A Novel Flow Cytometric HTS Assay Reveals Functional Modulators of ATP Binding Cassette Transporter ABCB6 Kishore Polireddy1., Mohiuddin Md. Taimur Khan2,3,4., Hemantkumar Chavan1, Susan Young 2 , Xiaochao Ma1, Anna Waller2, Matthew Garcia2, Dominique Perez2, Stephanie Chavez2, Jacob J. Strouse2, Mark K. Haynes2, Cristian G. Bologa2,3, Tudor I. Oprea2,3, George P. Tegos2,4,5,6*, Larry A. Sklar2,3,4*, Partha Krishnamurthy1* 1 Department of Pharmacology, Toxicology, and Therapeutics, The University of Kansas Medical Center, Kansas City, Kansas, United States of America, 2 Center for Molecular Discovery, University of New Mexico, Albuquerque, New Mexico, United States of America, 3 Division of Biocomputing, University of New Mexico Health Sciences Center, Albuquerque, New Mexico, United States of America, 4 Department of Pathology, University of New Mexico School of Medicine, Albuquerque, New Mexico, United States of America, 5 Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, Massachusetts, United States of America, 6 Department of Dermatology, Harvard Medical School, Boston, Massachusetts, United States of America Abstract ABCB6 is a member of the adenosine triphosphate (ATP)-binding cassette family of transporter proteins that is increasingly recognized as a relevant physiological and therapeutic target. Evaluation of modulators of ABCB6 activity would pave the way toward a more complete understanding of the significance of this transport process in tumor cell growth, proliferation and therapy-related drug resistance. In addition, this effort would improve our understanding of the function of ABCB6 in normal physiology with respect to heme biosynthesis, and cellular adaptation to metabolic demand and stress responses. To search for modulators of ABCB6, we developed a novel cell-based approach that, in combination with flow cytometric high-throughput screening (HTS), can be used to identify functional modulators of ABCB6. Accumulation of protoporphyrin, a fluorescent molecule, in wild-type ABCB6 expressing K562 cells, forms the basis of the HTS assay. Screening the Prestwick Chemical Library employing the HTS assay identified four compounds, benzethonium chloride, verteporfin, tomatine hydrochloride and piperlongumine, that reduced ABCB6 mediated cellular porphyrin levels. Validation of the identified compounds employing the hemin-agarose affinity chromatography and mitochondrial transport assays demonstrated that three out of the four compounds were capable of inhibiting ABCB6 mediated hemin transport into isolated mitochondria. However, only verteporfin and tomatine hydrochloride inhibited ABCB6’s ability to compete with hemin as an ABCB6 substrate. This assay is therefore sensitive, robust, and suitable for automation in a high-throughput environment as demonstrated by our identification of selective functional modulators of ABCB6. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel modulators of ABCB6 activity. Citation: Polireddy K, Khan MMT, Chavan H, Young S, Ma X, et al. (2012) A Novel Flow Cytometric HTS Assay Reveals Functional Modulators of ATP Binding Cassette Transporter ABCB6. PLoS ONE 7(7): e40005. doi:10.1371/journal.pone.0040005 Editor: Irina V. Lebedeva, Enzo Life Sciences, Inc., United States of America Received November 29, 2011; Accepted May 30, 2012; Published July 10, 2012 Copyright: 2012 Polireddy et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding for GPT and LAS was provided by the National Institutes of Health (NIH, 5U54MH084690-02, Center Driven Initiative 1) and PK by R03MH093193. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding received for this study. Competing Interests: The authors have declared that no competing interests exist. * E-mail: gtegos@salud.unm.edu (GPT); lsklar@salud.unm.edu (LAS); pkrishnamurthy@kumc.edu (PK) . These authors contributed equally to this work. Introduction Transporters perform essential roles in cellular metabolism and activity. They differ in membrane topology, energy coupling mechanisms, and most importantly in substrate specificities [1–4]. Based on their sequence similarity and structural homology, transporters are classified into six super-families [1,3]. The ATP binding cassette transporter superfamily is the largest, comprising seven subfamilies designated A to G [1,4]. ABC transporters are increasingly recognized as playing important roles in normal biology and therapeutic responses to medications in mammalian cells. The highly conserved ABC domains of ABC transporters provide the nucleotide-dependent engine that drives transport [1,2]. ABCB6 belongs to the B sub-family of ABC transporters, which includes the well-characterized human transporter ABCB1 that was the first ABC transporter implicated in multidrug resistance, the intracellular peptide transporters (TAP1 and TAP2) that function in major histocompatibility complex class I antigen presentation and ABCB5 which is essential for melanoma induction, as a doxorubicin efflux mediator in melanomas and xenotransplantation proliferation models [3,5]. ABCB6 is increas- ingly recognized as a relevant physiologic and therapeutic target. ABCB6 expression is upregulated in many tumor cell lines and in liver tumors where it appears to promote cell survival and tumor growth and proliferation [5–7]. ABCB6 gene is amplified in tumor cells with acquired chemotherapeutic resistance [8–13]. ABCB6 expression is also induced under cell stress, where it promotes cell survival [8,14–16]. Thus, ABCB6 expression could promote PLoS ONE | www.plosone.org 1 July 2012 | Volume 7 | Issue 7 | e40005