A Non‐Radioactive Cell‐Based Screening Assay to Identify Inhibitors of the Monocarboxylate Transporter Protein 1 (MCT1)
Authors: Bailey T. Liz , Nieto Ainhoa , McDonald Patricia H 
Published: June 2021

Solute carrier (SLC) transporters are a superfamily of membrane bound proteins with over 300 identified members. These proteins serve as key regulators of cellular homeostasis by facilitating substrate entry and by‐product elimination across the plasma membrane. Dysregulation of SLC transporter function contributes to numerous diseases such as diabetes, Parkinson's disease and cancer. As SLCs are associated with a disease phenotype they are considered ‘druggable’ i.e., they can be modulated by drugs. Most current drugs that modulate SLC transporters do so by inhibiting transporter activity. For diseases in which decreased transporter activity leads to a potentially beneficial effect, high‐throughput screening (HTS) of large compound libraries using cell lines that overexpress the transporter of interest can be used to discover small molecule inhibitors. However, this is not necessarily a straightforward endeavor for all SLCs. The monocarboxylate transporters (MCTs) are a sub family of SLC transporters comprised of 14 members among which MCT1‐4 facilitate the bidirectional proton‐coupled co‐transport of monocarboxylates such as ketone bodies, pyruvate and lactate. MCT1 is of particular interest for its roles in cancer. Not only is it upregulated in the tumor microenvironment, but has also been identified as a key regulator of angiogenesis, invasion, migration and immune escape. Targeting this protein, MCT1/2 inhibitors are currently in clinical trials as anticancer therapies. More recently, MCT1 has also been identified as a potential drug target for metabolic and CNS disorders. Although MCT1 inhibitors have been identified, they lack selectivity for any given MCT isoform, and have exhibited select off target effects. Typical for this protein class, inhibitors for MCT1 have been difficult to identify, because traditionally, methods for their identification rely on the use of radiolabeled substrate tracking. In addition to the safety concerns associated with radioactivity, this methodology is also expensive and time consuming. Herein, we present a novel, non‐radioactive, cell‐based HTS‐compatible assay for identifying MCT1 inhibitors. Our method utilizes a cell line that endogenously expresses MCT1, and an MCT1 selective cytotoxic substrate, 3‐bromopyruvate (3BrPA). In our assay construct, MCT1 expressing cells are treated with potential MCT1 inhibitors, and then incubated with 3BrPA. In this paradigm, compounds that protect cells from cytotoxicity are identified as MCT1 inhibitors, because only cells treated with an inhibitor have interrupted MCT1 mediated transport and remain viable in the presence of 3BrPA. The screening method described here is robust, reproducible and HTS amenable. Moreover, it establishes a novel technique to identify chemical probes to study the therapeutic potential of MCTs while providing the conceptual framework for further assay development to identify inhibitors for other members of the SLC family.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.