Cyclooxygenases - Part 3: Cyclooxygenase-2-Selective Inhibitors: Translating Pharmacology into Clinical Utility

Discovery

In 1988, the synthesis of a COX-like enzyme was shown to occur in response to interleukin (IL)-1 and bacterial lipopolysaccharide.(13,14) An induced form of COX was described that was immunologically distinct from a constitutive enzyme.(15) It was mitogenesis research, however, that led to the discovery of the COX-2 gene. In a study of gene activation in response to src, an mRNA expressed in Rous sarcoma virus-transformed cells was found that was homologous to COX.(16) COX-2 has been cloned from a variety of species, including humans.(17)

Similarities, differences, and interactions of COX enzymes

The ability of COX isoenzymes to orchestrate complex prostaglandin-mediated physiologic functions reflects an elaborate interplay between the 2 forms of the enzyme. Contributing to this balance are differences in their structure, level of expression, interaction with other enzymes, and feedback regulation.

In all species examined, COX-1 and COX-2 proteins have approximately 60% amino acid sequence identity.(18) The 3-dimensional structure of the COX enzymes are strikingly similar to each other.(19,20) COX isoenzymes are similar in active site structure. Both isozymes have an active site consisting of a hydrophobic channel, and amino acids in this region are nearly identical. Three amino acid differences, however, result in a larger and more accessible channel, in COX-2 (Figure 2).(21) Inside the hydrophobic channel of COX-2, substitution of a valine for isoleucine at residue 523 of COX-2 creates a "side pocket" that selectively allows certain agents to bind and inhibit this enzyme. (22)

Figure 2.
Structure of the COX-1 and COX-2 enzymes. (21)
Schematic showing active site similarities and differences. ILE = isoleucine; VAL - valine.

Although the overall structure and essential catalytic activity of the 2 COX isoenzymes are similar, there are vivid distinctions in their regulation and expression. The 2 enzymes are encoded on different chromosomes, and differ in translational and posttranslational regulation. In general, COX-1 is constitutive, and its expression is regulated by hormonal signals involved in maintaining physiologic homeostasis.

Consistent with the properties of "housekeeping" genes, COX-1 lacks a TATA box.(18) COX-1 is developmentally controlled, and little is known about how COX-1 expression is regulated. COX-1 is expressed in all tissues, albeit at different levels and not necessarily in all cells of a given tissue. Importantly, COX-1 but not COX-2 is constitutively expressed in the stomach, where it is involved in mucosal defense and repair.

Though COX-2 can also be constitutive in some tissues, COX-2 expression and activity is largely responsive to adverse stimuli, such as inflammation and physiologic imbalances. The COX-2 promoter has several putative regulatory regions that bind transcription factors. Although dozens of COX-2 stimulatory factors have been identified, those commonly seen in inflammation, and upregulated in the proinflammatory milieu, are key in regulation of COX-2 signaling pathways. These include transcription factors that respond to bacterial endotoxin, IL-1, and TNF-α such as NFkB, C/EBP, and protein kinases (ERK1/2 and MAPK).(23) The presence of a cyclic adenosine monophosphate (cAMP) response element (CRE) in the COX-2 promoter may allow for COX-2 expression to be directly regulated by feedback from prostaglandins through their influence on cellular cAMP levels.(24) The presence of cytokines stabilizes COX-2 transcripts. Control of COX-2 transcription and translation is thought to be the primary mechanism by which steroids such as cortisol and dexamethasone modulate this enzyme. Post-transcriptional factors also play a role in the expression of COX-2, an immediate early gene, whose expression is controlled by mRNA splicing and translational efficiency.(18,24)

Some prostaglandin-mediated physiologic activities are carried out by only one COX isoenzyme while other activities involve both isoenzymes. For example, COX-1 is essential for thromboxanemediated aggregation of human platelets and parturition, whereas COX-2 is essential to ovulation and nidation.(25) Other processes, such as inflammation and carcinogenesis, are mediated by both COX-1 and COX-2. In inflammation, COX-2 plays dual roles, both initiating and resolving inflammation.

Some of the segregated activities of COX-1 and COX-2 in cells simultaneously expressing both isoenzymes can be explained by the local concentration of arachidonic acid substrate. The level of enzyme expression itself also plays a role. The activity of each isoenzyme may be regulated in other ways. For example, COX-1 is subject to negative allosteric inhibition such that at lower concentrations of arachidonic acid, COX-2 may be exclusively active, despite the presence of COX-1.(23)

COX isoforms differ in their ability to interact with the terminal enzymes of prostaglandin synthesis. (23) For example, in the presence of COX-1, COX- 2 appears to selectively target specific prostaglandin synthases, resulting in a shift from the production of several prostaglandins to a preferential production of PGE2 and prostacyclin.(26)

The initial notion that COX-1 and COX-2 have unique and mutually exclusive functions has evolved to a concept incorporating multiple and complicated physiologic pathways and function. The view of COX-2 as the inducible COX enzyme is an oversimplification. While it is upregulated in response to certain stimuli, COX-2 is expressed constitutively in some tissues. In most tissues where COX-2 is constitutively expressed-notably the brain and kidney-the enzyme is involved in biologic response to physiologic stress. In the kidney, the macula densa is an important component of the renin-angiotensin system that orchestrates sodium balance and fluid volume by monitoring salt concentration. (27) COX-2 is constitutively expressed in the macula densa, and levels there are increased during salt deprivation, suggesting that prostaglandins produced by COX-2 are important in sodium reabsorption in response to volume contraction. (28) In the brain, prostaglandins are involved in nervous system functions such as sleep-waking cycles, fever induction, and pain transmission. While COX-2 is constitutively expressed in the brain, it is also upregulated in parallel with fever and in response to seizures.(29)

Cronstein BN. Cyclooxygenase-2-selective inhibitors: translating pharmacology into clinical utility. Cleve Clin J Med 2002;69:SI13-19.

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