Phosphorylation and function of OATP1B1 with tyrosine kinase inhibitors.

Abstract

IntroductionThe organic anion transporting polypeptide OATP1B1 regulates hepatic uptake of many therapeutics. As a result, drug‐drug interactions (DDI’s) are commonly mediated by OATP1B1 activity. Reduced drug elimination via this transporter can lead to increased rates of drug‐associated adverse reactions, such as statin‐induced rhabdomyolysis. Surprisingly, numerous tyrosine kinase inhibitors (TKIs) that are either weak or non‐substrates of OATP1B1 increase systemic concentrations of various known OATP1B1 substrates by an unknown mechanism. We hypothesize that OATP1B1 function is dependent on tyrosine phosphorylation, which can be reduced by specific TKIs.MethodsPrimary human hepatocytes and HEK293 cells transfected with OATP1B1, or tyrosine (Y) to phenylalanine (F) mutant variants of OATP1B1, were used to assess the uptake of 8‐[2‐[Fluoresceinyl]‐aminoethylthio)‐adenosine‐3′,5′‐cyclic‐monophosphate (5–40 μM) or estradiol‐b‐glucuronide (2 μM) in the presence or absence of FDA approved TKIs. A biotinylation labeling assay was utilized to measure surface expression of OATP1B1 treated with and without nilotinib (0–20 μM). Phosphorylation of OATP1B1 was assessed by Western blot and mass spectrometry‐based proteomic analysis. OATP1B1 protein was purified by immunoprecipitation from overexpressing cell lines in the presence or absence of nilotinib. siRNA was utilized to identify kinases that potentially mediate OATP1B1 function. Statistical analysis was performed with Graph‐pad Prism, and each experiment was repeated in triplicate.ResultsNine of 46 FDA approved TKIs inhibit OATP1B1 function over 80%, compared to untreated controls. Nilotinib, the most potent TKI among assessed, does not decrease OATP1B1 expression or membrane localization. Instead, it elicits inhibition of OATP1B1 through a non‐ or mixed‐competitive and reversible mechanism. Nilotinib also reduces OATP1B1‐mediated uptake in primary human hepatocytes (P < 0.001). Mass spectrometry analysis of OATP1B1 revealed a variety of tyrosine phosphorylation sites, among them, phosphorylation at position 625 is greatly reduced in the presence of nilotinib. Moreover, mutation of Y625F results in significantly reduced transport activity compared to the wild‐type OATP1B1 activity (P < 0.001). Finally, we confirmed that SiRNA knocking down of LYN kinase activity, a Src tyrosine kinase, significantly reduces OATP1B1 activity (P < 0.001).ConclusionWe found that OATP1B1 is tyrosine phosphorylated at numerous sites and that position 625 is highly sensitive to nilotinib exposure. The Y625F mutation of OATP1B1 results in decreased transport activity and similarly, loss of LYN expression also leads to lower OATP1B1 transport. These findings indicate that clinically relevant TKIs may promote drug‐drug interactions by interfering with a post‐translational event that is required for OATP1B1‐mediated transport.Support or Funding InformationUniversity at Buffalo School of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, Seed Support Program