Function-Altering SNPs in the Human Multidrug Transporter Gene ABCB1 Identified Using a Saccharomyces-Based Assay

Abstract

The human ABCB1 (MDR1)-encoded multidrug transporter P-glycoprotein (P-gp) plays a major role in disposition and efficacy of a broad range of drugs including anticancer agents. ABCB1 polymorphisms could therefore determine interindividual variability in resistance to these drugs. To test this hypothesis we developed a Saccharomyces-based assay for evaluating the functional significance of ABCB1 polymorphisms. The P-gp reference and nine variants carrying amino-acid–altering single nucleotide polymorphisms (SNPs) were tested on medium containing daunorubicin, doxorubicin, valinomycin, or actinomycin D, revealing SNPs that increased (M89T, L662R, R669C, and S1141T) or decreased (W1108R) drug resistance. The R669C allele's highly elevated resistance was compromised when in combination with W1108R. Protein level or subcellular location of each variant did not account for the observed phenotypes. The relative resistance profile of the variants differed with drug substrates. This study established a robust new methodology for identification of function-altering polymorphisms in human multidrug transporter genes, identified polymorphisms affecting P-gp function, and provided a step toward genotype-determined dosing of chemotherapeutics. Patients often show varied drug responses ranging from lack of therapeutic efficacy to life-threatening adverse drug reactions. Drug therapy would be greatly improved if it were possible to predict individual drug sensitivity and tailor drugs to patients' genetic makeup. Like all other organisms, humans have a set of transporters and enzymes to detoxify and eliminate foreign molecules including drugs. Understanding the function of genetic variants in these proteins is a key goal toward personalized medicine. To that end, we examined the functional consequences of naturally occurring genetic variants in P-glycoprotein, the most versatile human multidrug transporter. A novel method was developed and employed that can identify function-altering variants in human transporters. This methodology was robust and powerful in that the functional effect of genetic variants can be directly assessed in yeast where all confounding variables in humans are excluded. Surprisingly, the majority of single amino acid substitutions were found to cause alterations in resistance to three tested anticancer agents. This study extends the impact of yeast-based medical research to a new niche, pharmacogenomics.