Fluorescence-based assay for evaluating SLC49A4 function: Identification of acriflavine as a probe substrate

SLC49A4 is a lysosomal membrane transporter implicated in the export of cationic amphiphilic drugs (CADs), such as pyrilamine. By mediating lysosomal efflux of CADs, SLC49A4 may counteract lysosomal trapping, an important determinant of intracellular drug disposition and efficacy. Thus, its functional evaluation is pharmacokinetically relevant, particularly regarding lysosomal CAD accumulation. Using COS-7 cells transiently expressing a plasma membrane-localized mutant of SLC49A4, in which the lysosomal targeting dileucine motif was disrupted by alanine substitution, we identified acriflavine and proflavine-CAD-like fluorescent compounds-as SLC49A4 substrates, particularly under acidic conditions. Acriflavine exhibited a slightly higher transport activity and was selected for detailed functional characterization. Acriflavine uptake by SLC49A4 was saturable and exhibited high-affinity transport with the Michaelis constant of 22.7 μM. Pyrilamine competitively inhibited the acriflavine uptake with the inhibition constant of 54.7 μM, suggesting overlapping substrate recognition. Replacing Cl^- in the medium modestly reduced the acriflavine uptake, indicating partial Cl^- dependence. The profiles of inhibition of the acriflavine uptake by various compounds were in close agreement with those observed for pyrilamine, supporting the use of acriflavine as a reliable surrogate probe. These findings establish acriflavine as a high-affinity, fluorescent SLC49A4 substrate for rapid transport assays. This assay platform facilitates the screening of SLC49A4 inhibitors and provides insights into its role in lysosomal drug handling.