Type of Document Dissertation Author Hermanson, Daniel John URN etd-03212014-154440 Title Substrate-selective Inhibition of Cyclooxygenase-2: Molecular Determinants, Probe Development, and In Vivo Effects Degree PhD Department Chemistry Advisory Committee
Advisor Name Title Lawrence J. Marnett Committee Chair Bruce D. Carter Committee Member H. Alex Brown Committee Member Ned A. Porter Committee Member Sachin Patel Committee Member Keywords
Date of Defense 2014-03-14 Availability unrestricted AbstractSubstrate-selective Inhibition of Cyclooxygenase-2: Molecular Determinants, Probe Development, and In Vivo Effects.
Daniel John Hermanson
Dissertation under the direction of Professor Lawrence J. Marnett
Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid (AA) to form prostaglandins (PGs), but also inactivates endocannabinoids (eCBs) in vitro. We have identified and characterized a series of novel probes termed “substrate-selective” COX-2 inhibitors (SSCIs) that selectively inhibit eCB oxygenation with no impact on PG production. Using X-ray crystallography and site-directed mutagenesis we have elucidated the binding determinants of a series of SSCIs. The phenomenon of substrate-selective COX-2 inhibition arises from the fact that although COX-2 is a structural homodimer, it is a functional heterodimer. Kinetic analyses have revealed that substrate-selective COX-2 inhibitors bind in the allosteric COX-2 subunit and act as non-competitive inhibitors of eCB oxygenation in the catalytic subunit through a conformational change involving Leu-531.
We have explored the biological impact of SSCIs in multiple settings including stimulated dorsal root ganglion cells and the brain. These studies established that SSCIs act in a COX-2-dependent manner to decrease the production of eCB-derived PGs and increase the levels of eCBs while having no impact on PG production or AA levels. Importantly, the biochemical effects of SSCIs are unique relative to other modes of COX inhibition. Augmenting eCBs in the brain with SSCIs leads to cannabinoid receptor 1 activation and anxiolytic effects in multiple pre-clinical models of anxiety. Thus, COX-2 is a critical regulator of eCB signaling in vivo.
We have identified AA as a non-competitive inhibitor of eCB oxygenation by COX-2 in vitro. Inhibition of AA production in simulated macrophages through pharmacological or genetic inhibition of cytosolic phospholipase A2 (cPLA2) leads to a robust decrease in AA and PGs with increased eCB-derived PGs. Lipidomic analyses identified that this is due to enrichment of AA-containing phosphatidylinositols, diacylglycerols, and 2-arachidonoyl glycerol upon pharmacological or genetic inhibition of cPLA2. These studies classify the determinants of the oxygenation of AA and eCBs by COX-2 in stimulated macrophages.
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