Substrate Dependent Alterations of Organic Anion Transporting Polypeptide 1B3 (OATP1B3)

Abstract

Organic anion transporting polypeptides (OATPs) are multispecific transporters that mediate the uptake of numerous drugs and xenobiotics into cells. Alterations in the function of the liver–specific OATP1B1 and OATP1B3 have been shown to affect the disposition of drugs throughout the body. It has been proposed that new drug candidates should be screened for possible OATP inhibition using a prototypical substrate such as estradiol–17β–glucuronide. However, there is evidence that OATPs may have multiple binding sites, and therefore screening with a single compound may be ineffective. Therefore, I tested the hypothesis that OATP1B3 has multiple overlapping but distinct binding sites, which are affected in substrate–dependent ways. This hypothesis was tested via two specific aims: 1) to identify and characterize substrate–dependent effects of plant compounds on OATP1B3–mediated transport, and 2) to identify regions of OATP1B3 involved in the binding and/or translocation of individual model substrates. In the first specific aim, interacting compounds were identified by screening a library of plant compounds for inhibition or stimulation of OATP–mediated uptake of two model substrates. Completion of this specific aim identified two structurally similar compounds that produce substrate–dependent effects on OATP1B3–mediated transport. These compounds stimulate transport of estrone–3–sulfate by increasing substrate affinity. However, the compounds either inhibit or have no effect on the uptake of estradiol–17β–glucuronide. These results demonstrate that estrone–3–sulfate and estradiol–17β–glucuronide have distinct binding sites on OATP1B3. In specific aim two, thirty–three amino acids in the first transmembrane domain and extracellular loop of OATP1B3 were individually mutated to cysteines, and I determined the effect of these mutations on the transport of estradiol-17β-glucuronide and estrone–3–sulfate. Five of the cysteine–substituted OATP1B3 mutants produced different effects on transporter function depending upon the substrate tested. These results suggest that this region of OATP1B3 is involved in the recognition and translocation of individual model substrates. This dissertation demonstrates that OATP1B3 has distinct binding sites for estradiol–17β–glucuronide and estrone–3–sulfate. Furthermore, it shows that transport of these two model substrates is affected in different ways by the same compounds. This knowledge can be used to improve screening of drug candidates to prevent adverse drug–drug interactions prior to the occurrence of adverse events.