ABCB1 (P-glycoprotein/P-gp) is an ATP-binding cassette transporter well known for its association with multidrug resistance in cancer cells. Powered by the hydrolysis of ATP, it effluxes structurally diverse compounds. In this chapter, we discuss current views on the molecular basis of the substrate polyspecificity of P-gp. One of the features that accounts for this property is the structural flexibility observed in P-gp. Several X-ray crystal structures of mouse P-gp have been published recently in the absence of nucleotide, with and without bound inhibitors. All the structures are in an inward-facing conformation exhibiting different degrees of domain separation, thus revealing a highly flexible protein. Biochemical and biophysical studies also demonstrate this flexibility in mouse as well as human P-gp. Site-directed mutagenesis has revealed the existence of multiple transport-active binding sites in P-gp for a single substrate. Thus, drugs can bind at either primary or secondary sites. Biochemical, molecular modeling, and structure-activity relationship studies suggest a large, common drug-binding pocket with overlapping sites for different substrates. We propose that in addition to the structural flexibility, the molecular or chemical flexibility also contributes to the binding of substrates to multiple sites forming the basis of polyspecificity.
Melanoma is the most serious type of skin cancer with a high potential for metastasis and very low survival rates. The discovery of constitutive activation of the BRAF kinase caused by activating BRAF(V600E) kinase mutation in most melanoma patients led to the discovery of the first potent BRAF(V600E) signaling inhibitor, vemurafenib. Vemurafenib was effective in treating advanced melanoma patients and was proposed for the treatment of other BRAF(V600E) mutant cancers as well. Unfortunately, the success of vemurafenib was hampered by the rapid development of acquired resistance in different types of BRAF(V600E) mutant cancer cells. It becomes important to identify and evaluate all of the potential mechanisms of cellular resistance to vemurafenib. In this study, we characterized the interactions of vemurafenib with three major ATP-binding cassette (ABC) transporters, ABCB1, ABCC1 and ABCG2. We found that vemurafenib stimulated the ATPase activity and potently inhibited drug efflux mediated by ABCB1 and ABCG2. Vemurafenib also restored drug sensitivity in ABCG2-overexpressing cells. Moreover, we revealed that in the presence of functional ABCG2, BRAF kinase inhibition by vemurafenib is reduced in BRAF(V600E) mutant A375 cells. Taken together, our findings indicate that ABCG2 confers resistance to vemurafenib in A375 cells, suggesting involvement of this transporter in acquired resistance to vemurafenib. Thus, combination chemotherapy targeting multiple pathways could be an effective therapeutic strategy to overcome acquired resistance to vemurafenib for cancers harboring the BRAF(V600E) mutation.
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