Identification, K_i determination and CoMFA analysis of nuclear receptor ligands as competitive inhibitors of OATP1B1-mediated estradiol-17β-glucuronide transport

Abstract

Evidence shows that drug–drug interactions can occur at the level of drug transporters such as the organic anion transporting polypeptides (OATPs), a group of membrane solute carriers that mediate the sodium-independent transport of a wide range of amphipathic organic compounds. The polyspecific OATP1B1 is exclusively expressed at the basolateral membrane of hepatocytes and mediates uptake of amphipathic organic compounds from blood into hepatocytes. Nuclear receptors are ligand-activated transcription factors that play an important role in xenobiotic disposition and human diseases. Quite a few nuclear receptor ligands interact with transport proteins.

A high-resolution three-dimensional structure is critical to understand the polyspecificity of OATP1B1 to predict and prevent adverse drug–drug interactions. Unfortunately there are no crystal structures of OATPs/Oatps available to date. Therefore, in this study we attempted to elucidate the characteristics of the substrate binding site of OATP1B1 based on small molecules interacting with it. First, we identified inhibitors of the OATP1B1 model substrate estradiol-17β-glucuronide from about 40 nuclear receptor ligands. Among them, GW1929, paclitaxel and troglitazone were strong inhibitors, while 5α-androstane, 5α-androstane-3β, 17β-diol-17-hexahydrobenzoate and estradiol-3-benzoate were weak inhibitors. Then, we selected 25 compounds and performed inhibition kinetic studies to identify competitive inhibitors and determine their K_i values which ranged from submicromolar to submillimolar. Finally, we performed CoMFA analysis on the identified competitive inhibitors. The CoMFA results indicate that the substrate binding site of OATP1B1 consists of a large hydrophobic middle part with basic residues at both ends that could be very important for substrate binding.

Introduction

Severe adverse drug–drug interactions represent an increasing risk for patients taking several drugs at the same time. There is convincing evidence that besides at the level of drug metabolizing enzymes, drug–drug interactions also occur at the level of drug transporters. The human liver is the major organ for the uptake, metabolism and excretion of numerous drugs and other xenobiotics. The first step in hepatic clearance of such chemicals is the uptake of these substances from blood into liver mediated by transporters located on the basolateral membrane of hepatocytes. Organic anion transporting polypeptides (humans: OATPs; rodents: Oatps) are a group of membrane solute carriers that mediate the sodium-independent transport of a wide range of amphipathic organic compounds including numerous drugs and other xenobiotics [1], [2], [3]. So far, 11 human OATPs have been identified [2], [3]. The polyspecific OATP1B1 is exclusively expressed at the basolateral membrane of hepatocytes [4], [5], [6], [7] and is important for drug uptake.

Nuclear receptors are important ligand-activated transcription factors that strongly affect xenobiotic disposition. Furthermore, they are important drug targets for human diseases like diabetes, obesity and cancer [8], [9], [10], [11]. Nuclear receptors play important roles in the regulation of gene expression for metabolism, conjugation and transport of endogenous and exogenous compounds [12], [13], [14]. To exert their physiological effects, the ligands of nuclear receptors need to enter their target cells and transport proteins play a role during this process. For instance, the pregnane X receptor (PXR) ligand rifampicin [15], [16] and the farnesoid X receptor (FXR) ligand cholic acid [17], [18] are transported by OATP1B1 and 1B3; the thyroid hormones triiodothyronine (T₃) and l-thyroxine (T₄) are transported by OATP1 and OATP4 family members [4], [19], [20], [21], [22], [23]. Besides being transported directly, interactions of nuclear receptor ligands with transporters could result in unexpected potentially adverse effects. Previous studies showed that the peroxisome proliferator-activated receptor (PPAR) ligand troglitazone caused intracellular accumulation of bile salts and subsequent liver damage due to its inhibition of the canalicular bile salt export pump (BSEP) [24], and that the glucocorticoid receptor ligand dexamethasone inhibited OATP1A2-mediated dehydroepiandrosterone sulfate transport [25]. We recently demonstrated that the PXR ligand clotrimazole inhibited OATP1B1-mediated estradiol-17β-glucuronide transport, but stimulated OATP1B3-mediated estradiol-17β-glucuronide transport [26].

Given that adverse drug–drug interactions can be a result of inhibition at the uptake transporter level, there is a critical need to understand the polyspecificity of OATP1B1 to predict and prevent such adverse effects. Due to the difficulty of isolating and crystallizing membrane proteins, researchers attempted to elucidate the characteristics of the substrate binding sites of OATPs/Oatps based on the information of interacting small molecules. Chang et al. [27] used computational pharmacophore modeling to illuminate the key features for substrate interaction with rat Oatp1a1 and human OATP1B1 transporters. Yarim et al. [28] applied a three-dimensional quantitative structure–activity relationship (3D-QSAR) technique to obtain structural information of the substrate binding site of Oatp1a5. In these studies, the substrates used to derive theoretical models might bind to different sites as experimental evidence shows that OATPs/Oatps [26], [29], [30] as well as other transporters [31], [32] might have multiple substrate/ligand recognition sites. A computational model derived from compounds binding at the same site should be more reasonable and reliable. Therefore we decided to use competitive inhibitors of the OATP1B1 model substrate estradiol-17β-glucuronide which bind to the same site as the substrate to derive a model. From about 40 nuclear receptor ligands we identified inhibitors of OATP1B1-mediated transport, carried out kinetic studies to identify the competitive inhibitors and calculated their K_i values. Finally, we performed comparative molecular field analysis (CoMFA) on the identified competitive inhibitors to elucidate possible binding mechanism between OATP1B1 and its ligands.