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

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. Furthermore, 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 COXmore » 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.« less

Authors:

Xu, Shu  ^[1];

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Uddin, Md. Jashim ^[1]; Banerjee, Surajit ^[2]; Duggan, Kelsey ^[1]; Musee, Joel ^[1]; Kiefer, James R. ^[3]; Ghebreselasie, Kebreab ^[1]; Rouzer, Carol A. ^[1]; Marnett, Lawrence J. ^[1]

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+ Show Author Affiliations

1. Vanderbilt Univ. School of Medicine, Nashville, TN (United States)
2. Cornell Univ., Ithaca, NY (United States); Argonne National Lab. (ANL), Argonne, IL (United States). Northeastern Collaborative Access Team
3. Genentech, Inc., South San Francisco, CA (United States)

Publication Date:

Thu Apr 18 00:00:00 EDT 2019

Research Org.:

Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Org.:

National Institutes of Health (NIH); National Institute of General Medical Sciences (NIGMS); USDOE Office of Science (SC)

OSTI Identifier:

1530995

Grant/Contract Number:  

P30 GM124165; S10 RR029205; AC02-06CH11357; GM15431; CA089450

Resource Type:

Accepted Manuscript

Journal Name:

Journal of Biological Chemistry

Additional Journal Information:

Journal Volume: 294; Journal Issue: 22; Journal ID: ISSN 0021-9258

Publisher:

American Society for Biochemistry and Molecular Biology

Country of Publication:

United States

Language:

ENGLISH

Subject:

59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; cyclooxygenase (COX); enzyme structure; protein drug interaction; X-ray crystallography; enzyme kinetics; anticancer drug; inflammation chemotherapy; prostaglandin endoperoxide synthase