Chemical Recording of Pump‐Specific Drug Efflux in Living Cells

Abstract Drug efflux—a process primarily facilitated by efflux pumps such as multidrug resistance proteins (MRPs)—plays a pivotal role in cellular resistance to chemotherapies. Conventional approaches to assess drug efflux are predominantly conducted in vitro and often lack pump specificity. Here we report the bioorthogonal reporter inhibiting efflux (BRIEF) strategy, which enables the recording of pump‐specific drug efflux in living cells. In BRIEF, a specific substrate is engineered as a bioorthogonal efflux probe (BEP) for specific pumps. The cellular concentration and protein labeling level of the probe can be augmented when the test drug is transported by the same pumps. Serendipitously, we discovered that per‐ O ‐acetylated unnatural monosaccharides, initially designed for metabolic glycan labeling, are exported by some MRPs. Using Ac 4 GlcNAl as a BEP, we studied the structure‐efflux relationship of flavonoids and identified small molecules, including tannic acid, cholesterol and gallic acid, as novel MRP substrates in high‐throughput screening. Tannic acid, known for anti‐tumor and anti‐SARS‐CoV‐2 properties, showed increased efficacy upon MRP inhibition. Additionally, BRIEF was adapted to assess p‐glycoprotein‐mediated efflux using Rhodamine 123 as a BEP, leveraging its light‐activatable proximity labeling ability. BRIEF provides a versatile approach to investigate drug efflux and enhance chemotherapy strategies.