Role of Intracellular Lipid Binding Proteins in Xenobiotic and Retinoid Metabolism and Disposition

Abstract

Intracellular lipid binding proteins (iLBPs) are a family of small (~15 kDa) β-barrel proteins that bind and solubilize diverse endogenous lipids essential for life. iLBPs serve as cytosolic carriers facilitating the uptake of endogenous ligands into cells and targeting ligands to cellular sites of action. Xenobiotics have also been shown to bind to iLBPs, however, whether iLBPs influence xenobiotic disposition is poorly understood. Retinoid binding proteins and fatty acid binding proteins comprise the family of iLBPs. Retinoid binding proteins have high specificity toward the essential vitamin, A, and its metabolites (retinoids) and have been shown to channel retinoids directly to metabolic enzymes. Cellular retinoic acid binding proteins (CRABP1 and CRABP2) specifically interact with the cytochrome P450 (CYP) 26 family of enzymes to regulate the metabolism of the active form of vitamin A, all-trans-retinoic acid (atRA). Liver fatty acid binding protein (FABP1) is a highly expressed iLBP in the liver (0.7-1 mM) which is a major organ involved in the metabolic clearance of drugs. Yet, the role of FABP1 in the metabolism and disposition of drugs has not been defined, and it is unknown if FABP1 interacts with CYP enzymes in the liver to facilitate drug metabolism, similar to what has been observed for retinoid binding proteins. The overarching hypothesis for this thesis work was that CRABPs and FABPs facilitate the metabolism of their ligands via interactions with CYP enzymes in the liver. In this thesis work, I aimed to 1) define the interactions between CRABPs and CYP26A1, the major CYP26 enzyme in the liver, that influence the metabolism of atRA, 2) determine the binding affinities of drugs with FABP1 and the effect of FABP1 on the metabolism of diclofenac by CYP2C9 and 3) identify the major CYP enzymes responsible for the metabolism of Δ9-tetrahydrocannabinol (THC) and the effect of FABP1 on THC metabolism by these CYPs.The results presented in this thesis show that CRABPs and FABP1 alter the metabolism of their ligands by CYP enzymes. In chapter 2, kinetic modeling suggested that both CRABPs interact directly with CYP26A1 to regulate atRA metabolism similar to previous reports with CYP26B1 and CYP26C1. Based on the kinetic modeling, apo-CRABPs inhibited atRA metabolism by CYP26A1 while holo-CRABPs directly delivered atRA to CYP26A1. The findings in chapter 2 propose a mechanism by which CRABPs can fine tune cellular atRA levels via their interactions with CYP26A1. Similar to the findings in chapter 2, chapters 3 and 4 showed that FABP1 had a significant impact on the metabolism of drug ligands by several CYPs. The work in chapters 3 and 4 showed that a variety of drugs bound to FABP1 with binding affinities (Kds) ranging from 0.3-20 µM and these drugs formed ternary complexes with the fluorescent probe, DAUDA, and FABP1. In chapter 3, FABP1 had a significant impact on the metabolism of diclofenac by CYP2C9, decreasing the kcat of 4’-OH-diclofenac formation by ~50%. Chapter 4 showed similar effects of FABP1 with THC metabolism. Four major metabolites of THC were identified in human liver microsomes – 11-OH-THC and M1-M4 metabolites, and the major CYPs that contributed to the formation of these metabolites in HLMs were CYP2C9 (11-OH-THC), CYP2C19 (M3 and 11-OH-THC) and CYP3A4 (M2-M4). FABP1 altered the formation of these metabolites in an enzyme specific manner, altering both the Km and kcat of THC metabolite formation by these enzymes. Collectively, the data presented in this thesis suggest that iLBPs can modulate the metabolism of their ligands via interactions with CYP enzymes. Drugs are expected to be bound to FABP1 in the liver in vivo and the FABP1 may be a determinant of drug metabolism in the liver.