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

Nature Chemical Biology volume 7, pages 803–809 (2011)Cite this article

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Abstract

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.

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Figure 1: Crystal structure of (R)-naproxen within the mCOX-2 active site.
Figure 2: Binding of (R)-flurbiprofen within the mCOX-2 active site.
Figure 3: Analysis of DRGs.
Figure 4: Inhibition of eicosanoid synthesis in stimulated DRGs by (R)-flurbiprofen, (R)-naproxen and (R)-ibuprofen.
Figure 5: Comparison of the effects of (R)-flurbiprofen, (R)-naproxen and (R)-ibuprofen on substrate concentrations in basal versus stimulated DRGs.
Figure 6: The mechanism of COX-2 substrate-selective inhibition of endocannabinoid oxygenation by rapid, reversible inhibitors.

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Acknowledgements

This work was supported by the A.B. Hancock Jr. Memorial Laboratory for Cancer Research and by research (CA89450, GM15431, NS064278) and training grants (DA022873, DA031572) from the US National Institutes of Health. It is based upon research conducted at the Advanced Photon Source on the Northeastern Collaborative Access Team beamlines, which are supported by award RR-15301 from the National Center for Research Resources at the US National Institutes of Health. Use of the Advanced Photon Source is supported by the US Department of Energy, Office of Basic Energy Sciences, under contract no. DE-AC02-06CH11357. We are grateful to J. Harp for assistance with crystallography; K. Masuda, M. Brown, R. Stevens and B. Cravatt for a sample of FAAH; A. Brash for a sample of 15-lipoxygenase and J. Uddin for a sample of fluorocoxib A.

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Author notes

  1. Kelsey C Duggan and Daniel J Hermanson: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, USA

    Kelsey C Duggan, Joel Musee, Jeffery J Prusakiewicz, Jami L Scheib, Bruce D Carter & Lawrence J Marnett

  2. Department of Chemistry, Vanderbilt University, Nashville, Tennessee, USA

    Daniel J Hermanson & Lawrence J Marnett

  3. Northeastern Collaborative Access Team and Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York, USA, Argonne National Laboratory.,

    Surajit Banerjee

  4. Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, USA

    J A Oates

  5. Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA

    Lawrence J Marnett

  6. Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee, USA

    Lawrence J Marnett

  7. Vanderbilt Institute of Chemical Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA

    Lawrence J Marnett

  8. Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA

    J A Oates & Lawrence J Marnett

  9. Argonne National Laboratory, Argonne, Illinois, USA

    Surajit Banerjee

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Contributions

J.J.P., K.C.D. and L.J.M. originated the project. K.C.D. performed all in vitro wild-type COX-2 inhibition experiments. D.J.H. performed all in vitro mutant COX-2 inhibition experiments. K.C.D., J.M. and S.B. determined the COX-2–(R)-naproxen crystal structure, and K.C.D. and S.B. determined the (R)-flurbiprofen crystal structure. Primary DRGs were harvested and cultured by J.L.S. and D.J.H. in the laboratory of B.D.C. D.J.H. designed and executed COX-2 inhibition experiments in DRGs as well as the imaging studies. D.J.H. and J.L.S. performed western blot analysis of DRGs. J.A.O. reviewed the data and offered critical commentary, and L.J.M. oversaw the research and wrote the manuscript, which was reviewed and edited by all authors.

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Correspondence to Lawrence J Marnett.

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Duggan, K., Hermanson, D., Musee, J. et al. (R)-Profens are substrate-selective inhibitors of endocannabinoid oxygenation by COX-2. Nat Chem Biol 7, 803–809 (2011). https://doi.org/10.1038/nchembio.663

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