A Novel Flow Cytometric HTS Assay Reveals Functional Modulators of ATP Binding Cassette Transporter ABCB6

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

Liquid-Metal Synthesized Ultrathin SnS Layers for High-Performance Broadband Photodetectors

Atomically thin materials face an ongoing challenge of scalability, hampering practical deployment despite their fascinating properties. Tin monosulfide (SnS), a low-cost, naturally abundant layered material with a tunable bandgap, displays properties of superior carrier mobility and large absorption coefficient at atomic thicknesses, making it attractive for electronics and optoelectronics. However, the lack of successful synthesis techniques to prepare large-area and stoichiometric atomically thin SnS layers (mainly due to the strong interlayer interactions) has prevented exploration of these properties for versatile applications. Here, SnS layers are printed with thicknesses varying from a single unit cell (0.8 nm) to multiple stacked unit cells (approximate to 1.8 nm) synthesized from metallic liquid tin, with lateral dimensions on the millimeter scale. It is reveal that these large-area SnS layers exhibit a broadband spectral response ranging from deep-ultraviolet (UV) to near-infrared (NIR) wavelengths (i.e., 280-850 nm) with fast photodetection capabilities. For single-unit-cell-thick layered SnS, the photodetectors show upto three orders of magnitude higher responsivity (927 A W-1) than commercial photodetectors at a room-temperature operating wavelength of 660 nm. This study opens a new pathway to synthesize reproduceable nanosheets of large lateral sizes for broadband, high-performance photodetectors. It also provides important technological implications for scalable applications in integrated optoelectronic circuits, sensing, and biomedical imaging

Liquid-Metal Synthesized Ultrathin SnS Layers for High-Performance Broadband Photodetectors

Atomically thin materials face an ongoing challenge of scalability, hampering practical deployment despite their fascinating properties. Tin monosulfide (SnS), a low-cost, naturally abundant layered material with a tunable bandgap, displays properties of superior carrier mobility and large absorption coefficient at atomic thicknesses, making it attractive for electronics and optoelectronics. However, the lack of successful synthesis techniques to prepare large-area and stoichiometric atomically thin SnS layers (mainly due to the strong interlayer interactions) has prevented exploration of these properties for versatile applications. Here, SnS layers are printed with thicknesses varying from a single unit cell (0.8 nm) to multiple stacked unit cells (≈1.8 nm) synthesized from metallic liquid tin, with lateral dimensions on the millimeter scale. It is reveal that these large-area SnS layers exhibit a broadband spectral response ranging from deep-ultraviolet (UV) to near-infrared (NIR) wavelengths (i.e., 280-850 nm) with fast photodetection capabilities. For single-unit-cell-thick layered SnS, the photodetectors show upto three orders of magnitude higher responsivity (927 A W−1 ) than commercial photodetectors at a room-temperature operating wavelength of 660 nm. This study opens a new pathway to synthesize reproduceable nanosheets of large lateral sizes for broadband, high-performance photodetectors. It also provides important technological implications for scalable applications in integrated optoelectronic circuits, sensing, and biomedical imaging.The authors would like to acknowledge support from the ARC Discovery Project schemes DP180102752 (Y.L., M.J.S.S.), DP180104141 (S.B. and K.C.), DP170102138 (K.K.Z.) and the RMIT Vice Chancellor Fellowships (N.M. and S.W.). K.K.Z. also acknowledges support from the Australian Research Council Centre of Excellence FLEET

Selectivity and validation of HTS identified compounds by hemin agarose affinity chromatography.

<p>(a) verteporfin and (b) tomatine hydrochloride potently disrupt the interaction between ABCB6 and hemin-agarose compared with (c) succinylacetone. (d, e and f) image J analysis of ABCB6 band intensity treated with (d) verteporfin, (e) tomatine hydrochloride and (f) succinylacetone averaged over three independent experiments. Mitochondria isolated from K562 cells expressing ABCB6-Flag or the empty vector were incubated in the presence or absence of increasing concentration of the indicated compound and hemin-agarose and the resulting complex was immunoblotted using a monoclonal antibody to the flag-tag. Results are representative of 3 independent experiments. ‘*’ significantly different from untreated controls. P<0.05. ‘NS’ differences are non-significant compared to untreated control.</p

SDS-PAGE analysis of purified ABCB6 and selectivity and validation of HTS identified compounds by hemin-agarose affinity chromatography using purified ABCB6.

<p>(a) Purified ABCB6 sample was analyzed by SDS-PAGE. The figure shows coomassie brilliant blue staining of SDS gel (lane legends are 1, protein marker; 2, purified ABCB6-flag 2 µg protein; 3, purified ABCB6-flag 5 µg protein; 4, protein marker; and 5, bovine serum albumin control). b) verteporfin and (c) tomatine hydrochloride potently disrupt the interaction between purified ABCB6 protein and hemin-agarose compared with (d) succinylacetone. Three hundred nanograms of purified ABCB6-flag protein was incubated in the presence or absence of increasing concentration of the indicated compound and hemin-agarose and the resulting complex was immunoblotted using a monoclonal antibody to the flag-tag. Results are representative of three independent experiments.</p

Topology and homology model of ABCB6 dimer with the docked ligands.

<p>(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.</p

Verteporfin and tomatine hydrochloride are ABCB6 transport substrates.

<p>(a) verteporfin and (b) tomatine hydrochloride are transported by transport competent ABCB6 protein (ABCB6) in the presence of ATP which is significantly higher than transport by transport incompetent ABCB6 protein (ABCB6-MT) in the presence of ATP in mitochondria isolated from ABCB6 or ABCB6-MToverexpressing cells. Results are representative of three independent experiments. ‘*’ significantly different from ABCB6-MT and AMP treated ABCB6 expressing mitochondria; P<0.05. ‘**’ significantly different from ABCB6-MT and AMP treated ABCB6 expressing mitochondria; P<0.01. (c) and (d) vanadate sensitive ATPase activity (fold change relative to basal activity) was stimulated by (c) verteporfin and (d) tomatine hydrochloride in mitochondria from cells expressing transport competent ABCB6 protein (ABCB6) relative to mitochondria isolated from transport incompetent ABCB6 protein (ABCB6-MT). Values are means +/− SEM. ‘*’ significantly different from ABCB6-MT cells at each time point; P<0.05.</p

Toxicity profile for ABCB6 overexpressing and vector cells treated with the four compounds identified in the primary HTS screen.

<p>(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 +/− SD with n = 3.</p

Three of the four compounds identified in the primary HTS screen compete for ABCB6 mediated hemin transport.

<p>(a) verteporfin (c) tomatine hydrochloride and (e) benzethonium chloride compete for the transport of ¹⁴C-labeled hemin in the presence of ATP but not in the presence of AMP in mitochondria isolated from ABCB6-overexpressing cells. (b, d, and f) Line graph showing dose dependent competition of (b) verteporfin, (d) tomatine hydrochloride and (f) benzethonium chloride for ¹⁴C-labeled hemin in the presence of ATP. Results representative of 3 independent experiments. ‘*’ Significantly different from untreated controls. P<0.05.</p