Structural and biophysical analysis of the allosteric mechanisms regulating substrate oxygenation in the cyclooxygenase-2 dimer
Orlando, Benjamin J.
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The cyclooxygenase enzymes (COX-1 and COX-2) have remained the focus of intense research for greater than four decades. As the targets of nonsteroidal anti-inflammatory drugs (NSAIDs), the COX enzymes are recognized as two of the most common drug targets in modern history. Recent evidence has demonstrated that the COX-2 dimer functions with half of sites reactivity, and that certain NSAIDs inhibit COX-2 via an allosteric mechanism. However, the molecular details of allosteric regulation between monomers of COX-2 have remained largely elusive. The realization that certain NSAIDs selectively and allosterically inhibit endocannabinoid oxygenation by COX-2 has generated a strong impetus to fully understand the allosteric mechanisms governing substrate oxygenation in COX-2. The primary goal in this dissertation was to understand at the molecular level the mechanisms of allosteric regulation in COX-2. Through the use of x-ray crystallography, electron spin resonance, and various biochemical and biophysical assays, a more complete understanding of allosteric regulation and substrate selective inhibition has emerged. The research described herein demonstrates that allosteric regulation of endocannabinoid oxygenation is based on the catalytic mechanism of substrate oxygenation in COX-2, and the electron transfer properties of certain NSAIDs. These results have important implications for the design of the next generation of pharmaceuticals targeted at augmenting endocannabinoid levels through the inhibition of COX-2. We also provide a method for incorporating COX-2 into lipid bilayer nanodiscs, critical insight into the effect of detergent solubilization on the inhibition of COX-2 by NSAIDs, and the first publicly available crystal structures of human COX-2.