Characterization of OATP1B1 and OATP1B3 inhibition by Nilotinib

Abstract

IntroductionLiver uptake of numerous pharmaceutical drugs is mediated by the hepatic transport protein OATP1B1. Altered function of this protein, either by genetic variation or drug‐drug interaction is associated with systemic changes of drug concentrations that can lead to the onset of severe adverse events. We previously used mass‐spectrometric data from global phospho‐proteome databases to show that a substantial number of clinically‐relevant human transporters, including OATP1B1, have conserved tyrosine‐phosphorylation sites. We therefore hypothesize that OATP1B1 is regulated by tyrosine‐phosphorylation and that this form of modification is sensitive to specific tyrosine kinase inhibitors.MethodsUptake of Estradiol‐β‐glucuronide or 8‐(2‐[Fluoresceinyl]‐aminoethylthio)‐adenosine‐3′,5′‐cyclic‐monophosphate (8FcA), in the presence or absence of FDA approved tyrosine kinase inhibitors, was assessed using HEK293 cells transfected with either OATP1B1, tyrosine to phenylalanine mutant variations of OATP1B1, OATP1B3, or an empty vector. A high‐throughput siRNA screen of 779 protein kinases was performed to identify regulators of OATP1B1 function. A kinase HotSpot Assay was also carried out to confirm the presence of overlapping targets among OATP1B1 inhibitory TKIs. Statistical calculations were done using Graph‐pad Prism and each experiment was repeated in triplicate.ResultsOf the 32 FDA approved tyrosine kinase inhibitors assessed, nilotinib was identified as the most potent inhibitor of OATP1B1‐ and OATP1B3‐dependent uptake (p <0.001). Nilotinib‐induced inhibition was non‐competitive with a clinically relevant concentration of 1.3μM that inhibited 50% of OATP1B1 activity (IC50). Transporter activity was not immediately restored upon removal of nilotinib, although full function was recoverable after 24 hours. Following these findings, we mutated numerous predicted tyrosine phosphorylation sites to assess their role in regulating OATP1B1 function. We observed that mutation of the tyrosine residue at position 234, a site highly conserved across numerous OATP transporters including OATP1B3, reduced OATP1B1 function by more than 90% (p<0.0001). Additionally, our siRNA screen identified LYN kinase as a regulator of OATP1B1 and the LYN‐selective tyrosine kinase inhibitor bafetinib also significantly reduced OATP1B1 activity (p <0.005). Consistently, kinase assays confirmed that OATP1B1 inhibitory TKIs inhibit LYN kinase.ConclusionWe identified nilotinib as a significant, non‐competitive inhibitor of OATP1B1 at clinically relevant concentrations. Moreover, mutation of tyrosine 234 or loss of LYN kinase function also reduces OATP1B1 activity. These findings indicate that use of tyrosine kinase inhibitors, especially those that influence LYN, could make OATP1B1 substrates susceptible to phosphorylation‐dependent drug‐drug interactions and contribute to adverse events.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.