DISC766623_Supplemental_Material – Supplemental material for A High-Throughput Flow Cytometry Screen Identifies Molecules That Inhibit Hantavirus Cell Entry

<p>Supplemental material, DISC766623_Supplemental_Material for A High-Throughput Flow Cytometry Screen Identifies Molecules That Inhibit Hantavirus Cell Entry by Tione Buranda, Catherine Gineste, Yang Wu, Virginie Bondu, Dominique Perez, Kaylin R. Lake, Bruce S. Edwards and Larry A. Sklar in SLAS Discovery</p

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

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

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

Dose response analysis of active compounds identified in the primary HTS.

<p>(a) ABCB6 expressing cells showed dose dependent decrease in median channel fluorescence (representing PPIX fluorescence) in response to benzethonium chloride, verteporfin, and to a lesser extent piperlongumine. In contrast vector control cells did not show any change in median channel fluorescence of FL2 (representing PPIX fluorescence) in response to treatment with any of the four compounds. (b) EC₅₀ values for each of the compound and their corresponding hillslope values. Results represent mean +/− SD with n = 3. (c and d) compounds identified in the primary HTS screen do not alter ABCB6 expression in the ABCB6 over-expressing cells. (c) immunoblot analysis of ABCB6 expression in ABCB6 overexpressing cells treated with increasing concentration of benzethonium chloride (BCL). (d) immunoblot analysis of ABCB6 expression in ABCB6-flag overexpressing cells treated with increasing concentration of tomatine hydrochloride (THC). In these studies ABCB6 expression was measured using a monoclonal antibody against the flag-tag. Actin is used a loading control. Figure representative of three independent experiments.</p

Assay development at University of New Mexico Center for Molecular Discovery (UNMCMD).

<p>(a) Human erythroid leukemia cells (K562) expressing ABCB6 accumulate more porphyrins compared to the vector control cells as measured by flow cytometry. (b) Flow cytometric HTS of Prestwick Chemical Library (PCL). The figure depicts data from a representative PCL plate demonstrating decrease in FL2 fluorescence (representing PPIX fluorescence) mediated by verteporfin. The Y-axis displays the amount of fluorescence (FL2 log channel representing PPIX fluorescence). The X-axis displays each well. In the assays time bins were automatically drawn around the clusters by using IDLQuery software programs, each cluster corresponds to one well. The red square represents the PPIX fluorescence in untreated ABCB6 overexpressing cells while the white square represents the PPIX fluorescence in vector control cells. The arrow highlights the decrease in PPIX fluorescence of ABCB6 expressing cells in the presence of verteporfin.</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