Function-altering SNPs in the human multidrug transporter gene ABCB1 identified using a Saccharomyces-based assay

Area of Science:

• Pharmacogenomics
• Molecular Biology
• Drug Transport

Background:

• The ABCB1 gene encodes P-glycoprotein (P-gp), a crucial transporter influencing drug disposition and efficacy, particularly for anticancer agents.
• Interindividual variability in drug response is often linked to genetic variations (polymorphisms) in transporter genes like ABCB1.
• Understanding the functional impact of these polymorphisms is essential for optimizing drug therapy and predicting treatment outcomes.

Purpose of the Study:

• To develop and validate a Saccharomyces cerevisiae-based assay for assessing the functional significance of ABCB1 polymorphisms.
• To identify specific single nucleotide polymorphisms (SNPs) in ABCB1 that alter P-gp function and drug resistance.
• To provide a foundation for genotype-guided dosing strategies in chemotherapy.

Main Methods:

• A yeast assay system was engineered to express the human ABCB1 transporter, including a reference P-gp and nine variants with amino-acid-altering SNPs.
• The functional activity of these P-gp variants was evaluated by measuring drug resistance in yeast cells exposed to various substrates (daunorubicin, doxorubicin, valinomycin, actinomycin D).
• Phenotypic effects were correlated with specific SNPs, and protein levels/subcellular localization were assessed to rule out confounding factors.

Main Results:

• Several ABCB1 SNPs were identified that significantly altered P-gp-mediated drug resistance, including M89T, L662R, R669C, and S1141T (increased resistance) and W1108R (decreased resistance).
• The R669C variant exhibited highly elevated drug resistance, which was modulated when present with the W1108R variant.
• Observed functional changes could not be explained by alterations in protein expression levels or subcellular localization, indicating direct functional impact.
• The relative impact of identified polymorphisms varied depending on the specific drug substrate tested.

Conclusions:

• A robust yeast-based assay was established for functional screening of ABCB1 polymorphisms.
• Specific ABCB1 SNPs affecting P-gp function and drug resistance profiles were identified.
• These findings represent a significant step towards developing genotype-informed dosing strategies for chemotherapeutic agents, potentially improving treatment efficacy and reducing toxicity.