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Title page for ETD etd-03202018-132825


Type of Document Dissertation
Author Goodman, Michael Christopher
Author's Email Address michael.c.goodman@vanderbilt.edu
URN etd-03202018-132825
Title Investigations of the enzymatic mechanisms and inhibition of prostaglandin E2 biosynthesis
Degree PhD
Department Chemistry
Advisory Committee
Advisor Name Title
Lawrence J Marnett Committee Chair
Charles R Sanders Committee Member
Hassane S Mchaourab Committee Member
John A McLean Committee Member
Keywords
  • prostaglandins
  • cyclooxygenase
  • Inflammation
  • enzyme kinetics
Date of Defense 2018-03-15
Availability unrestricted
Abstract
Polyunsaturated fatty acids can be liberated from phospholipids in the membrane bilayer and enzymatically converted to oxygenated bioactive lipid compounds that contribute to the pathology, or in some cases, the resolution of inflammation. One of the most well-studied enzymatic pathways, the cyclooxygenase (COX) pathway, converts arachidonic acid (AA) to prostaglandins, prostacyclin, and thromboxane. The first step of this pathway, catalyzed by the COX enzymes is the enzymatic oxygenation of AA to produce prostaglandin H2, which serves as a substrate for various prostanoid synthases, including microsomal prostaglandin E2 synthase 1 (MPGES1). Consistent with their role in inflammation, the COX enzymes are therapeutic targets of the non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin and ibuprofen. In recent years, MPGES1 has become another therapeutic target to selectively inhibit production of the pro-inflammatory prostaglandin E2 (PGE2).

The studies described herein further investigate the molecular mechanisms of two enzymes involved in the biosynthesis of PGE2. COX-2 is a COX isoform whose expression is induced in the presence of various mediators such as cytokines, growth factors, and tumor promoters. Similarly, MPGES1 is induced by pro-inflammatory compounds and is co-localized with COX-2 in the perinuclear membrane and the endoplasmic reticulum. This coupled localization is associated with the induced production of PGE2.

Experiments sought to investigate the molecular basis of substrate-selective inhibition of COX-2 by the NSAIDs ibuprofen and mefenamic acid. The studies here suggest that inhibition by mefenamic acid is more dependent on blockade of peroxide-dependent enzyme activation than is inhibition by ibuprofen. Further inhibitor kinetics were investigated with a COX-2/fatty acid amide hydrolase (FAAH) dual inhibitor, ARN2508. Here, the (S)-enantiomer of ARN2508 was crystallized in complex with COX-2, and a unique binding mode, not observed for any previous NSAID, was observed.

For the investigation of MPGES1, it was revealed that two residues, Asp-49 and Arg-126, are crucial for activity, whereas Ser-127 is not. Based on these studies and further molecular refinement of the crystal structure, the molecular dynamics of the enzyme are better understood, setting the stage for the design of more potent MPGES1 inhibitors.

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