chronic arsenic compound poisoning. However, research has shown that low doses of arsenic can reduce the incidence of cancer. Arsenic compounds such as sodium arsenite, arsenic trioxide (ATO), and monomethylarsonic acid (MMA) can modulate the Keap1-Cul3 E3 ubiquitin ligase complex to activate Nrf2. Additionally, fluorescein-labeled bisarsenical thiol reagents (FLASH-EDT2) and phenylarsine oxide (PAO) can also activate Keap1-dependent Nrf2.

Selenium-Containing Compounds

Selenium and sulfur share similar electronegativity, and various organoselenium compounds exhibit cytoprotective activities. The selenium analogue of sulforaphane, SFN-isoSe, demonstrates superior Nrf2 induction activity compared to sulforaphane (SFN). Ebselen is an effective multifunctional antioxidant and anti-inflammatory agent that modulates the expression of Keap1-Nrf2-mediated detoxifying enzyme genes. The organoselenium compound 3-selena-1-dethiacephem serves as an activator of Nrf2-ARE and a direct scavenger of reactive oxygen species (ROS).

Hydroperoxides

When the production of free radicals and peroxides exceeds the biological system's ability to clear or repair them, oxidative stress is induced. The O-O bond in peroxides can easily break, releasing oxygen radicals that subsequently react with sulfhydryl groups to form sulfenic acids (RSO-). At low concentrations, hydrogen peroxide and organic peroxides such as tert-butyl hydroperoxide can act as inducers by oxidizing the sulfhydryl groups of Keap1, thereby activating Nrf2- and ARE-dependent genes.

Polyenes

Polyene compounds contain one or more alternating sequences of unsaturated single and double carbon-carbon bonds. Due to their high degree of unsaturation, these compounds are prone to biotransformation into electrophilic metabolites that can react with free sulfhydryl groups. Carotenoids, a class of polyene compounds, possess cancer-preventive properties. They can activate the ARE transcription system and induce the expression of antioxidant and cytoprotective enzymes. Lycopene, a red carotenoid pigment primarily found in tomatoes, and its potential oxidative metabolite 10,10’-diapocarotene-10,10’-dial contain Michael acceptor groups, which can covalently modify Keap1, activate Nrf2, and enhance the expression of ARE-mediated genes.

Heavy Metals and Metal Complexes

Humans require trace amounts of heavy metals such as iron, cobalt, copper, and zinc for survival, but excessive levels of heavy metals can be harmful. Some heavy metals, like mercury, cadmium, gold, and lead, are highly toxic. Certain heavy metals (e.g., mercury, cadmium, zinc) can induce the expression of ARE genes. Cadmium chloride, a known human carcinogen, can induce cancer through multiple mechanisms, including inducing abnormal gene expression, inhibiting DNA damage repair, inducing oxidative stress, and inhibiting apoptosis. Cadmium's ability to induce ARE gene expression can be viewed as a biological defense mechanism against the oxidative stress induced by cadmium. Auranofin, an anti-rheumatic drug containing gold, exerts its anti-inflammatory effects by activating the Keap1-Nrf2-ARE signaling pathway.

The Keap1-Nrf2-ARE signaling pathway is crucial for the antioxidant defense mechanism, as it activates the cellular adaptive response to counter various oxidative stress injuries. Keap1 serves as a negative regulator of Nrf2, which is the primary effector of the ARE system. By negatively regulating Keap1 protein to activate the ARE system, it can induce the expression of a series of antioxidant genes, which is essential for preventing oxidative damage, inflammation, and tumorigenesis. Therefore, activating the Keap1-Nrf2-ARE system holds significant value in the development of antioxidant, anti-inflammatory, and anticancer drugs. Over the past few decades, numerous Nrf2 activators have been developed, with some entering clinical trial phases. Both genetically and chemically induced upregulation of phase II metabolizing enzyme gene expression in vitro and in vivo can exert cytoprotective effects. Currently, several natural and synthetic small molecules have been identified as inducers of the ARE system, such as sulforaphane, bardoxolone methyl, oltipraz, and ebselen. These drugs are currently being used in clinical trials for various conditions, including breast cancer, prostate cancer, asthma, chronic obstructive pulmonary disease, chronic kidney disease, type 2 diabetes, and nonalcoholic fatty liver disease. Most known Keap1 inhibitors share the common characteristic of being electrophilic, which modifies and alters the cysteine sulfhydryl groups on Keap1, thereby activating ARE. The Keap1-Nrf2 interface has evolved into a molecular target for direct activation and reversible induction of ARE, providing research directions for the discovery and study of activators of the Keap1-Nrf2-ARE signaling pathway.

References:

[1] Yao Juan, Wu Ping'an, Li Yun, Li Yuefeng, Liu Dongling, Liu Xuefeng. Research Progress on Keap1-Nrf2-ARE Signaling Pathway and Its Activators [J]. Chinese Pharmacological Bulletin, 2019, 35(10): 1342-1346.

[2] Cheng Maojun, Guo Jie, Liu Jing, Xie Saisai. Research Progress on Nrf2 and Natural Product-Oriented Nrf2 Activators [J]. Natural Product Research and Development, 2021, 33(01): 165-177.

About the Author

Xiaonisha, a food technology professional holding a Master's degree in Food Science, is currently employed at a prominent domestic pharmaceutical research and development company. Her primary focus lies in the development and research of nutritional foods, where she contributes her expertise and passion to create innovative products.