Lipoxygenases are a class of non-heme iron-containing dioxygenases present in tissues and cells of both animals and plants. They catalyze the oxidation of arachidonic acid (AA), docosahexaenoic acid, and various polyunsaturated fatty acids (PUFAs). Lipoxygenases are mainly classified into six subtypes: 1-, 3-, 5-, 8-, 12-, and 15-lipoxygenases. Among them, 5-lipoxygenase (5-LOX) is the most common and well-studied non-heme iron-containing protein oxidative metabolic enzyme among the lipoxygenase isozymes. It serves as a key enzyme in the biosynthesis of many highly reactive oxidized lipids. The action of 5-LOX on AA oxidation occurs in two steps: the first step involves the oxidation of AA by 5-LOX to form 5-hydroperoxyeicosatetraenoic acid (5-HPETE), which is then converted into 5-hydroxyeicosatetraenoic acid (5-HETE). The second step catalyzes the conversion of 5-HPETE into leukotriene A4 (LTA4). LTA4 can either be converted into the inflammatory mediator leukotriene B4 (LTB4) or into leukotriene C4 (LTC4), which further generates leukotriene D4 (LTD4) and leukotriene E4 (LTE4), among other reactive substances that cause allergic reactions. Leukotrienes (LTs) are pro-inflammatory lipid mediators associated with various chronic inflammatory diseases such as asthma, chronic obstructive pulmonary disease, inflammatory bowel disease, arthritis, atherosclerosis, dermatitis, and cancer. Through further dioxygenation and synergistic oxidation of exogenous chemicals, LTs play a crucial role in the development and progression of diseases such as inflammation, asthma, cerebral ischemia, some types of malignant tumors, and Alzheimer's disease. 5-LOX inhibitors block the production of LTs by inhibiting the conversion of AA to LTs, thereby reducing inflammation. Therefore, the development of effective 5-LOX inhibitors is crucial for the treatment of these diseases.
Based on their mechanisms of action, 5-LOX inhibitors can be classified into direct inhibitors and indirect inhibitors.
Direct Inhibitors
Direct inhibitors are developed specifically targeting the structural characteristics of the 5-LOX molecule. They work by interfering with the normal conversion between Fe2+ and Fe3+ in the C-terminal catalytic domain of 5-LOX or by chelating with iron ions to inhibit the activity of 5-LOX. Direct inhibitors can be further classified into redox inhibitors, non-redox inhibitors, and iron ligand inhibitors.
Indirect Inhibitors, on the other hand, are primarily designed around FLAP (5-lipoxygenase-activating protein), which is required in the 5-LOX pathway. By inhibiting FLAP, the 5-LOX pathway can be blocked, thereby inhibiting the production of downstream inflammatory mediators.
1. Redox Inhibitors
Redox inhibitors are generally lipophilic small molecule aromatic compounds, such as phenolic compounds and quinone compounds. 1,4-Benzoquinone AA-861 was one of the first redox-type 5-LOX inhibitors, which has been proven through multiple studies to exhibit anti-inflammatory effects in vivo and has passed clinical trials for the prevention of seasonal allergic rhinitis. Quinone derivatives have been shown to be potent and active inhibitors of 5-LOX in human monocytes, neutrophils, and whole human blood. Experimental studies on two standardized animal models of LT-related inflammation have demonstrated that the novel 1,4-quinone RF-Id can inhibit the production of LTs in vivo, thereby suppressing the inflammatory response mediated by 5-LOX.
In addition to these, redox inhibitors also encompass several other classes of compounds, including nordihydroguaiaretic acid (NDGA), coumarins, and flavonoids, all of which have been proven to exhibit potent 5-LOX inhibitory effects in vitro. Redox inhibitors can reduce Fe3+ to Fe2+, thereby disrupting the normal catalytic cycle of iron ions and preventing the activation of 5-LOX, leading to the disruption of the 5-LOX pathway. Moreover, certain flavonoid compounds, such as quercetin and rutin, possess adjacent phenolic hydroxyl groups on their B-rings, enabling them to chelate with Fe3+, thereby enhancing their inhibitory effect on 5-LOX.
Furthermore, researchers have used fluorescence methods to determine the redox potential of redox inhibitors and have elucidated their mechanism of action. The presence of redox inhibitors reduces the conversion between Fe2+ and Fe3+, inhibiting the enzymatic catalytic reaction. Consequently, more lipid peroxides are required to oxidize Fe2+ into Fe3+, meaning that only a small amount of lipid peroxides are actually involved in the catalytic reaction, resulting in the inhibition of the enzymatic reaction.
2.Non-Redox Inhibitors
Non-redox inhibitors are competitive inhibitors that compete with the substrate AA for binding to the active center of 5-LOX, resulting in the blockage of the 5-LOX pathway. Compared to other inhibitors, they exhibit higher selectivity, but their effectiveness can be interfered with by phosphorylation and Ca2+. ZD2138 and ZM230487 are two representative examples of non-redox inhibitors belonging to the class of methoxytetrahydropyran derivatives. Clinical studies have shown that ZD2138 is effective in inhibiting arthritis and asthma. Experimental research has also demonstrated that some hybrids of natural products and ZD2138, such as L-697198, as well as its optimized derivatives L-708780 and L-739010, exhibit good bioavailability. However, subsequent animal experiments revealed multiple toxicities associated with this class of inhibitors, limiting their clinical use.
Additionally, L-739,010, CJ-13,610, and PF-419183 are other non-redox 5-LOX inhibitors that have shown low nanomolar IC50 values in cellular assays. L-739,010 reduces LT-mediated inflammatory responses, and its analgesic activity is correlated with reduced LT levels in inflamed brains. It exhibits excellent efficacy and pharmacokinetics in both acute carrageenan and chronic inflammation models but struggles to form covalent protein-bound metabolites. CJ-13,610 inhibits osteoarthritis pain in a rat medial meniscus transection model, and oral administration of CJ-13,610 reverses two pain modalities in this animal model, including tactile allodynia and weight-bearing asymmetry. PF-4191834 has been shown to inhibit LTB4 production in rat air pouch models and completed Phase II clinical trials for asthma. However, its Phase II clinical trials for knee osteoarthritis were terminated due to serious adverse events such as syncope, acute hepatitis, and gastric ulcer bleeding.
3.Iron Chelator Inhibitors
Iron chelator inhibitors can be broadly classified into two categories: hydroxamic acid derivatives and hydroxyurea derivatives. Among them, the hydroxamic acid derivative BWA4C and the hydroxyurea derivative Zileuton are effective oral 5-LOX inhibitors. BWA4C serves as a potent and selective inhibitor of LTB4 synthesis in colonic tissues from patients with ulcerative colitis. The use of BWA4C, a representative of acetylhydroxamic acid 5-lipoxygenase inhibitors, aids in evaluating the clinical significance of LTB4 in ulcerative colitis and offers a novel therapeutic approach for this disease.
Zileuton has been shown to improve acute and chronic airway function, reducing the need for β-agonists or glucocorticoids, and is approved in the United States for the treatment of asthma. However, its potential for treating allergic rhinitis, rheumatoid arthritis, and inflammatory bowel diseases is limited, and it is associated with numerous drawbacks such as hepatotoxicity and unfavorable pharmacokinetic properties. Based on the structure of Zileuton, a novel inhibitor named VIA-2291 has been designed, which exhibits five times greater potency than Zileuton in animal models of bronchospasm. With an oral half-life of 16 hours, VIA-2291 has demonstrated efficacy against exercise-induced bronchoconstriction in asthma patients. VIA-2291 has successfully completed Phase II clinical trials for atherosclerosis and cardiovascular diseases, positioning it as one of the leading 5-LOX inhibitors in clinical development.
RBX-7796, a urea derivative with a dodecyl chain, inhibits the in vitro synthesis of LTB4 in rat blood and exhibits activity in various animal models of inflammation and bronchoconstriction.
Indirect Inhibitors
Indirect inhibitors do not directly inhibit the activity of 5-LOX. Instead, they target FLAP, the 5-LOX activating protein, which binds to the substrate AA and transfers it to 5-LOX. In cells, 5-LOX and FLAP are co-expressed, and in the absence of FLAP, even with an adequate supply of AA, cells are unable to synthesize leukotrienes and related compounds. MK886 represents the first-generation of indirect inhibitors developed and is also the first of its kind to be used clinically. It competes with AA for binding sites on FLAP, preventing the interaction between 5-LOX and FLAP, thus blocking the 5-LOX pathway.
Additionally, the inhibitor AM803 is undergoing clinical trials and has completed Phase II trials in asthmatic patients. Its derivative, AM103, has also successfully passed Phase I clinical trials. However, some inhibitors under development, such as NGDA and MK0591, possess two major drawbacks. Firstly, they lack specificity towards 5-LOX, potentially interfering with normal human metabolism, thereby limiting their clinical applications. Secondly, their instability directly impacts the inhibitory efficacy of these inhibitors.
References
[1] Li Danfeng, Fan Tao, Lu Xinyu. Research Progress on 5-Lipoxygenase Inhibitors [J]. Chemistry of Life, 2023, 43(05): 666-673.
[2] Huang Kai, Jiang Shiyun, Chen Liujun. 5-Lipoxygenase and Its Inhibitors [J]. Chinese Journal of Biochemistry and Molecular Biology, 2014, 30(12): 1190-1196.
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.
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