Cellular Models and In Vitro Assays for the Screening of modulators of P-gp, MRP1 and BCRP

Abstract

Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are highly expressed in tumor cells, as well as in organs involved in absorption and secretion processes, mediating the ATP-dependent efflux of compounds, both endogenous substances and xenobiotics, including drugs. Their expression and activity levels are modulated by the presence of inhibitors, inducers and/or activators. In vitro, ex vivo and in vivo studies with both known and newly synthesized P-glycoprotein (P-gp) inducers and/or activators have shown the usefulness of these transport mechanisms in reducing the systemic exposure and specific tissue access of potentially harmful compounds. This article focuses on the main ABC transporters involved in multidrug resistance [P-gp, multidrug resistance-associated protein 1 (MRP1) and breast cancer resistance protein (BCRP)] expressed in tissues of toxicological relevance, such as the blood-brain barrier, cardiovascular system, liver, kidney and intestine. Moreover, it provides a review of the available cellular models, in vitro and ex vivo assays for the screening and selection of safe and specific inducers and activators of these membrane transporters. The available cellular models and in vitro assays have been proposed as high throughput and low-cost alternatives to excessive animal testing, allowing the evaluation of a large number of compounds.

Keywords: ATP-binding cassette transporters; P-glycoprotein; activators; breast cancer resistance protein; cell-based assays; cellular models; in vitro assays; inducers; membrane assays; multidrug resistance-associated protein 1.

Figure 1

Crystal structure (Protein Data Bank (PDB) ID: 3G61) [36] and general representation of human P-glycoprotein (P-gp). P-gp, a full-transporter, contains twelve transmembrane segments, split into two halves forming transmembrane domains, each with a nucleotide-binding domain. Adapted from [3,5].

Figure 2

Crystal structure (PDB ID: 2CBZ) [37] and general representation of human multidrug resistance-associated protein 1 (MRP1). MRP1, a full-transporter, has three transmembrane domains, including five extra transmembrane segments toward the N-terminus, and two nucleotide-binding domains. Adapted from [5].

Figure 3

General representation of human breast cancer resistance protein (BCRP). BCRP, a half-transporter, contains only six transmembrane segments (one transmembrane domain) and one nucleotide-binding domain. Adapted from [5].

Figure 4

Schematic overview of the main drug transporters expressed in brain capillary endothelial cells, as well as their localization. ADP, Adenosine 5′-diphosphate; BCRP, Breast cancer resistance protein; MRP, Multidrug resistance protein; OATP, Organic anion-transporting polypeptide. Adapted from [2,11].

Figure 5

Schematic overview of the main drug transporters expressed in hepatocytes, as well as their localization. ADP, Adenosine 5′-diphosphate; ATP, Adenosine 5′-triphosphate BSEP, Bile salt export pump; MATE, Multidrug and toxin extrusion transporter; NTCP, Sodium-taurocholate co-transporting polypeptide; OAT, Organic anion transporter; OATP, Organic anion-transporting polypeptide; OCT, Organic cation transporter; OST, Organic solute and steroid transporter. Adapted from [2,11].

Figure 6

Schematic overview of main drug transporters expressed in renal epithelial cells, as well as their localization. ADP, Adenosine 5′-diphosphate; ATP, Adenosine 5′-triphosphate; OAT, Organic anion transporter; OATP, Organic anion-transporting polypeptide; OCT, Organic cation transporter; OCTN, Organic Cation/Carnitine Transporter; MRP, Multidrug resistance protein; PEPT, Peptide transporter; P-gp, P-glycoprotein; URATE, Urate transporter. Adapted from [2,11].

Figure 7

Schematic overview of main drug transporters expressed in enterocytes, as well as their localization. * Also reported in the apical membrane [189]; ** OATP2B1 also reported in the basolateral membrane of neonatal, infantile and adolescent enterocytes [190]. ADP, Adenosine 5′-diphosphate; ATP, Adenosine 5′-triphosphate; BCRP, Breast cancer resistance protein; MCT, Monocarboxylate transporter; MRP, Multidrug resistance protein; OATP, Organic anion-transporting polypeptide; OCT, Organic cation transporter; OST, Organic solute and steroid transporter; PEPT, Peptide transporter; P-gp, P-glycoprotein. Adapted from [2,11].