HTS Assay for Functional Modulators of ABCB6 Figure 3. Toxicity profile for ABCB6 overexpressing and vector cells treated with the four compounds identified in the primary HTS screen. (a) Dose dependent toxicity of the four compounds in ABCB6 overexpressing and vector cells. (b) EC50 value for each of the compound and their corresponding hill slope values. Results represent mean +/2 SD with n = 3. doi:10.1371/journal.pone.0040005.g003 make quantitative predictions based on this docking model, we did notice a higher overlap between the endogenous substrate, coproporphyrinogen III, with tomatine and verteporfin, compared to the overlap with benzethonium and piperlongumine. Selectivity and Validation of Compounds Identified in the Primary HTS assay – Hemin Agarose Affinity Chromatography Using Mitochondrial Fractions The compounds identified in the primary HTS were evaluated for their ability to act as functional inhibitors of ABCB6 using hemin-agarose affinity chromatography and mitochondrial trans- port assay [17]. We have previously demonstrated that these two assays when used in combination have the ability to identify substrates and potential inhibitors of ABCB6 [17]. We found that following hemin-agarose affinity chromatography, verteporfin and tomatine hydrochloride showed a dose dependent displacement of ABCB6 from hemin agarose [.50% displacement of ABCB6 at 5 mM (Fig. 5)]. In contrast, neither succinylacetone (Fig. 5) a known inhibitor of porphyrin biosynthesis nor benzethonium chloride (data not shown) one of the compounds identified in the HTS screen were capable of displacing ABCB6 even at a concentration of 20 mM. 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. Selectivity and Validation of Compounds Identified in the Primary HTS Assay – Hemin Agarose Affinity Chromatography Using Purified Protein To confirm hemin interaction with ABCB6 specifically and to extend the studies described above we performed hemin-agarose affinity chromatography using purified ABCB6 protein. The PLoS ONE | www.plosone.org 7 July 2012 | Volume 7 | Issue 7 | e40005