Abstract
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.
Highlights
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
Whereas COX-1 is constitutively expressed in most tissues, expression of COX-2 is strongly induced in inflammatory and malignant sites. This property of COX-2 has led to the hypothesis that it can serve as a target for molecular imaging of cancer and/or inflammation [11]. Testing this hypothesis has been facilitated by the finding that many carboxylic acid– containing, isoform-nonselective nonsteroidal anti-inflammatory drugs (NSAIDs) can be converted to COX-2–selective inhibitors by esterification or amidation of the carboxyl group
In 1996, Luong et al [29] published the crystal structure of COX-2 complexed with an inhibitor comprising zomepirac attached to a p-iodophenyl group via an acyl sulfonamide linker. This COX-2–selective inhibitor had been created by amidation of the carboxyl group of the nonselective NSAID zomepirac in a similar fashion to our creation of COX-2– selective inhibitors by amidation or esterification of indomethacin
Summary
We initiated our studies by reassessing the activities of compounds 1 and 2 against both WT COX isoforms using an assay in which inhibitors were preincubated with enzyme for 15 min prior to addition of AA. Crystal structure data revealed that the Trp-89 residue fills a gap between helixes B and D of the MBD, changing the opening of the lobby from a C-shaped partial ring to a fully closed donut shape This mutation had minimal effect on the potency of indomethacin, we hypothesized that it might alter the potency of compounds 1 and/or 2 due to the positioning of the dansyl moiety within the lobby and formation of contacts with this residue. Mutation of Val-523 in COX-2 to isoleucine leads to a loss of potency of the diarylheterocycle inhibitors This mutation had minimal effect on the potency of indomethacin, compound 1, or compound 2, consistent with the structural data indicating that these inhibitors do not rely on the side pocket for binding (Table 1). The inhibition rate was too rapid for accurate evaluation of the binding or kinetic constants for compound 2’s interaction with R120A; these results suggest that the high potency of compound 2 for R120A COX-2 likely are the result of a much higher affinity for formation of the initial complex and/or a much more rapid conversion to the tightly bound complex
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