addition of 3.3 mM MgATP. The vanadate-sensitive ATPase activity stimulated by verteporfin and tomatine hydrochloride was measured as nanomoles of inorganic phosphate released per minute per milligram of protein. Results and Discussion Assay Development and HTS Flow Cytometry We developed an HTS flow cytometry assay to select functional modulators of ABCB6. The assay takes advantage of the unique ability of ABCB6 to regulate cellular porphyrin biosynthesis [17]. Cells over expressing ABCB6 exhibit increased porphyrin biosynthesis, which directly correlates with the level of ABCB6 expression (Fig. 1a and [17]). Furthermore, the increased cellular porphyrin synthesis requires a functional protein, because a non- functional transport mutant of ABCB6 is incapable of promoting porphyrin biosynthesis [17]. Accumulation of porphyrin in cells results in increased cell associated fluorescence intensity (Fig. 1a). Cellular porphyrin representative of porphyrin levels was con- firmed using succinylacetone (SA) as described [14,17]. SA is a potent inhibitor of cellular porphyrin biosynthesis and we have previously demonstrated that SA at a concentration of 200 mM completely blocks cellular porphyrin biosynthesis in less than 24 hours [14,17]. Feasibility of the HTS assay to identify compounds that functionally alter ABCB6 mediated porphyrin biosynthesis was evaluated using the PCL 2000 library. Although the central idea was to evaluate compounds that decreased the median channel fluorescent (MCF) intensity by FL2 (Fig. 1b, Fig. S5a, S5b and S5c), the assay also identified compounds that increased the MCF intensity (Fig. S5c) suggesting that the assay is capable of identifying both inhibitors and activators of ABCB6. The controls were K562 cells overexpressing ABCB6 treated with DMSO alone (negative control). Our analysis showed a tight data distribution with Z’ factors averaging 0.61+/20.08 and a hit rate of 0.4%. Calculations of percentage response were set to 100% at the positive control level and 0% at the negative control level. Cutoffs were set at three times higher than the standard deviation of mean fluorescence values, which corresponded to a percentage response of 58%. To minimize false positive hits we selected only compounds that satisfied the cutoff criteria because they concurrently decrease fluorescence. By using the criteria used in FL2, 12 compounds from the entire PCL set qualified as hits. However, 8 of these compounds were flagged as potentially fluorescent as the FL2 MCF in the vector was greater than average +2X standard deviation of DMSO control (which corresponded to the DMSO control value measured in the ABCB6 cells). Four compounds met the criteria (0.4% hit rate). The identified compounds were verteporfin, benzethonium chloride, tomatine hydrochloride and piperlongumine (Fig. 1b and Fig. S5a, S5b, S5c and S6). Confirmatory Dose Responses Compounds that displayed activity in the primary HTS assay were re-tested for dose response. The four hit compounds that decreased MCF intensity by FL2 in the primary screen were evaluated employing ABCB6 over-expressing and vector cells to identify compounds decreasing MCF by FL2 in a dose response manner (dose concentration ranging from 48.8 nM to 100 mM). All the four compounds (verteporfin, benzethonium chloride, tomatine hydrochloride and piperlongumine) showed a dose dependent ability to reduce ABCB6 mediated PPIX accumulation (Fig. 2). In contrast, none of the compounds had any effect on vector cells. Verteporfin exhibited the lowest EC50, while HTS Assay for Functional Modulators of ABCB6 piperlongumine exhibited the highest. Interestingly an increase in tomatine hydrochloride concentration more than ,1.0 mM leads to a rapid decrease of the MCF (Fig. 2). The differences in MFC at each dose of these compounds for three replicates were significant (p,0.05). To confirm the results from our primary HTS assay and to demonstrate that the decrease in PPIX fluorescence mediated by the identified compounds were not associated with their ability to alter ABCB6 expression per se we measured ABCB6 expression in ABCB6-overexpressing cells following treatment with each of the four compounds. As shown in Figure 2c and 2d tomatine hydrochloride and benzethonium chloride dose dependently reduced ABCB6-mediated PPIX fluorescence without affecting ABCB6 expression. Similar results were observed for verteporfin (data not shown). Benzethonium chloride is a broad range antiseptic with a variety of antimicrobial properties [28] and a tendency to be potentially toxic [29]. The plant steroidal alkaloid a-tomatine has a variety of reported activities spanning from antifungal to immune adjuvant [30–32]. Verteporfin (trade name Visudyne), is a benzoporphyrin derivative used as a photosensitizer for photodynamic therapy to eliminate the abnormal blood vessels in the eye associated with conditions such as the wet form of macular degeneration [33,34]. Piperlongumine has anti-cancer properties and selectively targets and kills cancer cells but leaving normal cells unharmed [35]. It is intriguing to note that the assay detected verteporfin, which is similar in structure to carboxylated porphyrin derivatives such as hemin and pheophorbide A, which have been shown to be ABCB6 substrates [17]. Cytotoxicity A follow-up cytotoxicity assay was performed employing ABCB6 overexpressing and vector cell lines (Fig. 3). This experiment aimed to identify in principal those compounds that are not toxic at the concentrations exhibiting their functional inhibitory role by decreasing the intracellular accumulation of porphyrin mediated by ABCB6. Interestingly, the normalized viability of vector cells was reduced more than 80% when the concentration of tomatine and benzethonium chloride was .390 nM. However, viability was not reduced more than 25% even at the highest concentration (100 mM) of verteporfin and piperlongumine. The differences in percent-normalized viability at each dose of these compounds for three replicates were significant (p,0.05). In case of ABCB6 overexpressing cells, benzethonium, verte- porfin, tomatine and piperlongumine exhibited EC50 values of 1.9, 0.17, 0.08 and 2.23 uM, respectively. It is true that both verteporfin and tomatine are more toxic to ABCB6 cells than the other two compounds (Fig. 3). However, the selection of a less toxic compound in between benzethonium and piperlongumine is also complex. The normalized viability of ABCB6 expressing cells was not reduced significantly up to a dose of 780 nM dose of benzethonium, which was reduced by 40% at this concentration of piperlongumine. Topology and Homology Modeling of ABCB6 ABC transporters have a characteristic architecture that consists minimally of four domains: two transmembrane domains (TMDs), and two cytoplasmic ATP Binding domains [1]. At the sequence level, the superfamily of ABC transporters is identified by a characteristic set of highly conserved motifs that are present in the ABCs. Although the detailed folds of the TMDs can vary, they all interact with the helical domains of the ABC domain through PLoS ONE | www.plosone.org 4 July 2012 | Volume 7 | Issue 7 | e40005