Arachidonic acid (AA) undergoes enzymatic and non-enzymatic oxidation to produce an array of lipid metabolites collectively known as eicosanoids. Many eicosanoids have central and diverse roles in tissue homeostasis, inflammation, platelet function, and smooth muscle contraction. Understanding the biological role of eicosanoids and finding ways to modify their formation and function has been the focus of intensive research for decades. Throughout these investigations, it has become clear that the AA cascade is a highly intertwined network of enzymes and metabolites with extensive crosstalk.

Click the links below to explore each section:

• COX Pathway

• LO Pathway

• Cytochrome P450 Pathway
  

• Products of Non-Enzymatic Reactions

• Interplay Between the AA Cascade & Endocannabinoids  

• Upsetting Balance in the AA Cascade

• Next Steps Towards Therapeutics

• Available from Cayman

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The Arachidonic Acid Cascade

Arachidonic acid (AA) is a 20-carbon ω-6 polyunsaturated fatty acid (PUFA) that is abundant in biological cell membranes where it is esterified into membrane phospholipids. Cytosolic phospholipase A₂ (PLA₂), a ubiquitous enzyme that is activated in response to various inflammatory stimuli, liberates AA and releases it into the cellular milieu, where AA can be shunted into three main enzymatic pathways: cyclooxygenase (COX), lipoxygenase (LO), and cytochrome P450 (CYP450) pathways. When investigating these pathways, it is important to consider: 1) activation of these pathways can either be beneficial or maladaptive, 2) these pathways do not occur in isolation, 3) there is a substantial amount of crosstalk, cooperation, and competition in the AA cascade, and 4) the net effect of any lipid metabolite depends on the amount produced, the presence or absence of other lipid metabolites, and its further conversion to inactive compounds.

Cyclooxygenase (COX) catalyzes the initial step that commits AA to the production of prostaglandins (PGs) and thromboxane A₂ (TXA₂), which are collectively known as prostanoids. There are two COX isoforms: COX-1 and COX-2. COX-1 is constitutively expressed in nearly all cells and tissues where it produces PGs that have homeostatic roles like inhibition of platelet aggregation and maintenance of cardiovascular, renal, and gastrointestinal systems. In contrast, COX-2 is inducible and transiently expressed in response to cytokine stimulation, LPS, and various growth factors, as well as tumor promoting agents. These PGs mediate inflammation by promoting inflammatory cell recruitment, amplifying cytokine signaling, and increasing vascular permeability, among other effects. Taken together, the presence of constitutive and inducible forms of COX represents a tailorable mechanism to harness a common set of lipid mediators for both homeostatic and inflammatory functions.

COX is a bifunctional enzyme, first generating the unstable intermediate PGG₂via its COX activity followed by the conversion of PGG₂ to PGH₂ by its peroxidase function. PGH₂ is the common precursor for PGs and TXs. PGH₂ can be isomerized into TXA₂, a vasoconstrictor with prothrombotic properties, or PGs like PGD₂, PGE₂, PGF_2α, or PGI₂, by their respective synthases. In some cases, more than one PG synthase is available, leading to the simultaneous formation of multiple PGs at a single site. The plethora of effects mediated by PGs and TXA₂ is mediated by the diversity of their G protein-coupled receptors (GPCRs): two PGD receptor subtypes (DP₁ and DP₂, also known as CRTH₂), four PGE receptor subtypes (EP₁-EP₄), the PGF receptor (FP), the PGI receptor (IP), and the TXA₂ receptor (TP). Prostanoid synthesis and their receptors are summarized in Figure 1. See our article on Eicosanoid Enzymology and Metabolism for a closer look at the formation of PGs and their receptors.

Figure 1. COX catalyzes the synthesis of various PGs and TXA₂. The biological activities of PGs and TXA₂ are mediated by their respective receptors.  

Agents that target the COX pathway are of interest for the treatment of inflammatory conditions and as anti-platelet therapeutics. COX inhibitors like aspirin and indomethacin are non-steroidal anti-inflammatory drugs (NSAIDs) that reduce PG production to decrease the cardinal signs of inflammation (redness, swelling, pain, and fever). In platelets, inhibition of COX-1 impairs TXA₂ synthesis, reducing platelet aggregation. PG analogs and receptor agonists have also found clinical use. The PG analogs  latanoprost, bimatoprost, and travoprost are used for the treatment of glaucoma. Novel applications for COX pathway inhibitors are continuing to be discovered. For example, Cayman Chemical is exploring the use of our selective EP₄ receptor agonist KMN-159 for dental implant osseointegration.

The lipoxygenase (LO) pathway produces leukotrienes (LTs), hydroxyeicosatetraenoic acids (HETEs), lipoxins (LXs), hepoxilins (HXs), and eoxins, leading to a myriad of biological effects including the promotion and resolution of inflammation, bronchoconstriction, platelet function, and tumorigenesis. Mammalian LOs catalyze the oxygenation of AA at the 5-, 12-, or 15-position to hydroperoxyeicosatetraenoic acid (HpETE) intermediates, which are reduced to HETEs by ubiquitous peroxidases or enzymatically converted to other bioactive lipid metabolites. LOs are widely expressed, especially in immune, epithelial, and tumor cells, as well as platelets.

5-LO, in concert with 5-LO activating protein (FLAP), catalyzes the formation of 5(S)-HpETE, which leads to 5(S)-HETE, a neutrophil chemoattractant, and leukotrienes (LTs). 5(S)-HpETE is converted to leukotriene A₄ (LTA₄) by the further action of 5-LO. LTA₄ is then converted to LTB₄ by LTA₄ hydrolase, which is ubiquitously expressed, or to the cysteinyl LTs. Cysteinyl LTs are a class of LTs produced when LTC₄ synthase, which is expressed primarily by innate immune cells, forms LTC₄ from LTA₄. LTC₄ is then converted sequentially to LTD₄ and LTE₄ by extracellular peptidases. LTs exert their biological activity through G protein-coupled receptors (Figure 2). LTB₄ has two receptors: BLT₁ and BLT₂, which mediate cell trafficking. BLT₁ has high affinity for LTB₄ and is expressed in leukocytes and platelets, whereas the low-affinity receptor BLT₂ is more ubiquitously expressed and has a wider range of ligand specificity. CysLTs also have two receptors: CysLT₁ and CysLT₂, which are co-expressed in most myeloid cells and notably, mediate bronchoconstriction and airway hyperresponsiveness. Zileuton (a reversible 5-LO inhibitor) as well as montelukast, pranlukast, and zafirlukast (CysLT receptor antagonists) are used clinically to treat asthma and allergic rhinitis.

Figure 2. 5-LO catalyzes the synthesis of 5(S)-HETE, LTB₄, and CysLTs. LTs exert their biological activity through BLT and CysLT receptors.  

12-LO is tightly regulated by transcription and its cellular distribution and reaction specificity is variable by species (Table 1). For simplicity, 12-LO herein refers to any LO with 12(S)-LO activity, including LOs encoded by human ALOX12 and ALOX15. We refer the reader to Kuhn et al. 2015 for an excellent review on species-specific variation in the cellular expression and product formation of these enzymes. 

Table 1. Reaction specificity of enzymes with 12-LO activity in human and mouse.

12(S)-HETE is produced from 12(S)-HpETE in large amounts by activated platelets in response to agents like ADP, TXA₂, thrombin, and collagen where it has roles in platelet aggregation and thrombus formation. It is also produced by leukocytes and acts as a chemoattractant, and in tumor cells, it is associated with cancer cell survival and tumorigenesis. GPR31, also known as the 12(S)-HETE receptor (12-HETER), is expressed mainly in platelets and is the primary receptor for 12(S)-HETE. However, 12(S)-HETE can also bind to TP and BLT₂. In the case of TP receptors, 12(S)-HETE induces vasodilation in arteries precontracted with carbocyclic TXA₂, a stable analog of TXA₂, thus acting as a competitive antagonist. Regarding BLT₂, 12(S)-HETE can act as a competitive antagonist or weak agonist. For example, 12(S)-HETE inhibits the binding of LTB₄ to BLT₂ in radioligand assays, and it also induces calcium mobilization in and chemotaxis of cells expressing BLT₂, albeit with reduced potency compared to LTB₄.

Isomerization of 12(S)-HpETE by HxA₃ synthase forms hepoxilin A₃, the founding member of the hepoxilin family. In general, hepoxilins promote inflammation and have been implicated in neutrophil transmigration across epithelial cells, stimulation of insulin secretion, and vascular relaxation. HxA₃ is further metabolized to its trioxilin 8,11,12-TriHETrE by epoxide hydrolase, a conversion that is coincident with loss of biological activity.

Figure 3. 12-LO catalyzes the formation of 12(S)-HETE and HxA₃. GPR31 has been identified as the primary receptor for 12(S)-HETE, though 12(S)-HETE can also bind BLT₂ and TP receptors. The weight of the arrow indicates the primary receptor.

There are two types of 15-LO in humans, 15-LO-1 (ALOX15) and 15-LO-2 (ALOX15B), both of which oxygenate AA to produce 15(S)-HpETE, the precursor for 15(S)-HETE, an angiogenic molecule that is a target for cancer treatments, eoxins, and lipoxins. 15-LO isozymes vary in their expression and reaction specificity. 15-LO-1 is mainly expressed in airway epithelial cells, eosinophils, and reticulocytes, whereas 15-LO-2 is expressed in the lung, prostate, skin, and cornea. Human 15-LO-1 produces 15(S)-HETE and, to a minor extent, 12(S)-HETE. In contrast, 15-LO-2 exclusively produces 15(S)-HETE (Figure 4). To further complicate matters, the enzyme encoded by Alox15, the murine ortholog of ALOX15, actually favors production of 12(S)-HETE over 15(S)-HETE. This discrepancy confounds the interpretation of mouse studies to human relevance. 

Figure 4. The two forms of 15-LO in humans have varying reaction specificity. The weight of the arrow indicates the primary product formed.

In eosinophils, further oxygenation of 15(S)-HpETE by 15-LOX-1 produces an alternative class of 14,15-LTs known as eoxins (Figure 5). The synthesis of the eoxins 14,15-LTC₄, 14,15-LTD₄, and 14,15-LTE₄ occurs in a manner identical to the formation of conventional 5-LO-produced LTs. In many instances, eoxins share similar functions with their LT counterparts. Eoxins are generally regarded as pro-inflammatory and contribute to asthma by inducing endothelial cell dysfunction and increasing vascular permeability.

Figure 5. 15-LOX-1 catalyzes the formation of 14,15-LTC₄, 14,15-LTD₄, and 14,15-LTE₄. These members form a class of LTs known as eoxins in eosinophils. 

Other metabolites are formed by the cooperative actions of multiple LOs. Lipoxins are a class of anti-inflammatory eicosanoid that are synthesized by at least three ways (Figure 6). In one route, 15-LO-generated 15(S)-HpETE is acted on by 5-LO to produce an epoxy intermediate, which is then converted by lipoxin hydrolases to lipoxin A₄ (LXA₄) and lipoxin B₄ (LXB₄). Another route uses LTA₄, the LT precursor, generated from 5-LO. LTA₄ is either converted by 12- or 15-LO to an epoxy intermediate that is converted to LTA₄ and LTB₄. This reaction can occur in cells that express both enzymes or via a transcellular mechanism. For example, platelets do not express 5-LO, but neutrophils do. Neutrophils produce LTA₄, which is transferred to platelets, which express 12-LO and can synthesize lipoxins. This eloquent mechanism represents a means by which cells that do not express the full repertoire of LO enzymes can utilize lipid metabolites for pro-inflammatory or anti-inflammatory functions. A third mechanism of synthesis produces the lipoxin epimers 15(R)-lipoxin A₄ and 15(R)-lipoxin B₄, which are also known as aspirin-triggered lipoxins. Aspirin acetylates COX-2, diverting COX-2 away from the production of prostaglandins and thromboxanes and redirecting it towards the production of 15(R)-HETE, a precursor for the formation of the aspirin-triggered LXs 15(R)-LXA₄ and 15(R)-LXB₄, which have a variety of anti-inflammatory and pro-resolving activities. LXs and aspirin-triggered LXs mediate their effects through formyl peptide receptor 2 (FPR2), a GPCR widely expressed in leukocytes as well as intestinal and airway epithelial cells.

Figure 6. Lipoxins can be formed by at least three mechanisms. The first two mechanisms proceed via the dual lipoxygenation of HpETE intermediates whereas the last mechanism occurs by aspirin-mediated acetylation of COX.

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Cytochrome P450 Pathway

The cytochrome P450 (CYP450) pathway is the third branch of the AA cascade and mainly produces epoxyeicosatrienoic acids (EETs) and HETEs (Figure 7). CYPs compromise a large family of membrane-bound enzymes that are expressed in the liver, brain, kidney, and lung as well as the cardiovascular system. CYPs with hydroxylase activity like CYP4A and CYP4F form 20-HETE and to a lesser extent 19-HETE, which have opposing effects on vasodilation. CYPs with epoxygenase activity like CYP2C and CYP2J catalyze the production of the four EET regioisomers 5(6)-, 8(9)-, 11(12)-, and 14(15)-EET, which are generally regarded as having anti-inflammatory and cardioprotective properties. These EETs are converted to their corresponding dihydroxyeicosatrienoic acids (DiHETs) 5(6)-, 8(9)-, 11(12)-, and 14(15)-DiHET by epoxide hydrolases, which significantly reduces their biological activity. Hence, increasing tissue accumulation of EETs by using epoxide hydrolase inhibitors is an attractive option for the treatment of various inflammatory conditions.

Figure 7. CYP450s catalyze the formation of EETs and HETE from AA.

CYP450 Pathway Research Tools Find small molecule inhibitors, isotope-labeled lipids, proteins, antibodies, and assay kits to study the CYP450 pathway.

Products of Non-Enzymatic Reactions

Non-enzymatically derived products of AA include isoprostanes and racemic forms of HpETEs and HETEs. The non-enzymatic peroxidation of AA produces a group of PG-like metabolites known as isoprostanes as well as racemic versions of HpETEs and their corresponding HETEs. A variety of isoprostane, HpETE, and HETE regio- and stereoisomers can be formed, depending on the position of oxygen insertion. As these compounds are formed by reactive oxygen species, they are generally regarded as pro-inflammatory lipid metabolites. Indeed, isoprostanes, especially 8-iso-PGF_2α, have been used as markers of oxidative stress and tissue injury.

HpETEs and their corresponding HETEs exist as racemic mixtures of R- and S-enantiomers (i.e., (±)5-, (±)8-, (±)9-, (±)11-, (±)12-, and (±)15-HETE). In general, the bioactivity of racemic HETEs is attributed to the S-enantiomer. An in-depth discussion on the mechanism of isoprostane and non-enzymatic HETE formation can be found here.

Downstream metabolites of AA can also undergo non-enzymatic reactions. PGE₂ and PGD₂ can undergo non-enzymatic dehydration to the cyclopentenone PGs PGA₂ and PGJ₂, respectively. PGA₂ is sequentially modified non-enzymatically to form PGC₂ and PGB₂. Similarly, PGJ₂ undergoes sequential non-enzymatic conversion into ∆¹²-PGJ₂ and 15-deoxy-∆^12,14-PGJ₂. 15-deoxy-∆^12,14-PGJ₂ is a potent PPARγ agonist that promotes a number of physiological effects, including anti-inflammatory and neuroprotective responses.

Interplay Between the AA Cascade & Endocannabinoids 

Endocannabinoids are endogenous lipids derived from AA that function in anti-inflammatory, neuroprotective, cardioprotective, and metabolic responses. Their effects are mediated mainly through the cannabinoid receptors CB₁ and CB₂. Arachidonoyl ethanolamine (AEA, also known as anandamide) and 2-arachidonoyl glycerol (2-AG) are the most well-known endocannabinoids. AEA and 2-AG can act as substrates for COX-2, producing a range of products mirroring those formed by COX-2-mediated metabolism of AA. The oxygenation of AEA and 2-AG by COX-2 produces prostaglandin ethanolamides (PG-EAs), also known as prostamides, and prostaglandin glyceryl esters (PG-Gs), respectively. Some oxygenated endocannabinoids like PGE₂-EA and PGD₂-G have anti-inflammatory activities, whereas others like PGF_2α-EA and PGE₂-G are regarded as being pro-inflammatory. In addition to COX-2, AEA and 2-AG can also be metabolized by CYP450s (specifically CYP3A4 and CYP2C19) as well as 12- and 15-LO to ethanolamide and glyceryl ester forms of EETs and HETEs (Figure 8).

The biological activities of AEA and 2-AG are terminated by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. FAAH and MAGL degrade AEA and 2-AG, liberating the AA component of these endocannabinoids in the process, making it freely available as substrate for COX-, LO-, or CYP450-mediated metabolism, marking yet another way that endocannabinoids and the AA cascade converge.

Figure 8. Metabolism of endocannabinoids by branches of the AA cascade produce ethanolamide (EA) and glycerol (G) ester forms of PGs, EETs, and HETEs. Catabolism of AEA and 2-AG leads to the release of AA, which acts as a substrate for the COX, LO, or CYP450.

Endocannabinoid Pathway Research Tools Find small molecule inhibitors, isotope-labeled endocannabinoids, proteins, antibodies, and assay kits to study the endocannabinoid pathway.

Upsetting Balance in the AA Cascade

Given the diversity of eicosanoids in several pathologies, small molecule inhibitors targeting COX, LO, and CYP450 pathways have been the focus of many investigations. Because of the significant crosstalk within the AA cascade, it is conceivable that altering one pathway invariably alters the other branches. Inhibition of one branch may shunt AA to a different branch, leading to the production of a different set of lipid metabolites that may oppose the action of those metabolites inhibited, leading to disappointing results. Indeed, unintended consequences can occur from upsetting balance in the AA cascade. Non-selective COX inhibitors inhibit both COX-1 and COX-2. Owing to the homeostatic role of COX-1 in the maintenance of gut epithelial integrity, non-selective COX inhibitors are associated with gastrointestinal side effects. To circumvent this off-target effect, selective COX-2 inhibitors, known as coxibs, were pursued as a means to target inflammatory PGs while sparing homeostatic PGs. Rofecoxib and valdecoxib were two COX-2 inhibitors that were approved - and then withdrawn - from U.S. markets because they were associated with an increased risk of adverse cardiovascular events. Under homeostatic conditions, COX-2 is induced by shear stress in blood vessels and is a key producer of PGI₂, an inhibitor of platelet aggregation and a vasodilator. Rofecoxib and valdecoxib inhibit COX-2-induced PGI₂ but spare COX-1-mediated TXA₂ formation, tipping the balance in the AA cascade toward TXA₂-mediated effects like vasoconstriction and thrombosis (Figure 9). Despite these contraindications, coxibs are a valuable therapeutic tool. Celecoxib is an FDA-approved COX-2 inhibitor approved for use in the treatment of pain and arthritis.

Figure 9. Inhibition of COX-2 shifts balance in the arachidonic acid cascade towards COX-1 mediated production of TXA₂, resulting in pro-thrombotic effects.

Next Steps Towards Therapeutics

Given the critical roles that eicosanoids play in various pathologies, studies that investigate the biological activity of eicosanoids and identify small molecule inhibitors targeting the AA cascade have been the focus of intensive research for decades. Advances in mass spectrometry-based lipidomics approaches are unraveling the complexity of the eicosanoid network in various pathologies. Because of the increasing appreciation for the interplay between branches in the AA cascade, many pharmaceutical efforts are moving away from a "one target" approach in favor of a dual inhibitor design to target multiple arms of the AA cascade.

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