#	Aladdin ID	Assay Type	Description	Organism	Compounds	Reference	BAO Format	Source
1.	ALA3431446	F	GSK_TCAKS: pIC50 Trypanosoma cruzi viability assay using beta-GAL activity. This assay was run at single shot at 5uM compound concentration per well. The biological reagents consisted of NIH-3T3 host cells and trypomastigotes parasitic cells; both cell types were set to a 3.3 * 10^5 cells/mL dilution in assay DMEM by means of a CASY cell counter device using a 60um capillary. 6 uL of the mixture were dispensed per well (about 1 * 10^3 host-cells and 1 * 10^3 parasites) with a Multidrop Combi dispenser. Previously, a solution of 1.67 * 10^5 trypomastigotes per mL in assay DMEM had been prepared and 6uL per well dispensed in the allotted control columns that defined 100% inhibition. The 0% inhibition of parasitic growth control was obtained by leaving the assay mixture untreated in its corresponding plate columns. In all wells, the percentage of DMSO never exceeded 0.5%. Plates incubated for four days at 37C, 5% CO2. The substrate used for the assay fluorescence intensity (FLINT) readout was resorufin-beta-D-galactopiranoside (Sigma-Aldrich) at 5uM per well. It was diluted in PBS supplemented with the soft detergent Igepal (Fluka). Upon addition to the plates, the substrate solution was incubated for 4h at room temperature and the signal read with the EnVision microtiter plate reader (Perkin-Elmer) using the corresponding set of filters (excitation/emission at 531/595nm).	Mus musculus	584	ALA3430912	organism-based format	GSK Kinetoplastid Screening
2.	ALA3431447	F	GSK_TCAKS: pIC50 Trypanosoma cruzi Imaging: Infected Cells. H9c2 cells were seeded in T-225 flasks (225cm^2 culture surface; Corning Inc., NY, USA) in DMEM-10% FBS for 4h to allow attachment. Cells were then washed once with PBS before infection. T. cruzi trypomastigotes, collected at days 5 to 8 after infection from LLC-MK2 parasite infected cultures, were allowed to swim out for 4h at 37C from a centrifuged pellet (2,500 rpm/10 min/room temperature). Trypomastigotes were then collected and counted in a CASY Cell Counter. Trypomastigotes, in supplemented DMEM, were added to H9c2 cultures in a multiplicity of infection of 1 and incubated for 18 h. Cells were washed once with PBS before incubation of the infected H9c2 monolayer with trypsin (Life-Technologies) to detach cells from the flask. Cells were counted in a CASY Cell Counter using a 150 um capillary and their density set at 5 * 10^4 cells per mL in supplemented assay DMEM. Infected H9c2 were dispensed into 384-well poly-lysine coated assay plates at 50uL per well using a Multidrop Combi dispenser. After seeding them, the plates were incubated at 37C, 5% CO2 for 72h. Cultures were then fixed and stained by addition of 50 uL of a solution containing 8% formaldehyde and 4 uM DRAQ5 DNA dye (BioStatus, UK) per well. Plates were kept light-protected and imaged one hour later in a Perkin-Elmer Opera microscope using a 20x air objective (NA 0.4) and the following acquisition set: a 635 nm laser excitation line and a 690/50 emission detection filter for DRAQ5 detection. Five images were collected per well for reliable statistical analysis. Automated image analysis was performed with a script developed on Acapella High Content Imaging and Analysis Software (PerkinElmer). Output: percentage of infected cells per well as a second infection marker.	Rattus norvegicus	580	ALA3430912	organism-based format	GSK Kinetoplastid Screening
3.	ALA3431448	F	GSK_TCAKS: pIC50 Trypanosoma cruzi Imaging: Amastigotes/Cell. H9c2 cells were seeded in T-225 flasks (225cm^2 culture surface; Corning Inc., NY, USA) in DMEM-10% FBS for 4h to allow attachment. Cells were then washed once with PBS before infection. T. cruzi trypomastigotes, collected at days 5 to 8 after infection from LLC-MK2 parasite infected cultures, were allowed to swim out for 4h at 37C from a centrifuged pellet (2,500 rpm/10 min/room temperature). Trypomastigotes were then collected and counted in a CASY Cell Counter. Trypomastigotes, in supplemented DMEM, were added to H9c2 cultures in a multiplicity of infection of 1 and incubated for 18 h. Cells were washed once with PBS before incubation of the infected H9c2 monolayer with trypsin (Life-Technologies) to detach cells from the flask. Cells were counted in a CASY Cell Counter using a 150 um capillary and their density set at 5 * 10^4 cells per mL in supplemented assay DMEM. Infected H9c2 were dispensed into 384-well poly-lysine coated assay plates at 50uL per well using a Multidrop Combi dispenser. After seeding them, the plates were incubated at 37C, 5% CO2 for 72h. Cultures were then fixed and stained by addition of 50 uL of a solution containing 8% formaldehyde and 4 uM DRAQ5 DNA dye (BioStatus, UK) per well. Plates were kept light-protected and imaged one hour later in a Perkin-Elmer Opera microscope using a 20x air objective (NA 0.4) and the following acquisition set: a 635 nm laser excitation line and a 690/50 emission detection filter for DRAQ5 detection. Five images were collected per well for reliable statistical analysis. Automated image analysis was performed with a script developed on Acapella High Content Imaging and Analysis Software (PerkinElmer). Output: number of amastigotes per cell as infection level measurement.	Rattus norvegicus	580	ALA3430912	organism-based format	GSK Kinetoplastid Screening
4.	ALA3431449	F	GSK_TCAKS: pIC50 H2C9 Imaging: H9c2 Host Cardiomyocytes. H9c2 cells were seeded in T-225 flasks (225cm^2 culture surface; Corning Inc., NY, USA) in DMEM-10% FBS for 4h to allow attachment. Cells were then washed once with PBS before infection. T. cruzi trypomastigotes, collected at days 5 to 8 after infection from LLC-MK2 parasite infected cultures, were allowed to swim out for 4h at 37C from a centrifuged pellet (2,500 rpm/10 min/room temperature). Trypomastigotes were then collected and counted in a CASY Cell Counter. Trypomastigotes, in supplemented DMEM, were added to H9c2 cultures in a multiplicity of infection of 1 and incubated for 18 h. Cells were washed once with PBS before incubation of the infected H9c2 monolayer with trypsin (Life-Technologies) to detach cells from the flask. Cells were counted in a CASY Cell Counter using a 150 um capillary and their density set at 5 * 10^4 cells per mL in supplemented assay DMEM. Infected H9c2 were dispensed into 384-well poly-lysine coated assay plates at 50uL per well using a Multidrop Combi dispenser. After seeding them, the plates were incubated at 37C, 5% CO2 for 72h. Cultures were then fixed and stained by addition of 50 uL of a solution containing 8% formaldehyde and 4 uM DRAQ5 DNA dye (BioStatus, UK) per well. Plates were kept light-protected and imaged one hour later in a Perkin-Elmer Opera microscope using a 20x air objective (NA 0.4) and the following acquisition set: a 635 nm laser excitation line and a 690/50 emission detection filter for DRAQ5 detection. Five images were collected per well for reliable statistical analysis. Automated image analysis was performed with a script developed on Acapella High Content Imaging and Analysis Software (PerkinElmer). Output: Number of H9c2 Host Cardiomyocytes nuclei to determine drug-related cytotoxicity.	Rattus norvegicus	580	ALA3430912	organism-based format	GSK Kinetoplastid Screening
5.	ALA3431450	F	GSK_TCAKS: pIC50 Leishmania donovani viability assay using fluorescence intensity. Assay plates (1536-well) were prepared by adding 30nL per well of compound. For single shot assay, the final compound concentration was 5uM. For potency determinations, eleven-point one in three dilution curves were generated with a top concentration of 50uM. Briefly, eGFP LdBOB axenic amastigotes were harvested, fixed and counted in a CASY cell counter (Roche-Applied Science). A final concentration of 1.5 * 10^5 cells per well were prepared in amastigote media containing 500 U/mL penicillin/streptomycin (Invitrogen) and 6uL was dispensed to each experimental well using a Multidrop Combi dispenser (Thermo Scientific). 6uL of assay media was dispensed to control columns used as reference for 100% compound response. Following incubation for 72 h at 37C, 5% CO2, 2 uL of resazurin was added to each well at a final concentration of 0.5 mM in phosphate buffered saline (PBS) with IGEPAL (Sigma-Aldrich), 0.05% (v/v). The plates were incubated for 4 h at room temperature and resorufin fluorescence was detected using a PerkinElmer EnVision plate reader with excitation at 528 nm and emission at 590 nm.	Leishmania donovani	590	ALA3430912	organism-based format	GSK Kinetoplastid Screening
6.	ALA3431451	F	GSK_TCAKS: pIC50 Leishmania donovani Imaging: Infected Macrophages. Assay plates (384-well) were prepared by adding 250nL of compound to each well. For single shot assay, the final compound concentration was 5 uM. For potency determinations, eleven-point one in three dilution curves were generated with a top concentration of 50 uM. Two exposure images were taken for each well using 405 nm and 488 nm laser excitation. Automated image analysis was performed with a script developed on Acapella High Content Imaging and Analysis Software (PerkinElmer). Output: Percent of infected macrophage cells were reported for each well.	Homo sapiens	592	ALA3430912	organism-based format	GSK Kinetoplastid Screening
7.	ALA3431452	F	GSK_TCAKS: pIC50 Leishmania donovani Imaging: Amastigotes/Macrophages. Assay plates (384-well) were prepared by adding 250nL of compound to each well. For single shot assay, the final compound concentration was 5 uM. For potency determinations, eleven-point one in three dilution curves were generated with a top concentration of 50 uM. Two exposure images were taken for each well using 405 nm and 488 nm laser excitation. Automated image analysis was performed with a script developed on Acapella High Content Imaging and Analysis Software (PerkinElmer). Output: Average number of amastigotes per macrophage cell were reported for each well.	Homo sapiens	588	ALA3430912	organism-based format	GSK Kinetoplastid Screening
8.	ALA3431453	F	GSK_TCAKS: pIC50 Leishmania donovani Imaging: THP-1 Host Macrophages. Assay plates (384-well) were prepared by adding 250nL of compound to each well. For single shot assay, the final compound concentration was 5 uM. For potency determinations, eleven-point one in three dilution curves were generated with a top concentration of 50 uM. Two exposure images were taken for each well using 405 nm and 488 nm laser excitation. Automated image analysis was performed with a script developed on Acapella High Content Imaging and Analysis Software (PerkinElmer). Output: THP-1 cell count were reported for each well.	Homo sapiens	591	ALA3430912	organism-based format	GSK Kinetoplastid Screening
9.	ALA3431454	F	GSK_TCAKS: pIC50 Trypanosoma brucei viability assay using fluorescence intensity. Assay plates (1536-well) were prepared by dispensing 25nL of compound solution per well using an Echo acoustic dispenser (Labcyte). For single shot assays the final compound concentration was 4.2uM. For potency determinations, eleven-point one in three dilution curves were generated and the top concentration was 42uM. Plates were incubated for 2h at room temperature and resorufin fluorescence was determined using a PerkinElmer ViewLux plate reader (excitation at 528 nm and emission at 590 nm).	Trypanosoma brucei	580	ALA3430912	organism-based format	GSK Kinetoplastid Screening
10.	ALA3431455	F	GSK_TCAKS: pIC50 Trypanosoma brucei viability assay using luminiscence. Assay plates (1536-well) were prepared. T. brucei cultures were harvested and counted in a CASY cell counter. A parasite suspension was prepared at a final concentration of 5 * 10^4 cells/mL in complete HMI9 medium and 6uL was dispensed to all wells containing compounds using a Multidrop Combi dispenser. 6uL of HMI9 media with 0.4% DMSO were dispensed to control columns. Plates were incubated for 24 h at 37C, 5% CO2 then tempered for 10 min at room temperature. ATP content was quantified by adding 4 uL of CellTiter-Glo Reagent (Promega) according to the manufacturer's instructions. Luminescence was measured using a ViewLux.	Trypanosoma brucei	572	ALA3430912	organism-based format	GSK Kinetoplastid Screening
11.	ALA3431456	T	GSK_TCAKS: pIC50 HepG2 human cell line viability assay using luminiscence. Actively growing HepG2 cells were removed from a T-175 TC flask using 5mL Eagle's MEM (containing 10% FBS, 1% NEAA, 1% penicillin/streptomycin) and dispersed in the medium by repeated pipetting. Seeding density was checked to ensure that new mono-layers were not more than 50% confluent at the time of harvesting. Cell suspension was added to 500 mL of the same medium at a final density of 1.2 * 10^5 cells/mL. This cell suspension was dispensed (25 uL, 3000 cells per well) into 384-well clear-bottom plates using a Multidrop Combi dispenser. Prior to addition of the cell suspension, the screening compounds (250 nL) were pre-dispensed into the plates with an Echo liquid handler. Plates were incubated for 48 h at 37C, 5% CO2. After incubation, plates equilibrated at room temperature for 30 min before proceeding to develop the luminescent signal. The signal developer, CellTiter-Glo Reagent, was allowed to equilibrate at room temperature for 30 min and added to the plates (25uL per well) using a Multidrop Combi dispenser. Plates were left for 10 min at room temperature for stabilization and then read using a ViewLux.	Homo sapiens	592	ALA3430912	cell-based format	GSK Kinetoplastid Screening
12.	ALA3431457	F	GSK_TCAKS: pIC50 NIH 3T3 cell line viability assay using luminiscence. Tissue culture surface treated assay plates (1536-well) were used. 5uL of a 2 * 10^5 cells per mL solution were dispensed per well (~10^3 host cells), into plates already containing compounds which had been pre-dispensed. The 100% cells survival control was determined exposing the host-cells to a 0.5% DMSO concentration, whereas the 100% toxicity control was achieved by seeding the cells in presence of Amphotericin B at 50uM. Plates were incubated for four days at 37C, 5% CO2. The assay readout was developed by addition of 5uL of CellTiter-Glo Reagent following the manufacturer's instructions. Luminiscence was measured using a ViewLux.	Mus musculus	222	ALA3430912	cell-based format	GSK Kinetoplastid Screening
13.	ALA3431458	F	GSK_TCAKS: pIC50 CYP51 from Trypanosoma cruzi.	Trypanosoma cruzi	584	ALA3430912	assay format	GSK Kinetoplastid Screening