Department of Pharmacology, Toxicology, and Therapeutics, The University of Kansas Medical Center, Kansas City, Kansas, United States of America.
PLoS One. 2012;7(7):e40005. doi: 10.1371/journal.pone.0040005. Epub 2012 Jul 10.
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.
ABCB6 是三磷酸腺苷（ATP）结合盒转运蛋白家族的成员，越来越多的研究表明它是一个相关的生理和治疗靶点。评估 ABCB6 活性调节剂将为更全面地了解该转运过程在肿瘤细胞生长、增殖和治疗相关药物耐药性中的意义铺平道路。此外，这一努力将有助于我们理解 ABCB6 在血红素生物合成、细胞适应代谢需求和应激反应等正常生理过程中的功能。为了寻找 ABCB6 的调节剂，我们开发了一种新的基于细胞的方法，该方法结合流式细胞术高通量筛选（HTS），可用于鉴定 ABCB6 的功能调节剂。野生型 ABCB6 表达 K562 细胞中荧光分子原卟啉的积累构成了 HTS 测定的基础。采用 HTS 测定法筛选 Prestwick 化学文库，发现了四种化合物，即苯扎氯铵、维替泊芬、盐酸番茄碱和胡椒碱，它们可以降低 ABCB6 介导的细胞卟啉水平。使用血红素琼脂糖亲和层析和线粒体转运测定法验证鉴定出的化合物，证明四种化合物中的三种能够抑制 ABCB6 将血红素转运到分离的线粒体中。然而，只有维替泊芬和盐酸番茄碱能够抑制 ABCB6 与血红素竞争作为 ABCB6 底物的能力。因此，该测定法灵敏、稳健，适合在高通量环境中自动化，正如我们鉴定出 ABCB6 的选择性功能调节剂所证明的那样。将该测定法应用于其他合成化合物文库和天然产物文库，预计将鉴定出 ABCB6 活性的新型调节剂。
