Luminogenic D‐Luciferin Derivatives as OATP1B1 and 1B3 Substrates in No‐wash Assays^†

Abstract

The human hepatic organic ion transporting polypeptides OATP1B1 and -1B3 are uptake transporters that influence the disposition of several small molecule drugs and perpetrate certain adverse drug-drug interactions. To predict these in vivo effects, in vitro systems are used to screen new drug entities as potential transporter substrates or inhibitors. To simplify such studies, we synthesized luminogenic derivatives of the OATP1B1 and -1B3 substrate D-luciferin to test as probe substrates in a rapid, no-wash optical approach for substrate and inhibitor identification and characterization. Each derivative is a pro-luciferin containing a selfimmolating trimethyl lock quinone linker that is sensitive to intracellular reducing environments that cause the release of free luciferin in proportion to the amount of probe taken up by the transporter. A subsequent luciferin-limited luciferase reaction produces light in proportion to transporter activity. We tested the derivatives in HEK293 cells that overexpress OATP1B1 or OATP1B3 by transient transfection or viral transduction. Derivatives were identified that showed OATPdependent uptake that was time and concentration dependent, saturable and sensitive to inhibition by known OATP1B1 and -1B3 substrates and inhibitors. These luminogenic transporter probes enabled an add-only multi-well plate protocol suitable for automation and high throughput screening.