Covalent Modification of Cyclooxygenase-2 (COX-2) by 2-Acetoxyphenyl Alkyl Sulfides, a New Class of Selective COX-2 Inactivators

Abstract

All of the selective COX-2 inhibitors described to date inhibit the isoform by binding tightly but noncovalently at the substrate binding site. Recently, we reported the first account of selective covalent modification of COX-2 by a novel inactivator, 2-acetoxyphenyl hept-2-ynyl sulfide (70) (Science 1998, 280, 1268-1270). Compound 70 selectively inactivates COX-2 by acetylating the same serine residue that aspirin acetylates. This paper describes the extensive structure-activity relationship (SAR) studies on the initial lead compound 2-acetoxyphenyl methyl sulfide (36) that led to the discovery of 70. Extension of the S-alkyl chain in 36 with higher alkyl homologues led to significant increases in inhibitory potency. The heptyl chain in 2-acetoxyphenyl heptyl sulfide (46) was optimum for COX-2 inhibitory potency, and introduction of a triple bond in the heptyl chain (compound 70) led to further increments in potency and selectivity. The alkynyl analogues were more potent and selective COX-2 inhibitors than the corresponding alkyl homologues. Sulfides were more potent and selective COX-2 inhibitors than the corresponding sulfoxides or sulfones or other heteroatom-containing compounds. In addition to inhibiting purified COX-2, 36, 46, and 70 also inhibited COX-2 activity in murine macrophages. Analogue 36 which displayed moderate potency and selectivity against purified human COX-2 was a potent inhibitor of COX-2 activity in the mouse macrophages. Tryptic digestion and peptide mapping of COX-2 reacted with [1-14C-acetyl]-36 indicated that selective COX-2 inhibition by 36 also resulted in the acetylation of Ser516. That COX-2 inhibition by aspirin resulted from the acetylation of Ser516 was confirmed by tryptic digestion and peptide mapping of COX-2 labeled with [1-14C-acetyl]salicyclic acid. The efficacy of the sulfides in inhibiting COX-2 activity in inflammatory cells, our recent results on the selectivity of 70 in attenuating growth of COX-2-expressing colon cancer cells, and its selectivity for inhibition of COX-2 over COX-1 in vivo indicate that this novel class of covalent modifiers may serve as potential therapeutic agents in inflammatory and proliferative disorders.

Inhibition of arachidonic acid metabolism and its implication on cell proliferation and tumour-angiogenesis

“…As previously mentioned, aspirin induces a covalent modification to COX by acetylating residue Ser530 of COX-1 and Ser516 of COX-2 located just below Tyr385 (Figure 7), thereby inhibiting its usual enzyme activity [124,100]. Furthermore, it has been noted that aspirin-acetylated COX-2 is able to synthesize an additional metabolite from AA, namely 15R-HETE, the enantiomer of 15S-HETE formed from AA by 15-LOX.…”

“…The majority of NSAIDs are considered to be competitive inhibitors of COX, since they require the same set of binding site interactions as the natural substrate AA, whereas aspirin is a covalent modifier of COX [100].…”

The origin of 15 R -prostaglandins in the Caribbean coral Plexaura homomalla : Molecular cloning and expression of a novel cyclooxygenase

“…Presently, the only known COXcatalyzed reaction with purely 15R stereospecificity is the biosynthesis of 15R-HETE by aspirin-inhibited COX-2 (24,(35)(36)(37)(38). Aspirin is known to inhibit COX enzymes by acetylating Ser-530 (39,40). In the case of COX-1, this eliminates all oxygenase activity.…”

Oxygenation of the Endocannabinoid, 2-Arachidonylglycerol, to Glyceryl Prostaglandins by Cyclooxygenase-2

“…Apoenzymes were reconstituted with hematin prior to activity assays. COX activity was quantified as described (24). Enzyme kinetics were analyzed by nonlinear regression using the computer program Enzyme Kinetics 1.5 (Trinity Software, Campton, NH).…”