Fluorescent indomethacin-dansyl conjugates utilize the membrane-binding domain of cyclooxygenase-2 to block the opening to the active site.

Many indomethacin amides and esters are cyclooxygenase-2 (COX-2)-selective inhibitors, providing a framework for the design of COX-2-targeted imaging and cancer chemotherapeutic agents. Although previous studies have suggested that the amide or ester moiety of these inhibitors binds in the lobby region, a spacious alcove within the enzyme's membrane-binding domain, structural details have been lacking. Here, we present observations on the crystal complexes of COX-2 with two indomethacin-dansyl conjugates (compounds 1 and 2) at 2.22-Å resolution. Both compounds are COX-2-selective inhibitors with IC50 values of 0.76 and 0.17 µm, respectively. Our results confirmed that the dansyl moiety is localized in and establishes hydrophobic interactions and several hydrogen bonds with the lobby of the membrane-binding domain. We noted that in both crystal structures, the linker tethering indomethacin to the dansyl moiety passes through the constriction at the mouth of the COX-2 active site, resulting in displacement and disorder of Arg-120, located at the opening to the active site. Both compounds exhibited higher inhibitory potency against a COX-2 R120A variant than against the WT enzyme. Inhibition kinetics of compound 2 were similar to those of the indomethacin parent compound against WT COX-2, and the R120A substitution reduced the time dependence of COX inhibition. These results provide a structural basis for the further design and optimization of conjugated COX reagents for imaging of malignant or inflammatory tissues containing high COX-2 levels.

Prostaglandin H synthase-2-catalyzed oxygenation of 2-arachidonoylglycerol is more sensitive to peroxide tone than oxygenation of arachidonic acid.

The endocannabinoid, 2-arachidonoylglycerol (2-AG), is a selective substrate for the inducible isoform of prostaglandin H synthase (PGHS), PGHS-2. Its turnover leads to the formation of glyceryl esters of prostaglandins (PG-Gs), a subset of which elicits agonism at unique, as yet unidentified, receptors. The k(cat)/K(m) values for oxygenation of arachidonic acid (AA) and 2-AG by PGHS-2 are very similar, but the sensitivities of the two substrates to peroxide-dependent activation have not been compared. 15-Hydroperoxy derivatives of AA and 2-AG were found to be comparable in their ability to serve as substrates for the peroxidase activities of PGHS-2, PGHS-1, and glutathione peroxidase (GPx). They also were comparable in the activation of AA oxygenation by cyanide-inhibited PGHS-2. However, oxygenation of 2-AG was significantly suppressed relative to AA by the presence of GPx and GSH. Furthermore, 2-AG oxygenation by peroxidase-deficient H388YmPGHS-2 was much less efficient than AA oxygenation. Wild-type rates of 2-AG oxygenation were restored by treatment of H388YmPGHS-2 with hydroperoxide derivatives of AA or 2-AG. RNAi silencing of phospholipid hydroperoxide-specific GPx (GPx4) in NIH/3T3 cells led to increases in cellular peroxidation and in the levels of the isoprostane product, 8-epi-PGF(2α). GPx4 silencing led to 2-4-fold increases in PG-G formation but no change in PG formation. Thus, cellular peroxide tone may be an important determinant of the extent of endocannabinoid oxygenation by PGHS-2.

(R)-Profens are substrate-selective inhibitors of endocannabinoid oxygenation by COX-2.

Cyclooxygenase-2 (COX-2) catalyzes the oxygenation of arachidonic acid and the endocannabinoids 2-arachidonoylglycerol and arachidonoylethanolamide. Evaluation of a series of COX-2 inhibitors revealed that many weak competitive inhibitors of arachidonic acid oxygenation are potent inhibitors of endocannabinoid oxygenation. (R) enantiomers of ibuprofen, naproxen and flurbiprofen, which are considered to be inactive as COX-2 inhibitors, are potent 'substrate-selective inhibitors' of endocannabinoid oxygenation. Crystal structures of the COX-2­(R)-naproxen and COX-2­(R)-flurbiprofen complexes verified this unexpected binding and defined the orientation of the (R) enantiomers relative to (S) enantiomers. (R)-Profens selectively inhibited endocannabinoid oxygenation by lipopolysaccharide-stimulated dorsal root ganglion (DRG) cells. Substrate-selective inhibition provides new tools for investigating the role of COX-2 in endocannabinoid oxygenation and a possible explanation for the ability of (R)-profens to maintain endocannabinoid tone in models of neuropathic pain.

Molecular basis for cyclooxygenase inhibition by the non-steroidal anti-inflammatory drug naproxen.

Naproxen ((S)-6-methoxy-α-methyl-2-naphthaleneacetic acid) is a powerful non-selective non-steroidal anti-inflammatory drug that is extensively used as a prescription and over-the-counter medication. Naproxen exhibits gastrointestinal toxicity, but its cardiovascular toxicity may be reduced compared with other drugs in its class. Despite the fact that naproxen has been marketed for many years, the molecular basis of its interaction with cyclooxygenase (COX) enzymes is unknown. We performed a detailed study of naproxen-COX-2 interactions using site-directed mutagenesis, structure-activity analysis, and x-ray crystallography. The results indicate that each of the pendant groups of the naphthyl scaffold are essential for COX inhibition, and only minimal substitutions are tolerated. Mutation of Trp-387 to Phe significantly reduced inhibition by naproxen, a result that appears unique to this inhibitor. Substitution of S or CH(2) for the O atom of the p-methoxy group yielded analogs that were not affected by the W387F substitution and that exhibited increased COX-2 selectivity relative to naproxen. Crystallization and x-ray analysis yielded structures of COX-2 complexed to naproxen and its methylthio analog at 1.7 and 2.3 Å resolution, respectively. The combination of mutagenesis, structure analysis, and x-ray crystallography provided comprehensive information on the unique interactions responsible for naproxen binding to COX-2.

Peroxidase active site activity assay.

Cyclooxygenase enzymes house spatially distinct cyclooxygenase- and peroxidase-active sites. The two-electron reduction of peroxides to their corresponding alcohols by the heme bound in the peroxidase-active site converts the heme to a ferryloxoprotoporyphrin cation radical, with a reductant providing the two electrons necessary to bring the heme back to its resting state. The ferryloxoprotoporyphrin cation radical can abstract a hydrogen atom from a tyrosine residue in the cyclooxygenase-active site, activating the oxygenase functionality. The tyrosyl radical subsequently abstracts a hydrogen atom from the cyclooxygenase substrate, arachidonic acid, leading to its oxygenation and the formation of a hydroperoxy endoperoxide intermediate, PGG(2). The peroxidase functionality reduces PGG(2) to the hydroxy endoperoxide, PGH(2), which serves as the precursor to downstream prostaglandins and thromboxane. The peroxidase activity of cycloxygenase enzymes can be assayed by quantifying the oxidation of a peroxidase reductant or the reduction of a hydroperoxide substrate. Here we describe a spectrophotometric assay used to measure the oxidation of a reductant, 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), as well as a high-performance liquid chromatography method for the measurement of the conversion of 5-phenyl-4-pentyl hydroperoxide (PPHP) to its corresponding alcohol. The first provides a continuous but indirect assay of peroxidase activity, whereas the second provides a discontinuous but direct assay.

Antiviral agents alter ability of HSV-2 to disrupt gap junctional intercellular communication between mammalian cells in vitro.

In cultured mammalian cells (Vero), different antiviral agents change to differing degrees the ability of HSV2 to down-regulate gap junctions, each agent having a specific effect. Measured by intracellular electrodes, control cell populations showed 49-51% coupling, uninfected populations treated with acyclovir or SDS averaged 43-51% coupling while populations infected with HSV2 had coupling reduced to 8%. The antiviral agent acyclovir (1 microg/ml), which suppresses viral replication, failed to prevent this down regulation (final coupling ratio of 11%). A plant extract (250 microg/ml) from Pilostigma thonningii offered slightly more protection (final coupling ratio of 22%), while sodium dodecyl sulfate (SDS) (50 microM) afforded nearly complete protection (final coupling ratio of 40%). With SDS there was an initial down regulation to only 16% coupling by 3 h post infection, followed by a recovery of intercellular communication to near control levels by 24 h. While SDS was originally believed to alter the viral coat and prevent entry into the cell, our data are in agreement with recent studies which indicate that SDS treated viruses can enter into host cells, but in a severely diminished condition. Our data also suggest that the gap junction antagonist is brought into the cells as part of the entering virus.