Luminogenic probes were designed and synthesized for the detection of uptake transporter activity in a lytic cell-based assay. These probes rely on a self-cleavable trimethyl lock quinone-cyanobenzothiazole (TMQ-CNBT) or trimethyl lock quinone-luciferin (TMQ-Luc) linked to the anion transporter substrate fluorescein. Upon cellular transport, the TMQ is reduced by viable cells, resulting in the facile intramolecular lactonization and rapid release of the bioluminescent reporter molecule. The uptake transporter activity can then be detected without removing and washing off the extracellular substrates. Six probes were tested with OATP1B1*1a and OATP1B3 overexpressing HEK293 cells, and all compounds showed up to 10.2-fold enhancement in uptake when compared to control cells. Uptake of TMQ-luciferin compounds 2, 4, and 6 increased linearly over time up to 30 min at a concentration ranging from 40 nM to 20 μM. The apparent Km values obtained at different time intervals up to 30 min were nearly identical for a given compound, which validates the 30 min window as appropriate for uptake transporter assays. The average apparent Km values ranged from 0.3 to 0.8 μM and 0.2 to 1.3 μM for OATP1B1*1a and OATP1B3, respectively, indicating good affinities to these anion transporters. Furthermore, uptake of compound 2 was inhibited by two inhibitors of OATP1B1*1a and OATP1B3: rifampicin and ritonavir. The preliminary results obtained from compound 2 exhibited a time-dependent, saturatable, and inhibitable nature of uptake, indicating the feasibility of using the probe for the detection of a transporter-mediated process. This add-and-read homogeneous assay may provide a convenient, rapid, and facile way to detect changes in transporter activity in a high-throughput format, and this assay design strategy could create a new platform for a general cell uptake assay for biomaterials in the future.
