Clickable Substrate Transport (CST): A High-Throughput Functional Assay for Solute Carrier Proteins

Solute carrier proteins (SLCs) are the largest family of membrane-associated transport proteins, facilitating the transport of a diverse range of substrates vital for cellular fitness and function. While the therapeutic potential of modulating SLC proteins is evident, with clinical-stage drugs targeting this superfamily well-documented, progress in drugging less well-described members has been hampered by challenges in working with these proteins in an in vitro setting. In this work, we present a novel strategy to enable the discovery and development of small molecules against a variety of transporters. Using the glucose transporter SLC5a1 as a model system, we have developed a cellular assay that reports on the transport of a substrate of interest through the application of a fluorogenic dye and bioorthogonal chemistry. This assay is robust, amenable to scale, and applicable across a range of substrates and targets of interest. We anticipate that this work will facilitate the discovery of novel modulators for this therapeutically significant protein family through expanding the limited catalog of available screening assays for these traditionally challenging targets.