Structural and Functional Studies of Organic Anion Transporting Polypeptides OATP1B1 and OATP1B3

Abstract

The liver plays a crucial role in numerous physiological processes, including blood detoxification and the maintenance of body homeostasis. Hepatocytes have evolved a complex enzymatic molecular machinery specializing in converting hydrophobic molecules into hydrophilic metabolites, which is a critical process for drug pharmacokinetics. Hepatic transporters are essential for the influx and efflux of drugs and their metabolites across cell membranes. The superfamily of organic anion-transporting polypeptides (OATPs), particularly OATP1B1, OATP1B3, and OATP2B1, is clinically involved in mediating drug absorption and distribution in humans, facilitating drug uptake across the basolateral membrane. OATP1B1 and OATP1B3 transporters recognize a wide range of structurally diverse amphipathic molecules and are linked to hepatic drug disposition and systemic drug clearance. Impaired function due to untargeted inhibition or nucleotide polymorphisms is linked to reduced drug clearance, systemic circulation, and associated adverse events. Drug-drug interactions (DDIs) are associated with adverse clinical events that can substantially affect patient health outcomes. Consequently, this can lead to increased healthcare costs and potential withdrawal of drugs from the market. Thus, numerous regulatory agencies recommend testing new molecular entities (NME) for hepatic uptake of OATP1B transporters to identify potential substrates or inhibitors and predict potential DDIs in the early stages of drug development. Owing to their significant clinical implications, it is essential to elucidate the molecular and structural basis of substrate selectivity and uptake mechanisms. The aim of this research project was to elucidate the substrate selectivity and transport mechanism of OATP1B transporters through structural and functional studies. To obtain high-resolution structures of OATP1B1 and OATP1B3 transporters, it was essential to establish a robust platform for the overexpression of functional proteins and purification of stable and homogenous samples. In Chapter 2, I describe the development and screening of different fusion constructs for heterologous expression and purification of OATP1B1 and OATP1B3, resulting in stable cell lines being the best expression system. To test the function of the fusion constructs expressed in stable cell lines, I established a cell-based transport assay using radioactively labeled substrates, as presented in Chapter 3. To facilitate the structural studies of OATP1B1 and OATP1B3 in lipid nanodiscs, in Chapter 4, I discuss the methods used to generate high-affinity conformational binders. In Chapter 5, I describe the determined cryo-EM structures of OATP1B1 and OATP1B3 in functionally distinct states, which allowed us to propose a transport mechanism. The structure of E1S-OATP1B1 was determined at a resolution of 3.67 Å, which allowed for the identification of the substrate-binding pocket. The results provide structural insight into transporter selectivity for large organic anions. Additionally, we observed a bicarbonate ion bound to OATP1B3 in a pocket formed by residues of the signature motif, and the ion was coordinated by a highly conserved histidine residue associated with pH-sensitivity in OATP transporters. The bicarbonate-bound OATP1B3 was determined at a resolution of 2.97 Å.