HTS Assay for Functional Modulators of ABCB6 Figure 4. Topology and homology model of ABCB6 dimer with the docked ligands. (a) far and (b) close view of docking poses of selected ligands to the human ABCB6 transporter. Coproporphyrinogen III – blue; verteporfin – green; benzethonium chloride– magenta; piperlongumine - light blue; and tomatine hydrochloride - yellow. The Gly426-Val429, and Phe545-Pro555 parts from one ABCB6 monomer were hidden in order to better see the ligands. doi:10.1371/journal.pone.0040005.g004 purification and characterization of ABCB6 is part of another manuscript that is currently in preparation and will not be discussed here in detail, except to show that the purified protein (confirmed by mass spectrometry) exists as two isforms of , 50 kDa and ,80 kDa proteins (Fig. 6a), both of which were recognized by the flag-antibody. Because the flag-antibody is fused to the c-terminal tail of ABCB6, it suggests that the two isoforms have different N-terminal sequences. We found that following hemin-agarose affinity chromatography, only the ,80 kDa isoform was detected in immunoblots (Fig. 6b, 6c and 6d and Fig. S7 and S8), suggesting isoform specific interaction with hemin. As with the isolated mitochondria, both verteporfin and tomatine hydrochloride showed a dose depen- dent displacement of purified ABCB6 from hemin-agarose, while no such competition was seen with succinylacetone. Taken together results presented in Fig. 6, and Fig.S7 and S8, demonstrate for the first time that hemin directly interacts with ABCB6. Despite these results it is still quite possible that hemin interaction with ABCB6 might require additional proteins that promote hemin interaction with ABCB6. However, from the results presented here it appears that this interaction might be transient and is potentially not required once the hemin-ABCB6 complex is formed, because no interacting proteins were seen following hemin-agarose-ABCB6 affinity chromatography (Fig. S7 and S8). In addition, these results suggest that verteporfin and tomatine hydrochloride could be potential substrates of ABCB6 and might be capable of interacting with the same substrate-binding site as hemin. The results from hemin-agarose affinity chromatography is further strengthened by the modeling studies described above which demonstrate that both tomatine and verteporfin show higher overlap with the endogenous substrate, coproporphyrino- gen III (Fig. 4). Further, given that verteporfin is a carboxylated porphyrin, similar to porphyrins that have previously been demonstrated to be transported by ABCB6, these data suggest that verteporfin is a substrate for ABCB6 that competes for the same site on ABCB6 as does hemin. Thus, the identification of verteporfin in this HTS assay highlights the capability of the assay to identify not only inhibitors of ABCB6 but also potential substrates. Selectivity and Validation of Compounds Identified in the Primary HTS Assay – Mitochondrial Transport Assay and ATPase Activity Assay To test further the specificity of the identified compounds to inhibit ABCB6 function we evaluated the ability of verteporfin, tomatine hydrochloride and benzethonium chloride to inhibit ABCB6 mediated transport of hemin into mitochondria. We have previously demonstrated that ABCB6 is capable of transporting radioactive labeled hemin into isolated mitochondria and that this assay is capable of distinguishing ABCB6 substrates from non- substrates [17]. As shown in Figure 7, hemin uptake by isolated mitochondria was inhibited by all three compounds with verteporfin showing the highest inhibition followed by tomatine hydrochloride and benzethonium chloride. The fact that ben- zethonium chloride acts as a weak inhibitor of ABCB6 mediated PLoS ONE | www.plosone.org 8 July 2012 | Volume 7 | Issue 7 | e40005