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

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.人类ABCB1（MDR1）编码的多药转运蛋白P-糖蛋白（P-glycoprotein）在包括抗肿瘤药物在内的多种药物的体内处置与疗效调控中发挥核心作用。ABCB1基因多态性或可决定个体间对这类药物耐药性的差异。为验证这一假说，我们开发了一种基于酿酒酵母（Saccharomyces）的功能检测方法，用以评估ABCB1基因多态性的生物学功能意义。本研究针对P-糖蛋白野生型参照株及9种携带氨基酸改变型单核苷酸多态性（single nucleotide polymorphisms, SNPs）的变异株，在分别添加道诺霉素（daunorubicin）、阿霉素（doxorubicin）、缬氨霉素（valinomycin）或放线菌素D（actinomycin D）的培养基中开展了检测。结果显示，部分SNPs可增强（M89T、L662R、R669C及S1141T）或削弱（W1108R）药物耐药性。当R669C等位基因与W1108R联合存在时，其原本大幅升高的耐药性会受到抑制。各变异株的蛋白表达水平及亚细胞定位均无法解释所观测到的表型差异。不同药物底物下，各变异株的相对耐药谱存在显著差异。本研究建立了一种稳健可靠的新型方法，用于鉴定人类多药转运蛋白基因中功能改变型多态性，明确了影响P-糖蛋白功能的多态性位点，为基于基因型的化疗药物个体化给药方案的研发迈出了关键一步。