A Yeast‐Based Assay for Inhibitors of <scp>l</scp>‐Lactate Transport Utilizing Fluorescent Biosensors

Inhibitors of ʟ-lactate transport are in development as a novel mode of action in antitumor therapy and malaria. Previously, we used radiolabeled ʟ-lactate to assay transport via the human monocarboxylate transporter 1, MCT1, and the structurally unrelated malaria parasite's transporter, PfFNT. We encountered a sensitivity limit at IC₅₀ around 100 nM possibly resulting from the required high cell number per sample. Here, we describe a sensitive background-free high-throughput assay in yeast based on fluorescent iLACCO biosensors. We used iLACCO for co-expression and fusions with the transporter protein. Uptake of ʟ-lactate produced strong intensiometric fluorescent responses that could be monitored in cell suspensions using a fluorometer and in individual cells by fluorescence microscopy. The signal decreased dose-dependently in the presence of specific MCT1 and PfFNT inhibitors. Re-evaluation of 36 PfFNT inhibitors yielded IC₅₀ values below 100 nM now matching previous data on K_i compound affinity to isolated transporter protein.