HTS Assay for Functional Modulators of ABCB6 Figure 2. Dose response analysis of active compounds identified in the primary HTS. (a) ABCB6 expressing cells showed dose dependent decrease in median channel fluorescence (representing PPIX fluorescence) in response to benzethonium chloride, verteporfin, and to a lesser extent piperlongumine. In contrast vector control cells did not show any change in median channel fluorescence of FL2 (representing PPIX fluorescence) in response to treatment with any of the four compounds. (b) EC50 values for each of the compound and their corresponding hillslope values. Results represent mean +/2 SD with n = 3. (c and d) compounds identified in the primary HTS screen do not alter ABCB6 expression in the ABCB6 over- expressing cells. (c) immunoblot analysis of ABCB6 expression in ABCB6 overexpressing cells treated with increasing concentration of benzethonium chloride (BCL). (d) immunoblot analysis of ABCB6 expression in ABCB6-flag overexpressing cells treated with increasing concentration of tomatine hydrochloride (THC). In these studies ABCB6 expression was measured using a monoclonal antibody against the flag-tag. Actin is used a loading control. Figure representative of three independent experiments. doi:10.1371/journal.pone.0040005.g002 coupling helices that are located in the loops between TM helices [36]. In addition to the sequence comparisons of the ABC domains, the structures of the binding proteins also suggest that the hydrophobic amino acid (HAA; such as Leu, Ile, Val system) and monosaccharide (MOS; such as ribose (RBS)) branch could exhibit a distinct TMD fold. The structures of the binding proteins for ABC transporters vary in their topological threading between domains [37–39] and can be classified into three distinct categories (designated I, II and III, which confusingly overlaps with the nomenclature used for the TMD folds). The homology modeling and docking results show that the best scoring poses of the docked ligands (Fig. 4a and 4b), are positioned in the upper part of the transmembrane region, close to the corresponding binding site of the QZ59 inhibitor in the ABCB1a crystal structure [40]. This is the site where verapamil is known to bind to the ABCB1 transporter [41,42]. While we do not intend to PLoS ONE | www.plosone.org 6 July 2012 | Volume 7 | Issue 7 | e40005