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Controlling Endocannabinoid Metabolism

 

By: Carol A. Rouzer, VICB Communications
Published:  February 11, 2015

 

 

The discovery of an endogenous cannabinoid metabolism potentiator sets the stage for new understanding of the regulation of cannabinoid signaling.

 

The endogenous cannabinoids (endocannabinoids), arachidonoyl ethanolamide (AEA) and arachidonoylglycerol (2-AG), are important lipid signaling molecules. They act at the cannabinoid receptors, CB1 and CB2, which also mediate the pharmacological effects of marijuana. Endocannabinoid action is terminated primarily by hydrolysis of AEA and 2-AG to yield their parent fatty acid, arachidonic acid (AA). However, recent studies show that oxygenation of endocannabinoids by the enzyme cyclooxygenase-2 (COX-2) may also be important in the regulation of endocannabinoid tone in vivo. Now, Vanderbilt Institute of Chemical Biology member Larry Marnett and his laboratory report the discovery of a novel potentiator of endocannabinoid oxygenation by COX-2, providing a vital new tool for the exploration of this interesting pathway [S. N. Kudalkar, et al. (2015) J. Biol. Chem., published online February 2, doi:10.1074/jbc.M114.634014].

 

The cyclooxygenase enzymes (COX-1 and COX-2) catalyze the first and second steps in the biosynthesis of a class of oxygen-containing lipid signaling molecules known as prostaglandins (PGs). The product of the COX reaction, PGH2, is further metabolized by other enzymes to yield five distinct final products (Figure 1). In addition, COX-2 can also use some ester and amide derivatives of AA, including AEA and 2-AG, as substrates. As shown in Figure 1, COX-2-mediated metabolism of 2-AG produces the glyceryl ester of PGH2 (PGH2-G), which then gives rise to four distinct glyceryl ester final products. The importance of PG biosynthesis in a range of physiological processes is highlighted by the fact that the widely used nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin, ibuprofen, and naproxen, act primarily by blocking the activity of the COX enzymes. In contrast, the role of COX-2-mediated endocannabinoid oxygenation is not fully understood, but increasing evidence suggests that it contributes to the modulation of endocannabinoid tone in the central nervous system.

 

 


Figure 1. (Left) Formation of prostaglandins (PGs) and thromboxane A2 (TXA2) from arachidonic acid. The cyclooxygenase enzymes catalyze the first two steps in the pathway, converting arachidonic acid to PGH2. Other enzymes then take over, producing four distinct PGs and TXA2. (Right) Arachidonoylglycerol is selectively metabolized by COX-2, which produces the glyceryl ester of PGH2 (PGH2-G). PGH2-G is then subject to further metabolism by all the same enzymes as PGH 2 with the exception of the enzyme that produces TXA2. The final products are prostaglandin glyceryl esters (PG-Gs).

 


COX-1 and COX-2 are homodimers, each comprising two subunits that are identical with regard to amino acid composition and overall structure. However, both enzymes behave as heterodimers, meaning that the two subunits appear to have different functions. Substantial evidence indicates that only one of the subunits contains the heme cofactor critical for enzymatic activity. Thus, this subunit is designated the catalytic subunit while the second subunit plays a regulatory role and is designated the allosteric subunit. Prior studies have demonstrated that various fatty acids that are not COX substrates can potentiate enzymatic activity through binding to the allosteric subunit. In addition, some NSAIDs interact strongly with the allosteric subunit of COX-2, resulting in inhibition of PG-G synthesis from 2-AG without having any effect on PG synthesis from AA. This phenomenon is known as substrate-selective inhibition (Figure 2).

 


Figure 2.
Mechanism of substrate-selective inhibition. (A) When 2-AG binds to the catalytic subunit of COX-2 (blue), the 2-AG is converted to PG-Gs. However, binding of a substrate-selective inhibitor (I) to the allosteric subunit leads to formation of a complex that is inactive with regard to PG-G formation. (B). When AA binds to the catalytic subunit of COX-2, the AA is converted to PGs. Binding of a substrate-selective inhibitor (I) to the allosteric subunit has no effect on PG formation from AA. However, the inhibitor may also bind to the catalytic site of COX-2, which competitively blocks PG production. Binding of inhibitor to the catalytic site is much weaker and requires higher concentrations than binding to the allosteric site. Thus, PG-G synthesis is much more readily blocked than PG synthesis.

 

 

AM-8138 (Figure 3) is the first example of a molecule that selectively potentiates PG-G synthesis while having no effect on PG synthesis. Its discovery was the result of a screen of AA analogs as substrates and/or modulators of COX-2 activity. AM-8138 was not a substrate for the enzyme, and it had no effect at all on PG formation from AA. However, it increased PG-G production from 2-AG, by up to five-fold, with maximal effects occurring at a concentration of 10 μM. X-Ray crystallography of a complex of COX-2 with AM-8138 revealed that the molecule binds in an “upside-down” orientation with regard to the productive binding mode of AA in the active site. It is interesting to note, however, that crystal structures of complexes of COX-2 and AA reveal the productive conformation of AA in only one monomer, while in the second monomer, AA adopts an upside-down orientation similar to that of AM-8138.

 

 

 

Figure 3.  Structure of AM-8138, 13(S)-methyl-arachidonic acid.

 

 

The crystal structure studies revealed amino acid residues of COX-2 that appear to be important for AM-8138 binding. The Marnett lab investigated the effects of mutation of these residues on the ability of AM-8138 to potentiate 2-AG oxygenation. These studies revealed that some mutations of tyrosine-355 and arginine-120 markedly suppressed PG-G formation while having little effect on PG synthesis. Surprisingly, AM-8138 completely restored 2-AG oxygenation in these mutants. In fact, most of the mutants investigated remained responsive to AM-8138, suggesting that the modified amino acids were not critical to its potentiating effects. The only exceptions were mutants of leucine-531. Most of these lost both AA and 2-AG oxygenating activity and were unresponsive to AM-8138.  The crystal structure revealed that binding of AM-8138 to COX-2 requires a rotation of the side chain of leucine-531. These results suggest that the position of this residue in the COX-2 active site is critical both for catalytic activity and for AM-8183 function.

 

Kinetic studies of wild-type COX-2 demonstrated that the enzyme is subject to substrate inhibition by 2-AG. This finding suggests that, at high concentrations, 2-AG binds to the allosteric site and suppresses its own oxygenation at the catalytic site. Addition of AM-8138 had two effects on PG-G biosynthesis. It reduced substrate inhibition, presumably by competing with 2-AG for binding to the allosteric site, and it raised the catalytic constant (kcat) for 2-AG oxygenation (Figure 4). This was also true for the mutant enzymes that responded to AM-8138-dependent potentiation.  The ability of AM-8138 to bind at the allosteric site was further supported by the finding that it could block the inhibitory effects of substrate-selective inhibitors of COX-2.

 

 

Figure 4. Mechanism of the allosteric potentiation of 2-AG oxygenation by AM-8138. In the absence of AM-8138, binding of 2-AG to the catalytic site of COX-2 alone leads to PG-G formation. At high concentrations, 2-AG can also bind to the allosteric site of COX-2, resulting in the formation of an inactive complex. In contrast, if AM-8138 is present, it binds to the allosteric site of COX-2, preventing the binding of 2-AG to that site and creating a complex that converts 2-AG to PG-Gs more efficiently than in its absence.

 

 

While 2-AG is metabolized almost as efficiently as AA by COX-2 in vitro, it is a very poor substrate for COX-1. Thus, the Marnett lab investigators were surprised to find that AM-8183 potentiated the oxygenation of 2-AG by COX-1 nearly as well as by COX-2. In fact, in the presence of AM-8138, the kinetic constants for the oxygenation of 2-AG by COX-1 were nearly the same as for COX-2 in the absence of AM-8183.

 

To see if AM-8138 could potentiate 2-AG oxygenation in cells, the investigators preincubated RAW264.7 macrophage-like cells with the potentiator, followed by Kdo2-lipidA, an inducer of COX-2 expression, and finally ionomycin to stimulate PG and PG-G biosynthesis. Cells pretreated with AM-8138 produced substantially more PG-Gs than those not exposed to the potentiator, while PG synthesis was unaffected. AM-8138 had no effect on the levels of AA or 2-AG in the cells.

 

Together, the results demonstrate that allosteric regulation of COX-2 or COX-1 can selectively increase PG-G biosynthesis. These findings reveal new insights into the allosteric regulation of COX-2 and provide a valuable tool for the exploration of PG-G biosynthesis in the cellular environment. They also raise the interesting possibility that similar potentiators are produced endogenously in vivo to regulate the activity of the COX enzymes for their various substrates. Accumulating evidence suggests that metabolism of endocannabinoids by COX-2 regulates neural functions related to emotional states. Thus, a better understanding of processes that regulate COX-2’s ability to metabolize endocannabinoids may lead to better approaches to treat psychiatric disease related to anxiety and depression.

 

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