Gunaratne GS , Johns ME , Hintz HM , Walseth TF , Marchant JS .

R01 GM088790 NIGMS NIH HHS

	Fig. 1. Drugs that inhibit NAADP-evoked Ca2+ release block MERS translocation. A, Ca2+ release in sea urchin egg homogenate as resolved by fluo-3 fluorescence measurements. Ca2+ liberation was measured in the absence (solid circles, top trace) or presence of fangchinoline (open circles, 10μM) in response to NAADP (blue, 70 nM), IP3 (red, 200 nM) or cADPR (green, 100 nM). Data represent values from a minimum of three independent experiments and are expressed as mean ± SEM. B, correlation plot comparing the extent of inhibition of NAADP- (blue) IP3- (red) or cADPR-evoked Ca2+ release (green) observed with individual ligands (10μM) correlated with the extent of inhibition of MERS-pseudovirus translocation evoked by the same ligands (at the same concentration, 10μM). None of these tested ligands evoked Ca2+ release by themselves. Solid (NAADP) and dotted lines (IP3, cADPR) represent linear regression of datapoints. Ligand key: 1 = DMSO, 2=Cycleanine, 3=Tubocurarine, 4=Nimodipine, 5=Procaine, 6=Chondocurine, 7=Benzocaine, 8=Hernandezine, 9=Berbamine, 10=Nicardipine, 11=Verapamil, 12=Tetrandrine, 13=Fangchinoline, 14=Amitriptyline, 15=Loperamide, 16=Ned-19, 17=Bafilomycin, 18=U18666 A, 19 = YM201636, 20=Fluoxetine, 21=Citalopram, 22=Desipramine, 23=Siramesine. MERS-pseudovirus infectivity was measured using a luciferase-based cell entry assay, assay methodology and inhibition of NAADP-evoked Ca2+ shown in this figure are described in detail in the companion paper [25].
	Fig. 2. Schematic of workflow for identification of inhibitors of NAADP-evoked Ca2+ release and screening assay validation. A, Illustration of screening workflow. Small molecule libraries (1534 compounds) were screened for inhibitors of NAADP-evoked Ca2+ release in sea urchin egg homogenate. Primary screen hits were validated and characterized prior to testing against Ca2+ signals evoked by NAADP microinjection and pseudotyped MERS-CoV cell entry in mammalian cells. B, Representative traces of fluo-3 fluorescence for negative control in response to addition of vehicle (1st injection) and then NAADP (2nd injection, 167 nM) at indicated timepoints (arrows). C, Representative traces showing example of positive control (initial injection with NAADP, 167 nM). D, Representative traces of fluo-3 fluorescence used to measure Z’ after the 2nd injection timepoint (arrows).
	Fig. 3. Identification of top hits from primary drug screen. A, Scatter plot of average peak amplitude of NAADP-evoked Ca2+ release in the presence of drug (25μM) from LOPAC®1280 (left, numbered 1–1280) or Selleck (GPCR) compound library (right, numbered 1281–1534). B, results from both libraries were combined and compounds were ranked by amplitude of response from greatest inhibition (rank #1, left) to potentiation (rank #1534, right). The majority of compounds were in a range ±25% of control response (shaded box). Compounds that exhibited >80% inhibition of NAADP-evoked Ca2+ release were prioritized (red box) and selected for further characterization (Table 1). C, raw data from the primary screen for selected compounds – PPADS, PPNDS, thapsigargin, A23187, thio-NADP and vehicle control (DMSO). D, enlargement of red box from ‘B’ showing ranking of top eighteen hits after pruning, which displayed >80% inhibition of NAADP-evoked Ca2+ release. Pruned compounds were thio-NADP and A23187 (4th and 5th top hits), shown in grey. E, traces of NAADP-evoked Ca2+ release in the presence of the two top ranked candidates (#1, PF-543; #2, SKF96365; coloured lines, 25μM).
	Fig. 4. Secondary validation of candidates in sea urchin egg homogenate. A, Full concentration response curves resolving effects of selected primary screen candidates on Ca2+ release in sea urchin egg homogenates evoked by NAADP (70 nM). Current NAADP antagonists (PPADS, ned-19 and naringenin) are also included. IC50 values for the entire dataset are provided in Table 1. B, Averaged peak Ca2+ release in the presence of drug (30μM, 5 min preincubation time) relative to vehicle controls, in response to EC70 concentrations of NAADP (70 nM), cADPR (100 nM), or IP3 (200 nM). C, Analysis of effects of all 18 candidates on specific 32P-NAADP radioligand binding levels in the presence of drug (30μM), relative to vehicle controls. p-values: ** p < 0.01 relative to DMSO controls.
	Fig. 5. Characterization of candidate inhibitors on lysosomal Ca2+ stores in U2OS cells. A, representative traces of GPN induced Ca2+ release (top) and lysosomal disruption (bottom). U2OS cells were loaded with Fluo-4 AM and LysoTracker® Red (LTR). Baseline fluorescence values were recorded for 15 min before addition of test compounds (30μM). Fluorescence was then monitored for 40 min before addition of GPN (300μM) to release lysosomal Ca2+. Measurements were made to quantify GPN evoked Ca2+ release (top) and loss of LTR fluorescence following initial drug addition (bottom) by assessing fluorescence ratio values in the regions highlighted by the green (peak F/Fo ratio) and red bars (minimal F/Fo ratio). PIC, protease inhibitor cocktail; BafA1, bafilomycin A1. B, Correlation plot depicting relationship between measurements of GPN induced calcium release (y-axis, green) and drug-induced lysosome disruption (x-axis red). Values are replotted from highlighted regions in (A). The cluster of non-lysomotropic compounds is highlighted (box). Data represents peak fluo-4 fluorescence ratios (Ffluo/F0, where ‘Ffluo’ represents fluo-4 fluorescence at peak, and F0 represents fluorescence at time = 0) and minimum LysoTracker® fluorescence ratios (FLTR/F0, where ‘FLTR’ represents minimum LysoTracker® Red fluorescence prior to GPN addition, and F0 represents fluorescence at time = 0). Compounds are labeled according to ranking # in Table 1.
	Fig. 6. Validation of NAADP-inhibitors in mammalian cells. A, Ca2+ traces resolved by fluo-4 fluorescence in response to NAADP microinjection (100 nM pipette concentration) in U2OS cells treated with indicated drugs (10μM, 10 min pretreatment). Individual traces shown in red, averaged response shown in black. B, Quantification of peak amplitude of NAADP-evoked Ca2+ transients in microinjected U2OS cells relative to control (blue bars) following preincubation with indicated drugs as shown in (A). Red bars report luciferase levels in a MERS-pseudovirus cell translocation assay in Huh7 cells relative to controls (H2O, DMSO) following treatment with the same panel of drugs (10μM for 1 h prior to exposure to MERS-pseudovirus for a 5 h period). MERS-pseudovirus cell entry was detected 3 days post infection by measuring luciferase activity as described fully in the companion paper [25]. p-values: * p < 0.05, ** p < 0.01 relative to DMSO controls.

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