title: Novel No-Wash Luminogenic Probes for the Detection of Transporter Uptake Activity.
abstract: 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.
authors.affiliation: Promega Biosciences LLC , 277 Granada Drive, San Luis Obispo, California 93401, United States.
authors.forename: Dana
authors.initials: D
authors.lastname: Mustafa
authors.affiliation: Promega Corporation , 2800 Woods Hollow Road, Madison, Wisconsin 53711-5399, United States.
authors.forename: Dongping
authors.initials: D
authors.lastname: Ma
authors.affiliation: Promega Biosciences LLC , 277 Granada Drive, San Luis Obispo, California 93401, United States.
authors.forename: Wenhui
authors.initials: W
authors.lastname: Zhou
authors.affiliation: Promega Biosciences LLC , 277 Granada Drive, San Luis Obispo, California 93401, United States.
authors.forename: Poncho
authors.initials: P
authors.lastname: Meisenheimer
authors.affiliation: Promega Corporation , 2800 Woods Hollow Road, Madison, Wisconsin 53711-5399, United States.
authors.forename: James J
authors.initials: JJ
authors.lastname: Cali
mesh_terms: D052160:Benzothiazoles / Q000737:chemistry; D001692:Biological Transport / Q000187:drug effects; D060326:Chemistry Techniques, Synthetic; D049454:Firefly Luciferin / Q000031:analogs & derivatives / Q000737:chemistry / Q000493:pharmacokinetics; D019793:Fluorescein / Q000737:chemistry; D005452:Fluoresceins / Q000737:chemistry / Q000493:pharmacokinetics; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D027381:Liver-Specific Organic Anion Transporter 1 / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D049408:Luminescent Agents / Q000737:chemistry*; D008163:Luminescent Measurements / Q000379:methods; D057054:Molecular Imaging / Q000379:methods*; D015335:Molecular Probes / Q000138:chemical synthesis / Q000737:chemistry* / Q000493:pharmacokinetics; D009570:Nitriles / Q000737:chemistry; D029362:Organic Anion Transporters, Sodium-Independent / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D000074542:Solute Carrier Organic Anion Transporter Family Member 1B3
chemical_list: C578355:2-cyanobenzothiazole; D052160:Benzothiazoles; D005452:Fluoresceins; D027381:Liver-Specific Organic Anion Transporter 1; D049408:Luminescent Agents; D015335:Molecular Probes; D009570:Nitriles; D029362:Organic Anion Transporters, Sodium-Independent; C503999:SLCO1B1 protein, human; C421209:SLCO1B3 protein, human; D000074542:Solute Carrier Organic Anion Transporter Family Member 1B3; C000624442:fluorescein-PEG2-TMQ-Luc; D049454:Firefly Luciferin; D019793:Fluorescein