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BioAssay: AID 602175

Developing isoenzyme selective inhibitors of phospholipase D as an antiviral therapeutic - Secondary Assay Panel

Phospholipase D's (PLD) involvement in enumerate cellular functions and pathways through the generation of the second messenger phosphatidic acid. Phosphatidic acid (PtdOH) is a critical signaling lipid that generated by the hydrolysis of phosphatidylcholine and other amine containing phospholipids. Phosphatidic acid participates in both G protein-coupled receptor and receptor tyrosine kinase more ..
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 Tested Compounds
 Tested Compounds
All(3)
 
 
Unspecified(3)
 
 
 Tested Substances
 Tested Substances
All(3)
 
 
Unspecified(3)
 
 
 Related BioAssays
 Related BioAssays
AID: 602175
Data Source: Southern Research Specialized Biocontainment Screening Center (PLD_Panel_01)
BioAssay Type: Panel
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-01-06
Hold-until Date: 2013-01-03
Modify Date: 2013-01-03

Data Table ( Complete ):           View All Data
Tested Compounds:
Related Experiments
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AIDNameTypeProbeComment
588854Summary AID for PLD inhibitor development projectSummary3 depositor-specified cross reference
602143Cellular PLD2 isoenzyme activity assays at a set concentrationScreening same project related to Summary assay
602144Cellular PLD1 isoenzyme activity assays at set concentrationScreening same project related to Summary assay
602147Cellular PLD2 concentration responseConfirmatory same project related to Summary assay
602149PLD2 purified enzyme concentration responseConfirmatory same project related to Summary assay
602150PLD1 purified enzyme concentration responseConfirmatory same project related to Summary assay
602151Cellular PLD1 concentration responseConfirmatory same project related to Summary assay
602153ML270, ML271, ML272 Competition in Radioligand Binding assays (Ricerca)Other same project related to Summary assay
623933ML298, ML299 Competition in Radioligand Binding assays (Ricerca)Other same project related to Summary assay
624000PLD2 purified enzyme concentration response (PLD2_Assay_3B_Lib32)Confirmatory same project related to Summary assay
624001PLD1 purified enzyme concentration response (PLD1_Assay_3A_Lib32)Confirmatory same project related to Summary assay
624003PLD1 Cellular concentration response (PLD1_Assay_2A_Lib32)Confirmatory same project related to Summary assay
624004PLD2 Cellular concentration response (PLD2_Assay_2B_Lib32)Confirmatory same project related to Summary assay
624008PLD1 Cellular isoenzyme activity (PLD1_Assay_1A_Lib32)Screening same project related to Summary assay
624009PLD2 Cellular isoenzyme activity (PLD2_Assay_1B_Lib32)Screening same project related to Summary assay
Description:
Phospholipase D's (PLD) involvement in enumerate cellular functions and pathways through the generation of the second messenger phosphatidic acid. Phosphatidic acid (PtdOH) is a critical signaling lipid that generated by the hydrolysis of phosphatidylcholine and other amine containing phospholipids. Phosphatidic acid participates in both G protein-coupled receptor and receptor tyrosine kinase signal transduction networks. Two mammalian isoforms of PLD have been identified, PLD1 and PLD2, which share 53% sequence identity and are subject to different regulatory mechanisms (1). In addition to a role for PLD in cancer cell survival it has been shown that this enzyme is the major source of signaling PtdOH in many cells, including neutrophils, macrophages, and airway epithelial cells that play key roles in defense against infection. PLD1 was recently confirmed as a primary regulator of Fc-gamma receptor-stimulated ROS production in neutrophils (4) using a strategic combination of genetic knockouts and pharmacological inhibitors. A recent report in Nature identified PLD2 as a gene that when downregulated drastically reduced viral replication of a panel of influenza strains including avian H5N1, endemic H1N1 and the pandemic swine flu of 2009 (5), although two related reports (6,7) using similar approaches failed to identify PLD isoenzymes as being relevant in influenza. Also because of PLD's role in vesicular trafficking, more specifically the link between PLD activity and influenza virus haemagglutinin (HA) trafficking to the plasma membrane (8), we hypothesize that this enzyme is a critical target to combat influenza infection. Current therapeutics strategies rely on vaccines or molecules targeting viral neuraminidase, both of which are subject to rapid resistance development.

In preliminary findings, PLD1 and PLD2 have been strongly implicated as playing essential roles in the entry of influenza virus into human airway epithelial cells. Given that PLD1-and PLD2-knockout animals are viable and appear to lack any obvious deficits, we hypothesize that PLD is a novel and therapeutically attractive target for blocking influenza infection. We propose to optimize potency, isoenzyme selectivity, and drug metabolism/pharmacokinetic parameters to develop chemical tool compounds into a preclinical lead compound for therapeutic development.

The development of these tool compounds will be essential in understanding the roles played by PLD in modulating cell responses to oxidative stress that is involved in a variety of human diseases including cancer, infectious disease, cardiovascular diseases and other conditions that are consequences of chronic, pathological inflammation. The development of these tool compounds has empowered the PLD signaling field. Still, major challenges remain to develop more potent and selective compounds that differentiate between PLD1 and PLD2 isoenzymes.

Following the primary screen, the hits were evaluated in a panel virus screen to evaluate the potential to modulate viral replication, similar to the reduced viral replication of influenza strains.
Panel Information
PLD_01
    Data Table(All)Show more
PID§NameSubstancePanel TargetsDescriptionAdditional Information
ActiveInactive
1Influenza A3Influenza A/Puerto Rico/8/1934 (H1N1)Taxonomy id: 211044
2Influenza B3Influenza B/Lee/40Taxonomy id: 107412
3Dengue Virus 212Dengue Virus 2 (New Guinea Strain C)Taxonomy id: 11060
4Herpes Simplex Virus Type 121Herpes Simplex Virus Type 1Taxonomy id: 10298
5Adenovirus Type 13Adenovirus Type 1Taxonomy id: 10535
6Human immunodeficiency virus 33Human immunodeficiency virus 3Taxonomy id: 35274
7Parainfluenza Type 321Parainfluenza Type 3Taxonomy id: 11216
8Respiratory syncytial virus21Human respiratory syncytial virus A strain LongTaxonomy id: 11260
9Human coxsackievirus A721Human coxsackievirus A7Taxonomy id: 42787
10Human coxsackievirus B321Human coxsackievirus B3Taxonomy id: 12072
11Human enterovirus 713Human enterovirus 71 (strain BRCR)Taxonomy id: 69153
12Human poliovirus 112Human poliovirus 1Taxonomy id: 12080
13Human rhinovirus 1B3Human rhinovirus 1BTaxonomy id: 12129
14Venezuelan equine encephalitis3Venezuelan equine encephalitis virus (strain TC-83)Taxonomy id: 11037
15Vaccinia virus WR3Vaccinia virus WRTaxonomy id: 10254

§ Panel component ID.
Protocol
Briefly, virus and cells are mixed in the presence of test compound and incubated for the required assay duration as specified. Each virus is pre-titered such that control wells exhibit 85 to 95% loss of cell viability due to virus replication. Therefore, antiviral effect or cytoprotection is observed when compounds prevent virus replication. A standardized plate format is used for cytoprotection assays. Each plate contains cell control wells (cells only), virus control wells (cells plus virus), drug cytotoxicity wells (cells plus drug only), positive control drug wells (cells plus virus plus literature reported drug control) and experimental wells (drug plus cells plus virus). Samples are evaluated for antiviral efficacy with triplicate measurements at 6 - 10 concentrations in order to determine EC50 values and with duplicate measurements to determine cytotoxicity, if detectable. The standard plate format for testing compounds concentrations used a representative high-test concentration of 100 muM. A positive control antiviral compound is included with each assay. The end point was read after the predetermined incubation time using either Promega's CellTiter 96 Reagent or Cell TiterGlo. Individual protocol descriptions are listed below:

Dengue Virus Type 2: Huh7 cells were cultured in complete DMEM (Dulbecco's Modified Eagle's Medium supplemented with 10% FBS, 2.0mM L-Glutamine, 100 units/ml Penicillin, 100ug/ml Streptomycin and .1mM non-essential amino acids) and diluted to a concentration of 5 x 10;3 cells/well. The diluted cells were dispensed to the cell control wells and the drug cytotoxicity wells. The Huh7 cells were are mixed with Dengue Type 2 New Guinea C strain and dispensed to the virus control wells, the positive drug control wells (Ribavirin) and the experimental wells. The plates were incubated for 7 days at 37C, 5% CO2. At assay termination, the assay plates are stained with the soluble tetrazolium-based dye MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; CellTiter 96 Reagent, Promega) to determine cell viability and quantify compound toxicity. MTS is metabolized by the mitochondrial enzymes of metabolically active cells to yield a soluble formazan product, allowing the rapid quantitative analysis of cell viability and compound cytotoxicity. This reagent is a stable, single solution that does not require preparation before use. At termination of the assay, 10-25 uL of MTS reagent is added per well (10% final concentration based on volume) and the microtiter plates are then incubated for 4-6 hrs at 37C, 5% CO2 to assess cell viability. Adhesive plate sealers are used in place of the lids, the sealed plate are inverted several times to mix the soluble formazan product and the plate is read spectrophotometrically at 490/650 nm with a Molecular Devices Vmax or SpectraMax Plus plate reader.#

Herpes Simplex Virus - 1 (HSV-1): Vero cells were cultured in complete DMEM (Dulbecco's Modified Eagle's Medium supplemented with 10% FBS, 2.0mM L-Glutamine, 100 units/ml Penicillin, 100ug/ml Streptomycin and .1mM non-essential amino acids) and diluted to a concentration of 1 x 10;4 cells/well. The diluted cells were dispensed to the cell control wells and the drug cytotoxicity wells. The Vero cells were mixed with HSV-1 strain HF and dispensed to the virus control wells, the positive drug control wells (Acyclovir) and the experimental wells. The plates were incubated for 5 days at 37C, 5% CO2. At assay termination, the assay plates are stained with the soluble tetrazolium-based dye MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; CellTiter 96 Reagent, Promega) to determine cell viability and quantify compound toxicity. MTS is metabolized by the mitochondrial enzymes of metabolically active cells to yield a soluble formazan product, allowing the rapid quantitative analysis of cell viability and compound cytotoxicity. This reagent is a stable, single solution that does not require preparation before use. At termination of the assay, 10-25 uL of MTS reagent is added per well (10% final concentration based on volume) and the microtiter plates are then incubated for 4-6 hrs at 37C, 5% CO2 to assess cell viability. Adhesive plate sealers are used in place of the lids, the sealed plate are inverted several times to mix the soluble formazan product and the plate is read spectrophotometrically at 490/650 nm with a Molecular Devices Vmax or SpectraMax Plus plate reader.#

Adenovirus Type 1: HeLa cells were cultured in complete DMEM (Dulbecco's Modified Eagle's Medium supplemented with 5% FBS, 2.0mM L-Glutamine, 100 units/ml Penicillin, 100ug/ml Streptomycin and .1mM non-essential amino acids) and diluted to a concentration of 5 x 10;3 cells/well. The diluted cells were dispensed to the cell control wells and the drug cytotoxicity wells. The HeLa cells were mixed with Adenovirus Type 1 and dispensed to the virus control wells, the positive drug control wells (Ribavirin) and the experimental wells. The plates were incubated for 7 days at 37C, 5% CO2. At assay termination, the assay plates are stained with the soluble tetrazolium-based dye MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; CellTiter 96 Reagent, Promega) to determine cell viability and quantify compound toxicity. MTS is metabolized by the mitochondrial enzymes of metabolically active cells to yield a soluble formazan product, allowing the rapid quantitative analysis of cell viability and compound cytotoxicity. This reagent is a stable, single solution that does not require preparation before use. At termination of the assay, 10-25 uL of MTS reagent is added per well (10% final concentration based on volume) and the microtiter plates are then incubated for 4-6 hrs at 37C, 5% CO2 to assess cell viability. Adhesive plate sealers are used in place of the lids, the sealed plate are inverted several times to mix the soluble formazan product and the plate is read spectrophotometrically at 490/650 nm with a Molecular Devices Vmax or SpectraMax Plus plate reader.

Influenza A/PR/8/34 (H1N1): A549 cells were cultured in complete F-12K (Kaighn's Modification of Ham's F-12 Media supplemented with 10% FBS, 2.0mM L-Glutamine, 100 units/ml Penicillin, 100ug/ml Streptomycin, .1mM non-essential amino acids and 1.0mM sodium pyruvate) and diluted to a concentration of 1 x 10;4 cells/well. The diluted cells were dispensed to the cell control wells and the drug cytotoxicity wells. The A549 cells were mixed with Influenza A/PR/8/34 and dispensed to the virus control wells, the positive drug control wells (Ribavirin and Zanamivir) and the experimental wells. The plates were incubated for 7 days at 37C, 5% CO2. At assay termination, the assay plates are stained with the soluble tetrazolium-based dye MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; CellTiter 96 Reagent, Promega) to determine cell viability and quantify compound toxicity. MTS is metabolized by the mitochondrial enzymes of metabolically active cells to yield a soluble formazan product, allowing the rapid quantitative analysis of cell viability and compound cytotoxicity. This reagent is a stable, single solution that does not require preparation before use. At termination of the assay, 10-25 uL of MTS reagent is added per well (10% final concentration based on volume) and the microtiter plates are then incubated for 4-6 hrs at 37C, 5% CO2 to assess cell viability. Adhesive plate sealers are used in place of the lids, the sealed plate are inverted several times to mix the soluble formazan product and the plate is read spectrophotometrically at 490/650 nm with a Molecular Devices Vmax or SpectraMax Plus plate reader.

Influenza B/Lee/40: A549 cells were cultured in complete F-12K (Kaighn's Modification of Ham's F-12 Media supplemented with 10% FBS, 2.0mM L-Glutamine, 100 units/ml Penicillin, 100ug/ml Streptomycin, .1mM non-essential amino acids and 1.0mM sodium pyruvate) and diluted to a concentration of 1 x 10;4 cells/well. The diluted cells were dispensed to the cell control wells and the drug cytotoxicity wells. The A549 cells were mixed with Influenza B/Lee/40 and dispensed to the virus control wells, the positive drug control wells (Ribavirin) and the experimental wells. The plates were incubated for 7 days at 37C, 5% CO2. At assay termination, the assay plates are stained with the soluble tetrazolium-based dye MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; CellTiter 96 Reagent, Promega) to determine cell viability and quantify compound toxicity. MTS is metabolized by the mitochondrial enzymes of metabolically active cells to yield a soluble formazan product, allowing the rapid quantitative analysis of cell viability and compound cytotoxicity. This reagent is a stable, single solution that does not require preparation before use. At termination of the assay, 10-25 uL of MTS reagent is added per well (10% final concentration based on volume) and the microtiter plates are then incubated for 4-6 hrs at 37C, 5% CO2 to assess cell viability. Adhesive plate sealers are used in place of the lids, the sealed plate are inverted several times to mix the soluble formazan product and the plate is read spectrophotometrically at 490/650 nm with a Molecular Devices Vmax or SpectraMax Plus plate reader.
Parainfluenza Type 3: Vero cells were cultured in complete DMEM (Dulbecco's Modified Eagle's Medium supplemented with 10% FBS, 2.0mM L-Glutamine, 100 units/ml Penicillin, 100ug/ml Streptomycin and .1mM non-essential amino acids) and diluted to a concentration of 1 x 10;4 cells/well. The diluted cells were dispensed to the cell control wells and the drug cytotoxicity wells. The Vero cells were are mixed with Parainfluenza Type 3 strain C 243 and dispensed to the virus control wells, the positive drug control wells (Enviroxime) and the experimental wells. The plates were incubated for 7 days at 37C, 5% CO2. At assay termination, the assay plates are stained with the soluble tetrazolium-based dye MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; CellTiter 96 Reagent, Promega) to determine cell viability and quantify compound toxicity. MTS is metabolized by the mitochondrial enzymes of metabolically active cells to yield a soluble formazan product, allowing the rapid quantitative analysis of cell viability and compound cytotoxicity. This reagent is a stable, single solution that does not require preparation before use. At termination of the assay, 10-25 uL of MTS reagent is added per well (10% final concentration based on volume) and the microtiter plates are then incubated for 4-6 hrs at 37C, 5% CO2 to assess cell viability. Adhesive plate sealers are used in place of the lids, the sealed plate are inverted several times to mix the soluble formazan product and the plate is read spectrophotometrically at 490/650 nm with a Molecular Devices Vmax or SpectraMax Plus plate reader.#

Respiratory Syncitial Virus (RSV): A549 cells were cultured in complete F-12K (Kaighn's Modification of Ham's F-12 Media supplemented with 10% FBS, 2.0mM L-Glutamine, 100 units/ml Penicillin, 100ug/ml Streptomycin, .1mM non-essential amino acids and 1.0mM sodium pyruvate) and diluted to a concentration of 1 x 10;4 cells/well. The diluted cells were dispensed to the cell control wells and the drug cytotoxicity wells. The A549 cells were mixed with RSV Long strain and dispensed to the virus control wells, the positive drug control wells (Ribavirin) and the experimental wells. The plates were incubated for 7 days at 37C, 5% CO2. At assay termination, the assay plates are stained with the soluble tetrazolium-based dye MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; CellTiter 96 Reagent, Promega) to determine cell viability and quantify compound toxicity. MTS is metabolized by the mitochondrial enzymes of metabolically active cells to yield a soluble formazan product, allowing the rapid quantitative analysis of cell viability and compound cytotoxicity. This reagent is a stable, single solution that does not require preparation before use. At termination of the assay, 10-25 uL of MTS reagent is added per well (10% final concentration based on volume) and the microtiter plates are then incubated for 4-6 hrs at 37C, 5% CO2 to assess cell viability. Adhesive plate sealers are used in place of the lids, the sealed plate are inverted several times to mix the soluble formazan product and the plate is read spectrophotometrically at 490/650 nm with a Molecular Devices Vmax or SpectraMax Plus plate reader.

Coxsackie A7 virus: MRC-5 cells were cultured in complete EMEM (Eagle's Minimal Essential Medium supplemented with 10% FBS, 2.0mM L-Glutamine, 100 units/ml Penicillin, 100ug/ml Streptomycin and .1mM non-essential amino acids) and diluted to a concentration of 1 x 10;4 cells/well. The diluted cells were dispensed to the cell control wells and the drug cytotoxicity wells. The MRC-5 cells were mixed with Coxsackie A7 virus and dispensed to the virus control wells, the positive drug control wells (Ribavirin) and the experimental wells. The plates were incubated for 7 days at 37C, 5% CO2. At assay termination, the assay plates are stained with the soluble tetrazolium-based dye MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; CellTiter 96 Reagent, Promega) to determine cell viability and quantify compound toxicity. MTS is metabolized by the mitochondrial enzymes of metabolically active cells to yield a soluble formazan product, allowing the rapid quantitative analysis of cell viability and compound cytotoxicity. This reagent is a stable, single solution that does not require preparation before use. At termination of the assay, 10-25 uL of MTS reagent is added per well (10% final concentration based on volume) and the microtiter plates are then incubated for 4-6 hrs at 37C, 5% CO2 to assess cell viability. Adhesive plate sealers are used in place of the lids, the sealed plate are inverted several times to mix the soluble formazan product and the plate is read spectrophotometrically at 490/650 nm with a Molecular Devices Vmax or SpectraMax Plus plate reader.#

Coxsackie B3 Virus: Vero cells were cultured in complete DMEM (Dulbecco's Modified Eagle's Medium supplemented with 10% FBS, 2.0mM L-Glutamine, 100 units/ml Penicillin, 100ug/ml Streptomycin and .1mM non-essential amino acids) and diluted to a concentration of 1 x 10;4 cells/well. The diluted cells were dispensed to the cell control wells and the drug cytotoxicity wells. The Vero cells were mixed with Coxsackie B3 Virus and dispensed to the virus control wells, the positive drug control wells (Enviroxime) and the experimental wells. The plates were incubated for 7 days at 37C, 5% CO2. At assay termination, the assay plates are stained with the soluble tetrazolium-based dye MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; CellTiter 96 Reagent, Promega) to determine cell viability and quantify compound toxicity. MTS is metabolized by the mitochondrial enzymes of metabolically active cells to yield a soluble formazan product, allowing the rapid quantitative analysis of cell viability and compound cytotoxicity. This reagent is a stable, single solution that does not require preparation before use. At termination of the assay, 10-25 uL of MTS reagent is added per well (10% final concentration based on volume) and the microtiter plates are then incubated for 4-6 hrs at 37C, 5% CO2 to assess cell viability. Adhesive plate sealers are used in place of the lids, the sealed plate are inverted several times to mix the soluble formazan product and the plate is read spectrophotometrically at 490/650 nm with a Molecular Devices Vmax or SpectraMax Plus plate reader.#

Enterovirus 71: RD cells were cultured in complete DMEM (Dulbecco's Modified Eagle's Medium supplemented with 10% FBS, 2.0mM L-Glutamine, 100 units/ml Penicillin, 100ug/ml Streptomycin and .1mM non-essential amino acids) and diluted to a concentration of 5 x 10;3 cells/well. The diluted cells were dispensed to the cell control wells and the drug cytotoxicity wells. The RD cells were mixed with Enterovirus 71Virus and dispensed to the virus control wells, the positive drug control wells (Enviroxime) and the experimental wells. The plates were incubated for 7 days at 37C, 5% CO2. At assay termination, the assay plates are stained with the soluble tetrazolium-based dye MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; CellTiter 96 Reagent, Promega) to determine cell viability and quantify compound toxicity. MTS is metabolized by the mitochondrial enzymes of metabolically active cells to yield a soluble formazan product, allowing the rapid quantitative analysis of cell viability and compound cytotoxicity. This reagent is a stable, single solution that does not require preparation before use. At termination of the assay, 10-25 uL of MTS reagent is added per well (10% final concentration based on volume) and the microtiter plates are then incubated for 4-6 hrs at 37C, 5% CO2 to assess cell viability. Adhesive plate sealers are used in place of the lids, the sealed plate are inverted several times to mix the soluble formazan product and the plate is read spectrophotometrically at 490/650 nm with a Molecular Devices Vmax or SpectraMax Plus plate reader.

Polio Virus Type 1: Vero cells were cultured in complete DMEM (Dulbecco's Modified Eagle's Medium supplemented with 10% FBS, 2.0mM L-Glutamine, 100 units/ml Penicillin, 100ug/ml Streptomycin and .1mM non-essential amino acids) and diluted to a concentration of 1 x 10;4 cells/well. The diluted cells were dispensed to the cell control wells and the drug cytotoxicity wells. The Vero cells were mixed with Polio Virus Type 1 Chat strain and dispensed to the virus control wells, the positive drug control wells (Enviroxime) and the experimental wells. The plates were incubated for 7 days at 37C, 5% CO2. At assay termination, the assay plates are stained with the soluble tetrazolium-based dye MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; CellTiter 96 Reagent, Promega) to determine cell viability and quantify compound toxicity. MTS is metabolized by the mitochondrial enzymes of metabolically active cells to yield a soluble formazan product, allowing the rapid quantitative analysis of cell viability and compound cytotoxicity. This reagent is a stable, single solution that does not require preparation before use. At termination of the assay, 10-25 uL of MTS reagent is added per well (10% final concentration based on volume) and the microtiter plates are then incubated for 4-6 hrs at 37C, 5% CO2 to assess cell viability. Adhesive plate sealers are used in place of the lids, the sealed plate are inverted several times to mix the soluble formazan product and the plate is read spectrophotometrically at 490/650 nm with a Molecular Devices Vmax or SpectraMax Plus plate reader.#

Rhinovirus Type 1B: HeLa cells were cultured in complete DMEM (Dulbecco's Modified Eagle's Medium supplemented with 5% FBS, 2.0mM L-Glutamine, 100 units/ml Penicillin, 100ug/ml Streptomycin and .1mM non-essential amino acids) and diluted to a concentration of 5 x 10;3 cells/well. The diluted cells were dispensed to the cell control wells and the drug cytotoxicity wells. The HeLa cells were mixed with Rhinovirus Type 1B and dispensed to the virus control wells, the positive drug control wells (Enviroxime) and the experimental wells. The plates were incubated for 7 days at 37C, 5% CO2. At assay termination, the assay plates are stained with the soluble tetrazolium-based dye MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; CellTiter 96 Reagent, Promega) to determine cell viability and quantify compound toxicity. MTS is metabolized by the mitochondrial enzymes of metabolically active cells to yield a soluble formazan product, allowing the rapid quantitative analysis of cell viability and compound cytotoxicity. This reagent is a stable, single solution that does not require preparation before use. At termination of the assay, 10-25 uL of MTS reagent is added per well (10% final concentration based on volume) and the microtiter plates are then incubated for 4-6 hrs at 37C, 5% CO2 to assess cell viability. Adhesive plate sealers are used in place of the lids, the sealed plate are inverted several times to mix the soluble formazan product and the plate is read spectrophotometrically at 490/650 nm with a Molecular Devices Vmax or SpectraMax Plus plate reader.

Human Immunodeficiency Virus Type 1 (HIV-1): CEM-SS cells were cultured in complete RPMI (Rosewell Park Memorial Institute medium with 10% FBS, 2.0mM L-Glutamine, 100 units/ml Penicillin, 100ug/ml Streptomycin and .1mM non-essential amino acids) and diluted to a concentration of 2.5 x 10;3 cells/well. The diluted cells were dispensed to the cell control wells and the drug cytotoxicity wells. The CEM-SS cells were mixed with HIV-1 IIIB and dispensed to the virus control wells, the positive drug control wells (Zidovudine) and the experimental wells. The plates were incubated for 6 days at 37C, 5% CO2. At assay termination, the assay plates are stained with the soluble tetrazolium-based dye MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; CellTiter 96 Reagent, Promega) to determine cell viability and quantify compound toxicity. MTS is metabolized by the mitochondrial enzymes of metabolically active cells to yield a soluble formazan product, allowing the rapid quantitative analysis of cell viability and compound cytotoxicity. This reagent is a stable, single solution that does not require preparation before use. At termination of the assay, 10-25 uL of MTS reagent is added per well (10% final concentration based on volume) and the microtiter plates are then incubated for 4-6 hrs at 37C, 5% CO2 to assess cell viability. Adhesive plate sealers are used in place of the lids, the sealed plate are inverted several times to mix the soluble formazan product and the plate is read spectrophotometrically at 490/650 nm with a Molecular Devices Vmax or SpectraMax Plus plate reader.

Venezuelan Equine Encephalitis Virus (VEEV): Vero76 cells were cultured in complete EMEM (Eagle's Minimal Essential Medium supplemented with 5% FBS, 1% L-Glutamine/ Penicillin/ Streptomycin and 1% sodium pyruvate) and diluted to a concentration of 1.8 x 10;5 cells/ml. The diluted cells were dispensed to the cell control wells and the drug cytotoxicity wells. The Vero76 cells were mixed with VEEV TC-83 strain and dispensed to the virus control wells, the positive drug control wells (Mycophenolic Acid 10uM) and the experimental wells. The plates were incubated for 3 days at 37C, 5% CO2. The assay plates were equilibrated to room temperature for 30 minutes and then an equal volume of CellTiter-Glo reagent (Promega Inc.) was added to each well. Plates were incubated for 10 min at room temperature and luminescence was measured using a Perkin Elmer Envision multi-label reader.

Vaccinia Virus: JC cells were cultured in complete RPMI (Rosewell Park Memorial Institute medium with 10% FBS, 2.0mM L-Glutamine, 100 units/ml Penicillin, 100ug/ml Streptomycin and .1mM non-essential amino acids) and diluted to a concentration of 8 x 10;4 cells/ml. The diluted cells were dispensed to the cell control wells and the drug cytotoxicity wells. The JC cells were mixed with Vaccinia Western Reserve strain and dispensed to the virus control wells, the positive drug control wells (Cidofovir 25uM) and the experimental wells. The plates were incubated for 5 days at 37C, 5% CO2. The assay plates were equilibrated to room temperature for 30 minutes and then an equal volume of CellTiter-Glo reagent (Promega Inc.) was added to each well. Plates were incubated for 10 min at room temperature and luminescence was measured using a Perkin Elmer Envision multi-label reader.
BHK21 cells were cultured in complete EMEM (Eagle's Minimal Essential Medium supplemented with 5% FBS, 1% L-Glutamine/ Penicillin/ Streptomycin and 1% sodium pyruvate) and diluted to a concentration of 2 x 10;4 cells/ml. The diluted cells were dispensed to the cell control wells and the drug cytotoxicity wells. The BHK21 cells were mixed with Vaccinia Western Reserve strain and dispensed to the virus control wells, the positive drug control wells (Cidofovir 25uM) and the experimental wells. The plates were incubated for 5 days at 37C, 5% CO2. The assay plates were equilibrated to room temperature for 30 minutes and then an equal volume of CellTiter-Glo reagent (Promega Inc.) was added to each well. Plates were incubated for 10 min at room temperature and luminescence was measured using a Perkin Elmer Envision multi-label reader.

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Comment
Data Analysis: Confirmatory assays were performed in triplicate for each assay in the panel as were cytotoxicity assays. IC50 and TC50 values were calculated for each replicate. Compounds were considered active in a panel if an IC50 of <100 was reached and the TC50 was not reached (>100).

Comment: Possible artifacts in this assay include, but are not limited to, compounds that interfere with the formazan reaction, absorb luminescence, or precipitate.
Result Definitions
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TIDNameDescriptionPID§Panel TargetsHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1Influenza A Outcome1Outcome
2Influenza A IC50 Rep 1*1FloatμM
3Influenza A IC50 Rep 21FloatμM
4Influenza A IC50 Rep 31FloatμM
5Influenza A TC50 Modifier Rep 11String
6Influenza A TC50 Rep 11FloatμM
7Influenza A TC50 Modifier Rep 21String
8Influenza A TC50 Rep 21FloatμM
9Influenza A TC50 Modifier Rep 31String
10Influenza A TC50 Rep 31FloatμM
11Influenza B Outcome2Outcome
12Influenza B IC50 Rep 1*2FloatμM
13Influenza B IC50 Rep 22FloatμM
14Influenza B IC50 Rep 32FloatμM
15Influenza B TC50 Modifier Rep 12String
16Influenza B TC50 Rep 12FloatμM
17Influenza B TC50 Modifier Rep 22String
18Influenza B TC50 Rep 22FloatμM
19Influenza B TC50 Modifier Rep 32String
20Influenza B TC50 Rep 32FloatμM
21Dengue Virus 2 Outcome3Outcome
22Dengue Virus 2 IC50 Modifier Rep 13String
23Dengue Virus 2 IC50 Rep 1*3FloatμM
24Dengue Virus 2 IC50 Modifier Rep 23String
25Dengue Virus 2 IC50 Rep 23FloatμM
26Dengue Virus 2 IC50 Modifier Rep 33String
27Dengue Virus 2 IC50 Rep 33FloatμM
28Dengue Virus 2 TC50 Modifier Rep 13String
29Dengue Virus 2 TC50 Rep 13FloatμM
30Dengue Virus 2 TC50 Modifier Rep 23String
31Dengue Virus 2 TC50 Rep 23FloatμM
32Dengue Virus 2 TC50 Modifier Rep 33String
33Dengue Virus 2 TC50 Rep 33FloatμM
34Herpes Simplex Virus Type 1 Outcome4Outcome
35Herpes Simplex Virus Type 1 IC50 Modifier Rep 14String
36Herpes Simplex Virus Type 1 IC50 Rep 1*4FloatμM
37Herpes Simplex Virus Type 1 IC50 Rep 24FloatμM
38Herpes Simplex Virus Type 1 IC50 Modifier Rep 34String
39Herpes Simplex Virus Type 1 IC50 Rep 34FloatμM
40Herpes Simplex Virus Type 1 TC50 Modifier Rep 14String
41Herpes Simplex Virus Type 1 TC50 Rep 14FloatμM
42Herpes Simplex Virus Type 1 TC50 Modifier Rep 24String
43Herpes Simplex Virus Type 1 TC50 Rep 24FloatμM
44Herpes Simplex Virus Type 1 TC50 Modifier Rep 34String
45Herpes Simplex Virus Type 1 TC50 Rep 34FloatμM
46Adenovirus Type 1 Outcome5Outcome
47Adenovirus Type 1 IC50 Modifier Rep 15String
48Adenovirus Type 1 IC50 Rep 1*5FloatμM
49Adenovirus Type 1 IC50 Modifier Rep 25String
50Adenovirus Type 1 IC50 Rep 25FloatμM
51Adenovirus Type 1 IC50 Modifier Rep 35String
52Adenovirus Type 1 IC50 Rep 35FloatμM
53Adenovirus Type 1 TC50 Modifier Rep 15String
54Adenovirus Type 1 TC50 Rep 15FloatμM
55Adenovirus Type 1 TC50 Modifier Rep 25String
56Adenovirus Type 1 TC50 Rep 25FloatμM
57Adenovirus Type 1 TC50 Modifier Rep 35String
58Adenovirus Type 1 TC50 Rep 35FloatμM
59Human immunodeficiency virus 3 Outcome6Outcome
60Human immunodeficiency virus 3 IC50 Modifier Rep 16String
61Human immunodeficiency virus 3 IC50 Rep 1*6FloatμM
62Human immunodeficiency virus 3 IC50 Modifier Rep 26String
63Human immunodeficiency virus 3 IC50 Rep 26FloatμM
64Human immunodeficiency virus 3 IC50 Modifier Rep 36String
65Human immunodeficiency virus 3 IC50 Rep 36FloatμM
66Human immunodeficiency virus 3 TC50 Rep 16FloatμM
67Human immunodeficiency virus 3 TC50 Rep 26FloatμM
68Human immunodeficiency virus 3 TC50 Rep 36FloatμM
69Parainfluenza Type 3 Outcome7Outcome
70Parainfluenza Type 3 IC50 Modifier Rep 17String
71Parainfluenza Type 3 IC50 Rep 1*7FloatμM
72Parainfluenza Type 3 IC50 Modifier Rep 27String
73Parainfluenza Type 3 IC50 Rep 27FloatμM
74Parainfluenza Type 3 IC50 Modifier Rep 37String
75Parainfluenza Type 3 IC50 Rep 37FloatμM
76Parainfluenza Type 3 TC50 Modifier Rep 17String
77Parainfluenza Type 3 TC50 Rep 17FloatμM
78Parainfluenza Type 3 TC50 Modifier Rep 27String
79Parainfluenza Type 3 TC50 Rep 27FloatμM
80Parainfluenza Type 3 TC50 Modifier Rep 37String
81Parainfluenza Type 3 TC50 Rep 37FloatμM
82Respiratory syncytial virus Outcome8Outcome
83Respiratory syncytial virus IC50 Modifier Rep 18String
84Respiratory syncytial virus IC50 Rep 1*8FloatμM
85Respiratory syncytial virus IC50 Modifier Rep 28String
86Respiratory syncytial virus IC50 Rep 28FloatμM
87Respiratory syncytial virus IC50 Modifier Rep 38String
88Respiratory syncytial virus IC50 Rep 38FloatμM
89Respiratory syncytial virus TC50 Modifier Rep 18String
90Respiratory syncytial virus TC50 Rep 18FloatμM
91Respiratory syncytial virus TC50 Modifier Rep 28String
92Respiratory syncytial virus TC50 Rep 28FloatμM
93Respiratory syncytial virus TC50 Modifier Rep 38String
94Respiratory syncytial virus TC50 Rep 38FloatμM
95Human coxsackievirus A7 Outcome9Outcome
96Human coxsackievirus A7 IC50 Modifier Rep 19String
97Human coxsackievirus A7 IC50 Rep 1*9FloatμM
98Human coxsackievirus A7 IC50 Modifier Rep 29String
99Human coxsackievirus A7 IC50 Rep 29FloatμM
100Human coxsackievirus A7 IC50 Modifier Rep 39String
101Human coxsackievirus A7 IC50 Rep 39FloatμM
102Human coxsackievirus A7 TC50 Modifier Rep 19String
103Human coxsackievirus A7 TC50 Rep 19FloatμM
104Human coxsackievirus A7 TC50 Modifier Rep 29String
105Human coxsackievirus A7 TC50 Rep 29FloatμM
106Human coxsackievirus A7 TC50 Modifier Rep 39String
107Human coxsackievirus A7 TC50 Rep 39FloatμM
108Human coxsackievirus B3 Outcome10Outcome
109Human coxsackievirus B3 IC50 Modifier Rep 110String
110Human coxsackievirus B3 IC50 Rep 1*10FloatμM
111Human coxsackievirus B3 IC50 Modifier Rep 210String
112Human coxsackievirus B3 IC50 Rep 210FloatμM
113Human coxsackievirus B3 IC50 Modifier Rep 310String
114Human coxsackievirus B3 IC50 Rep 310FloatμM
115Human coxsackievirus B3 TC50 Modifier Rep 110String
116Human coxsackievirus B3 TC50 Rep 110FloatμM
117Human coxsackievirus B3 TC50 Modifier Rep 210String
118Human coxsackievirus B3 TC50 Rep 210FloatμM
119Human coxsackievirus B3 TC50 Modifier Rep 310String
120Human coxsackievirus B3 TC50 Rep 310FloatμM
121Human enterovirus 71 Outcome11Outcome
122Human enterovirus 71 IC50 Rep 1*11FloatμM
123Human enterovirus 71 IC50 Rep 211FloatμM
124Human enterovirus 71 IC50 Rep 311FloatμM
125Human enterovirus 71 TC50 Modifier Rep 111String
126Human enterovirus 71 TC50 Rep 111FloatμM
127Human enterovirus 71 TC50 Modifier Rep 211String
128Human enterovirus 71 TC50 Rep 211FloatμM
129Human enterovirus 71 TC50 Modifier Rep 311String
130Human enterovirus 71 TC50 Rep 311FloatμM
131Human poliovirus 1 Outcome12Outcome
132Human poliovirus 1 IC50 Modifier Rep 112String
133Human poliovirus 1 IC50 Rep 1*12FloatμM
134Human poliovirus 1 IC50 Modifier Rep 212String
135Human poliovirus 1 IC50 Rep 212FloatμM
136Human poliovirus 1 IC50 Modifier Rep 312String
137Human poliovirus 1 IC50 Rep 312FloatμM
138Human poliovirus 1 TC50 Modifier Rep 112String
139Human poliovirus 1 TC50 Rep 112FloatμM
140Human poliovirus 1 TC50 Modifier Rep 212String
141Human poliovirus 1 TC50 Rep 212FloatμM
142Human poliovirus 1 TC50 Modifier Rep 312String
143Human poliovirus 1 TC50 Rep 312FloatμM
144Human rhinovirus 1B Outcome13Outcome
145Human rhinovirus 1B IC50 Rep 1*13FloatμM
146Human rhinovirus 1B IC50 Rep 213FloatμM
147Human rhinovirus 1B IC50 Rep 313FloatμM
148Human rhinovirus 1B TC50 Modifier Rep 113String
149Human rhinovirus 1B TC50 Rep 113FloatμM
150Human rhinovirus 1B TC50 Modifier Rep 213String
151Human rhinovirus 1B TC50 Rep 213FloatμM
152Human rhinovirus 1B TC50 Modifier Rep 313String
153Human rhinovirus 1B TC50 Rep 313FloatμM
154Venezuelan equine encephalitis Outcome14Outcome
155Venezuelan equine encephalitis IC50 Modifier Rep 114String
156Venezuelan equine encephalitis IC50 Rep 1*14FloatμM
157Venezuelan equine encephalitis IC50 Modifier Rep 214String
158Venezuelan equine encephalitis IC50 Rep 214FloatμM
159Venezuelan equine encephalitis IC50 Modifier Rep 314String
160Venezuelan equine encephalitis IC50 Rep 314FloatμM
161Venezuelan equine encephalitis TC50 Modifier Rep 114String
162Venezuelan equine encephalitis TC50 Rep 114FloatμM
163Venezuelan equine encephalitis TC50 Modifier Rep 214String
164Venezuelan equine encephalitis TC50 Rep 214FloatμM
165Venezuelan equine encephalitis TC50 Modifier Rep 314String
166Venezuelan equine encephalitis TC50 Rep 314FloatμM
167Vaccinia virus WR Outcome15Outcome
168Vaccinia virus WR IC50 Modifier Rep 115String
169Vaccinia virus WR IC50 Rep 1*15FloatμM
170Vaccinia virus WR IC50 Modifier Rep 215String
171Vaccinia virus WR IC50 Rep 215FloatμM
172Vaccinia virus WR IC50 Modifier Rep 315String
173Vaccinia virus WR IC50 Rep 315FloatμM
174Vaccinia virus WR TC50 Rep 115FloatμM
175Vaccinia virus WR TC50 Rep 215FloatμM
176Vaccinia virus WR TC50 Rep 315FloatμM

* Activity Concentration. § Panel component ID.
Additional Information
Grant Number: P01 ES013125

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