HTS Assay for Functional Modulators of ABCB6 Figure 6. SDS-PAGE analysis of purified ABCB6 and selectivity and validation of HTS identified compounds by hemin-agarose affinity chromatography using purified ABCB6. (a) Purified ABCB6 sample was analyzed by SDS-PAGE. The figure shows coomassie brilliant blue staining of SDS gel (lane legends are 1, protein marker; 2, purified ABCB6-flag 2 mg protein; 3, purified ABCB6-flag 5 mg protein; 4, protein marker; and 5, bovine serum albumin control). b) verteporfin and (c) tomatine hydrochloride potently disrupt the interaction between purified ABCB6 protein and hemin-agarose compared with (d) succinylacetone. Three hundred nanograms of purified ABCB6-flag protein was incubated in the presence or absence of increasing concentration of the indicated compound and hemin-agarose and the resulting complex was immunoblotted using a monoclonal antibody to the flag-tag. Results are representative of three independent experiments. doi:10.1371/journal.pone.0040005.g006 hemin interaction with ABCB6 suggesting that these compounds could be potential substrates of ABCB6. To confirm our assertion above and to demonstrate that verteporfin and tomatine hydrochloride are potential substrates of ABCB6 we performed transport assays using these compounds in functional mitochondria isolated from cells expressing either a transport competent ABCB6 protein (referred to as ABCB6 in the figure legends and Fig. 8) or a transport incompetent ABCB6 protein (referred to as ABCB6-MT) as described above in methods and [17]. As shown in Figure 8 both verteporfin (Fig. 8a) and tomatine hydrochloride (Fig. 8b) were taken up in an ATP dependent manner into functional mitochondria isolated from ABCB6 expressing cells which was significantly higher than the uptake observed using functional mitochondria isolated from ABCB6-MT cells. We have previously demonstrated that ABCB6 mediated transport of substrates is an energy-dependent process, and that ABCB6 is a mitochondrial membrane ATPase that functions as an active drug transporter [17]. Based on these observations we hypothesized that ABCB6 might function as an ATPase during the transport of verteporfin and tomatine hydrochloride. To test this hypothesis we measured the ATPase activity of ABCB6 in the presence and absence of ATP, and the drug, using mitochondria isolated from ABCB6 expressing or ABCB6-MT expressing cells. As shown in Fig. 8 both verteporfin (Fig. 8c) and tomatine hydrochloride (Fig. 8d) stimulated vanadate-sensitive mitochon- drial ATPase activity in ABCB6 expressing cells but not in ABCB6-MT expressing or vector control cells. Together results presented in Figures 7 and 8 demonstrate that verteporfin and tomatine hydrochloride could be potential substrates of ABCB6 and highlights the robustness of the HTS assay to identify not only potential inhibitors but also potential substrates of ABCB6. There is increasing evidence that ABCB6 localizes to multiple cellular compartments including the plasma membrane [9]. Although the precise identity and the substrate specificity of ABCB6 localized to the plasma membrane are not defined, there is a potential possibility that the differentially localized ABCB6 might have overlapping substrate specificity [9]. This could lead to a possible erroneous generalization of the identified compounds as either inhibitors or substrates of ABCB6 based on the primary HTS assay. Although this is a concern that has to be considered, the secondary assays of specificity described here can eliminate these discrepancies in most cases as these secondary assays utilize isolated mitochondria and purified ABCB6 protein as opposed to whole cells used in the primary HTS assay. Further, it is interesting to note that the purified ABCB6 protein exists as two isoforms (Fig. 6a), very similar to what has been described by Paterson et al [9] as the mitochondrial and plasma membrane PLoS ONE | www.plosone.org 10 July 2012 | Volume 7 | Issue 7 | e40005