Genetic variants in ABC transporters have led to chemotherapeutic resistance by exporting cytotoxic drugs from tumors, decreasing efficacy of cancer treatments. ABCG2 is a polyspecific exporter that transports a broad range of xenobiotics. Variants in cancer lead to the emergence of chemotherapeutic resistance. Human mutations, particularly in R482, which does not directly interact with substrates, have been observed to drastically increase cancer drug resistance and enhance transport ability of doxorubicin, a poor substrate for WT ABCg2. In order to understand the evolution of substrate specificity in transporters, we created a library of all possible single genetic variants and performed in-cell screenings. We screen this library against three cancer drugs, SN-38, mitoxantrone, and doxorubicin. Our screen revealed multiple additional hot spots beyond R482 distal to the substrate and ATP binding pockets that are altering transport substrate specificity through unknown mechanisms. It is likely that these single mutations are not altering global structure; therefore, to understand how substrate specificity diverges in chemotherapeutic resistance, we must understand how variants change dynamics. We will use smFRET to observe how variants with divergent effects on substrate specificity may be altering dynamics of the transport cycle. By combining transporter functional genomics and dynamics studies, we will reveal mechanisms of how chemotherapeutic resistance emerges and how substrate specificity diverges.
O'Donnell et al. (Sun,) studied this question.