|Abstract:||Lipid mediators are a recently discovered class of endogenous molecules that perform a variety of functions in the body. Specifically, polyunsaturated fatty acids (PUFAs) are transformed by three main pathways: the cyclooxygenase, lipoxygenase, and cytochrome P450 (CYP) epoxygenase pathways. Arachidonic acid is the canonical lipid mediator and it is transformed by the cyclooxygenase and lipoxygenase pathways into pro-inflammatory mediators at the onset of inflammation. For this reason, many drugs, particularly NSAIDs, have been developed to combat inflammation by inhibiting these enzymes. The CYP pathway, contrariwise, converts arachidonic acid into anti-inflammatory epoxide mediators known as epoxyeicosatrienoic acids (EETs). Additionally, these compounds regulate cardiovascular function, and importantly EETs are cardioprotective. The cyclooxygenase and lipoxygenase pathways were discovered first and have been extensively studied. There currently is not a lot known regarding the CYP pathway and how it is regulated. The CYPs responsible for producing EETs metabolize other PUFAs, lipids, and drugs. Complex substrate-substrate interactions have been observed with drug-metabolizing CYPs and is thought to mediate some of the known drug-drug interactions. Therefore, the aim of this work is to gain an enzymological understanding into lipid-drug interactions concerning the predominant CYP epoxygenase in the heart, CYP2J2. We aim to understand how multiple-ligand binding events effect the endogenous lipid metabolism by CYP2J2 in order to understand how they may effect cardiovascular and cerebrovascular function. We have developed a combined approach of kinetic and in silico molecular dynamic (MD) analysis in order to determine the nuances of multiple ligands binding to CYP2J2.
CYP2J2 is the most highly expressed CYP of the heart and has high levels of expression in the brain and endothelial tissues. All CYPs are membrane-bound proteins requiring the assistance of a membrane-bound redox partner, cytochrome P450 reductase (CPR). The hydrophobicity of CYP2J2, CPR, and the substrates have made studying this system challenging. We have been able to circumvent many of these challenges by recombinantly expressing CYP2J2 and CPR in E. coli and incorporating them into Nanodiscs. Nanodiscs are a membrane mimic composed of a lipid bilayer with two membrane scaffold proteins shielding the hydrophobic core. We then developed LC-MS/MS detection methods in order to quantify EETs and other lipid mediators. In Chapter 2, we study how arachidonic acid competes for metabolism by CYP2J2 within a pool of PUFA substrates. In Chapter 3, we investigate how a cardiotoxic drug, doxorubicin, modulates arachidonic acid metabolism by CYP2J2.
PUFAs can be further transformed in the body into endocannabinoids, typically by conjugating the carboxylate headgroups with a variety of biological amines. These endocannabinoids modulate many of the functions associated with cannabinoids derived from marijuana (phytocannabinoids), including appetite, mood, and inflammation. Endocannabinoids are also substrates of CYP2J2, and the epoxide metabolites of these endocannabinoids have been shown to be anti-inflammatory. In Chapter 4, we determine that phytocannabinoids from cannabis plant are substrates of CYP2J2 and inhibit CYP2J2 mediated endocannabinoid metabolism. Particularly, Δ9-THC, the most psychoactive and often the most predominant cannabinoid in marijuana, potently shuts down endocannabinoid metabolism through a non-competitive model. In Chapter 5, we characterize a new class of endocannabinoids called endovanilloid epoxides. These molecules are derived from the conjugation of arachidonic acid with dopamine and serotonin and bind to the transient receptor potential vanilloid 1 (TRPV1). We determine that endovanilloids are also substrates of CYP2J2. The epoxides of these endovanilloids are potently anti-inflammatory and have distinct properties compared to the parent compounds. Particularly, an epoxide of N-arachidonoyl-serotonin is a potent TRPV1 antagonist and cannabinoid receptor 1 agonist, thereby making it a promising molecule to regulate pain and inflammation. Finally, we determine that the endocannabinoid, anandamide, potentiates endovanilloid metabolism in BV2 microglia and directly through substrate-substrate interactions in CYP2J2.
From these studies, we can build a map of substrates binding to CYP2J2 in the active and understand how multiple-ligand binding events modulate CYP2J2 activity. We are able to see how ligands compete, inhibit, or potentiate the binding of other ligands to CYP2J2 (Chapter 6).