External ID: bumalcp250

Protocol: ADST Assay Protocol Summary:

The 1536-well SYBR Green viability assay was based on the method of Bennett et al (2004) and described previously in 1536-well format (Yuan J., et al. 2009). For screening 3 ul culture medium was dispensed into 1536 well black clear bottom plates (Aurora Biotechnologies) using a Multidrop Combi (Thermo Fisher Scientific Inc.), 23 nL compounds in DMSO were added by a pin transfer (Cleveland, P.H. and Koutz, P.J, 2005) using a pin tool-based delivery system (Kalypsys), and 5 ul of erythrocytes infected with P. falciparum one of five lines (cp250, Dd2, HB3, 7G8 or GB4, 0.3% parasitemia, 2.5% hematocrit final concentration) were added. The plates were incubated at 37C in a humidified incubator in 5% CO2 for 72 h, and 2 ul lysis buffer (20 mM Tris HCl, 10 mM EDTA, 0.16% saponin, 1.6% triton X, 10X SYBR Green I (supplied as 10,000X final concentration by Invitrogen) was added to each well. The plates were mixed for 25 sec with gentle shaking and incubated overnight at room temperature in the dark. The following morning, fluorescence intensity at 485(14) nm excitation and 535(25) nm emission wavelengths was measured on an EnVision (Perkin Elmer) plate reader. Plate reads were normalized relative to the control inhibitor (0.29 uM artemisinin) and vehicle (DMSO) wells present on each plate and then corrected by an algorithm using vehicle-only control plates at the beginning and end of the compound plate stack.

References
Shah, S. (2010). The Fever: how malaria has ruled humankind for 500,000 years. New York, Sarah Crichton Books.
CDC web site, http://www.cdc.gov/malaria/about/biology/index.html

Mu, J., Myers, R.A., Jiang, H., Liu, S., Ricklefs, S., Waisberg, M., Chotivanich, K., Wilairatana, P., Krudsood, S., White, N.J., et Plasmodium falciparum genome-wide scans for positive selection, recombination hot spots and resistance to antimalarial drugs Nature Genetics 2010, 42, pp 268-271

Bennett TN, Paguio, M., Gligorijevic, B., Seudieu, C., Kosar, A. D., Davidson, E., and Roepe, P. D. Novel, rapid, and inexpensive cell-based quantification of antimalarial drug efficacy Antimicrobial Agents Chemother. 2004, 48(5): pp 1807-10

Yuan J, Johnson RL, Huang RL, Wichterman J, Jiang HY, Hayton K, Fidock DA, Wellems TE, Inglese J, Austin CP, Su XZ. Genetic mapping of targets mediating differential chemical phenotypes in Plasmodium falciparum. Nature Chem Biol 2009, 5(10), pp 765-771.

Cleveland, P.H. and Koutz, P.J. Nanoliter Dispensing for uHTS Using Pin Tools, ASSAY and Drug Devel Technol 2005, 3(2), pp, 213-225

Comment: Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.

External ID: adst-hNprA-bu-o2

Protocol: PROTOCOL TABLES (as described by Inglese J, Shamu CE and Guy RK. 2007)

SEQUENCE No. (e.g., 1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE and DESCRIPTION.
GLOSENSOR PROTOCOL
1. Cells. Seed 1500 HEK293 cGMP-sensor cells expressing hNpr1 per well in 4uL into Greiner 1536-well white/solid bottom high base, tissue culture-treated plates.
2. Incubation. Incubate plated cells at 37C in 5% CO2, 95% RH overnight.
3. Reagent. Add 1uL per well GloSensor reagent from Promega (2% final concentration) or CO2 independent media vehicle control to respective columns of Greiner 1536-well plates.
4. Incubation. Incubate plated cells at room temperature for 2 h, protect from light.
5. Compounds. Library compounds and A-71915 control added at 23 nL per well, transferred by Pin Tool.
6. Incubation. Incubate plated cells for 30 min. at room temperature, protect from light.
7. Detector. GloSensor baseline pre-read luminescence measured using CCD-based camera (Viewlux).
8. Compound. Add 1uL of hNppA peptide to 0.1 nM final concentration to stimulate hNpr1 or CO2 independent media as vehicle control.
9. Incubation. Incubate treated cells at room temperature for 20 min., protect from light.
10. Detector. GloSensor post-hNppA luminescence measured using CCD-based camera (Viewlux).

NOTES (numbers refer to Sequence numbers above)

1. HEK293 cells expressing hNpr1 and GIP/pGS-40F grown to 85-95% confluence (48-72 h) in T175 culture flasks (CO2 independent media supplemented with 10% FBS, 1% Pen/Strep antibiotics and 1 ug/mL puromycin). Trypsinize and harvest cells from T175 culture flasks and filter through 40 um cell strainer. Cells counted with Invitrogen Countess (30,000,000 - 50,000,000 cells/flask). Cells diluted to a final concentration of 375 cells/uL in CO2 independent media and a Millipore multidrop used to dispense 1500 cells per well (4uL/well) into 13 1536-well Greiner White/Solid bottom, high base tissue-culture treated (TC) plates and one Aurora 1536-well Black/Clear bottom, low base, TC plate (for visual inspection of cell health throughout the duration of the assay).

2. Cells incubated overnight at ambient temperature, 24 h. at 37C in 95% RH incubator protected from light, covered with metal lid with gas-exchange holes.

3. Prepare GloSensor Reagent (Promega): Reconstitute GloSensor luciferase substrate by adding 8.2mL Hepes buffer to 250mg substrate, mix well, protect from light and stored at -80C. Prepare a fresh 12% GloSensor solution in CO2 independent supplemented plating media (see above). Mix well and store at ambient temperature, protected from light. 1uL 12% GloSensor Luciferase Reagent added (2% final concentration) to respective columns of 13 x 1536-well Greiner plate with BioRaptr, 1uL CO2 independent plating media added to respective columns as vehicle control with BioRaptr.

4. Cells incubated for 2 h. at ambient temperature, protected from light with metal lid with gas-exchange holes.

5. A-71915 stock solution prepared (10 mM in DMSO). Working dilution for dispense = 10 mM stock dilution (38.3 uM final concentration; 16-pt 1:2 titration in duplicate = final concentration range of 38.3 uM - 1.17 nM). 23nl of library compounds, A-71915 antagonist control or DMSO vehicle control transferred to the 13 cell-containing Greiner white/solid bottom high base, tissue culture-treated plates in 11 interplate 1:3 titrations (see Yasgar, A. et al. 2008) with Pintool.

6. Cells incubated for 30-50 min. at ambient temperature protected from light with metal lid with gas-exchange holes.

7. Read cellular baseline GloSensor luminescence in presence of A-71915 antagonist or test library compounds on the ViewLux [Exposure = 10 s.; Gain = High; Speed = Slow; and Binning = 2X.]

8. hNppA peptide agonist solution prepared at 250 uM (in H2O) stock concentration. From this a working dilution was prepared at 0.6 nM in CO2 Independent media. 1uL of 0.6 nM hNppA agonist (0.1 nM final concentration) or CO2 Independent media vehicle control dispensed into respective columns of 13 x Greiner 1536-well white/solid bottom, tissue culture plates with BioRaptr.

9. Cells incubated with agonist for 20 min. at ambient temperature, protected from light, covered with metal lid with gas-exchange holes.

10. Read final Luminescence on the ViewLux. [Exposure = 10 s.; Gain = High; Speed = Slow; and Binning = 2X.]

REFERENCES:

Inglese J, Shamu CE and Guy RK, Reporting data from high throughput screening of small molecule libraries, Nature Chemical Biology, 2007, 3(8): 438-441. doi.org/10.1038/nchembio0807-438

Yasgar A, Shinn P, Jadhav A, Auld DS, Michael S, Zheng W, Austin CP, Inglese J and Simeonov A, Compound Management for Quantitative High-Throughput Screening. J. Assoc. Lab. Auto., 2008, 13: 79-89. doi: 10.1016/j.jala.2007.12.004

Comment: Compound Ranking:
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, with an activity curve class of 4, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. For activity curve class = -1.1, score = 80+abs((log_ac50+4.5)*inf_activity/20). For activity.curve_class == -1.2 && abs(activity.max_response) > 6*10, score = 60+abs((log_ac50+4.5)*inf_activity/20). For activity.curve_class == -2.1 || ( activity.curve_class==-2.2 && abs(activity.max_response) > 6*10), score = 40+abs((log_ac50+4.5)*inf_activity/20). For activity.curve_class == -1.2 || activity.curve_class == -2.2, score = 20+abs((log_ac50+4.5)*inf_activity/20). Inconclusive compounds, with a preread curve class other than 4, have PUBCHEM_ACTIVITY_SCORE of 10.

External ID: bumalhb3

Protocol: ADST Assay Protocol Summary:

The 1536-well SYBR Green viability assay was based on the method of Bennett et al (2004) and described previously in 1536-well format (Yuan J., et al. 2009). For screening 3 ul culture medium was dispensed into 1536 well black clear bottom plates (Aurora Biotechnologies) using a Multidrop Combi (Thermo Fisher Scientific Inc.), 23 nL compounds in DMSO were added by a pin transfer (Cleveland, P.H. and Koutz, P.J, 2005) using a pin tool-based delivery system (Kalypsys), and 5 ul of erythrocytes infected with P. falciparum one of five lines (cp250, Dd2, HB3, 7G8 or GB4, 0.3% parasitemia, 2.5% hematocrit final concentration) were added. The plates were incubated at 37C in a humidified incubator in 5% CO2 for 72 h, and 2 ul lysis buffer (20 mM Tris HCl, 10 mM EDTA, 0.16% saponin, 1.6% triton X, 10X SYBR Green I (supplied as 10,000X final concentration by Invitrogen) was added to each well. The plates were mixed for 25 sec with gentle shaking and incubated overnight at room temperature in the dark. The following morning, fluorescence intensity at 485(14) nm excitation and 535(25) nm emission wavelengths was measured on an EnVision (Perkin Elmer) plate reader. Plate reads were normalized relative to the control inhibitor (0.29 uM artemisinin) and vehicle (DMSO) wells present on each plate and then corrected by an algorithm using vehicle-only control plates at the beginning and end of the compound plate stack.

References
Shah, S. (2010). The Fever: how malaria has ruled humankind for 500,000 years. New York, Sarah Crichton Books.
CDC web site, http://www.cdc.gov/malaria/about/biology/index.html

Mu, J., Myers, R.A., Jiang, H., Liu, S., Ricklefs, S., Waisberg, M., Chotivanich, K., Wilairatana, P., Krudsood, S., White, N.J., et Plasmodium falciparum genome-wide scans for positive selection, recombination hot spots and resistance to antimalarial drugs Nature Genetics 2010, 42, pp 268-271

Bennett TN, Paguio, M., Gligorijevic, B., Seudieu, C., Kosar, A. D., Davidson, E., and Roepe, P. D. Novel, rapid, and inexpensive cell-based quantification of antimalarial drug efficacy Antimicrobial Agents Chemother. 2004, 48(5): pp 1807-10

Yuan J, Johnson RL, Huang RL, Wichterman J, Jiang HY, Hayton K, Fidock DA, Wellems TE, Inglese J, Austin CP, Su XZ. Genetic mapping of targets mediating differential chemical phenotypes in Plasmodium falciparum. Nature Chem Biol 2009, 5(10), pp 765-771.

Cleveland, P.H. and Koutz, P.J. Nanoliter Dispensing for uHTS Using Pin Tools, ASSAY and Drug Devel Technol 2005, 3(2), pp, 213-225

Comment: Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.

External ID: bumal7g8

Protocol: ADST Assay Protocol Summary:

The 1536-well SYBR Green viability assay was based on the method of Bennett et al (2004) and described previously in 1536-well format (Yuan J., et al. 2009). For screening 3 ul culture medium was dispensed into 1536 well black clear bottom plates (Aurora Biotechnologies) using a Multidrop Combi (Thermo Fisher Scientific Inc.), 23 nL compounds in DMSO were added by a pin transfer (Cleveland, P.H. and Koutz, P.J, 2005) using a pin tool-based delivery system (Kalypsys), and 5 ul of erythrocytes infected with P. falciparum one of five lines (cp250, Dd2, HB3, 7G8 or GB4, 0.3% parasitemia, 2.5% hematocrit final concentration) were added. The plates were incubated at 37C in a humidified incubator in 5% CO2 for 72 h, and 2 ul lysis buffer (20 mM Tris HCl, 10 mM EDTA, 0.16% saponin, 1.6% triton X, 10X SYBR Green I (supplied as 10,000X final concentration by Invitrogen) was added to each well. The plates were mixed for 25 sec with gentle shaking and incubated overnight at room temperature in the dark. The following morning, fluorescence intensity at 485(14) nm excitation and 535(25) nm emission wavelengths was measured on an EnVision (Perkin Elmer) plate reader. Plate reads were normalized relative to the control inhibitor (0.29 uM artemisinin) and vehicle (DMSO) wells present on each plate and then corrected by an algorithm using vehicle-only control plates at the beginning and end of the compound plate stack.

References
Shah, S. (2010). The Fever: how malaria has ruled humankind for 500,000 years. New York, Sarah Crichton Books.
CDC web site, http://www.cdc.gov/malaria/about/biology/index.html

Mu, J., Myers, R.A., Jiang, H., Liu, S., Ricklefs, S., Waisberg, M., Chotivanich, K., Wilairatana, P., Krudsood, S., White, N.J., et Plasmodium falciparum genome-wide scans for positive selection, recombination hot spots and resistance to antimalarial drugs Nature Genetics 2010, 42, pp 268-271

Bennett TN, Paguio, M., Gligorijevic, B., Seudieu, C., Kosar, A. D., Davidson, E., and Roepe, P. D. Novel, rapid, and inexpensive cell-based quantification of antimalarial drug efficacy Antimicrobial Agents Chemother. 2004, 48(5): pp 1807-10

Yuan J, Johnson RL, Huang RL, Wichterman J, Jiang HY, Hayton K, Fidock DA, Wellems TE, Inglese J, Austin CP, Su XZ. Genetic mapping of targets mediating differential chemical phenotypes in Plasmodium falciparum. Nature Chem Biol 2009, 5(10), pp 765-771.

Cleveland, P.H. and Koutz, P.J. Nanoliter Dispensing for uHTS Using Pin Tools, ASSAY and Drug Devel Technol 2005, 3(2), pp, 213-225

Comment: Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.

External ID: bumalgb4

Protocol: ADST Assay Protocol Summary:

The 1536-well SYBR Green viability assay was based on the method of Bennett et al (2004) and described previously in 1536-well format (Yuan J., et al. 2009). For screening 3 ul culture medium was dispensed into 1536 well black clear bottom plates (Aurora Biotechnologies) using a Multidrop Combi (Thermo Fisher Scientific Inc.), 23 nL compounds in DMSO were added by a pin transfer (Cleveland, P.H. and Koutz, P.J, 2005) using a pin tool-based delivery system (Kalypsys), and 5 ul of erythrocytes infected with P. falciparum one of five lines (cp250, Dd2, HB3, 7G8 or GB4, 0.3% parasitemia, 2.5% hematocrit final concentration) were added. The plates were incubated at 37C in a humidified incubator in 5% CO2 for 72 h, and 2 ul lysis buffer (20 mM Tris HCl, 10 mM EDTA, 0.16% saponin, 1.6% triton X, 10X SYBR Green I (supplied as 10,000X final concentration by Invitrogen) was added to each well. The plates were mixed for 25 sec with gentle shaking and incubated overnight at room temperature in the dark. The following morning, fluorescence intensity at 485(14) nm excitation and 535(25) nm emission wavelengths was measured on an EnVision (Perkin Elmer) plate reader. Plate reads were normalized relative to the control inhibitor (0.29 uM artemisinin) and vehicle (DMSO) wells present on each plate and then corrected by an algorithm using vehicle-only control plates at the beginning and end of the compound plate stack.

References
Shah, S. (2010). The Fever: how malaria has ruled humankind for 500,000 years. New York, Sarah Crichton Books.
CDC web site, http://www.cdc.gov/malaria/about/biology/index.html

Mu, J., Myers, R.A., Jiang, H., Liu, S., Ricklefs, S., Waisberg, M., Chotivanich, K., Wilairatana, P., Krudsood, S., White, N.J., et Plasmodium falciparum genome-wide scans for positive selection, recombination hot spots and resistance to antimalarial drugs Nature Genetics 2010, 42, pp 268-271

Bennett TN, Paguio, M., Gligorijevic, B., Seudieu, C., Kosar, A. D., Davidson, E., and Roepe, P. D. Novel, rapid, and inexpensive cell-based quantification of antimalarial drug efficacy Antimicrobial Agents Chemother. 2004, 48(5): pp 1807-10

Yuan J, Johnson RL, Huang RL, Wichterman J, Jiang HY, Hayton K, Fidock DA, Wellems TE, Inglese J, Austin CP, Su XZ. Genetic mapping of targets mediating differential chemical phenotypes in Plasmodium falciparum. Nature Chem Biol 2009, 5(10), pp 765-771.

Cleveland, P.H. and Koutz, P.J. Nanoliter Dispensing for uHTS Using Pin Tools, ASSAY and Drug Devel Technol 2005, 3(2), pp, 213-225

Comment: Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.

External ID: bumaldd2

Protocol: ADST Assay Protocol Summary:

The 1536-well SYBR Green viability assay was based on the method of Bennett et al (2004) and described previously in 1536-well format (Yuan J., et al. 2009). For screening 3 ul culture medium was dispensed into 1536 well black clear bottom plates (Aurora Biotechnologies) using a Multidrop Combi (Thermo Fisher Scientific Inc.), 23 nL compounds in DMSO were added by a pin transfer (Cleveland, P.H. and Koutz, P.J, 2005) using a pin tool-based delivery system (Kalypsys), and 5 ul of erythrocytes infected with P. falciparum one of five lines (cp250, Dd2, HB3, 7G8 or GB4, 0.3% parasitemia, 2.5% hematocrit final concentration) were added. The plates were incubated at 37C in a humidified incubator in 5% CO2 for 72 h, and 2 ul lysis buffer (20 mM Tris HCl, 10 mM EDTA, 0.16% saponin, 1.6% triton X, 10X SYBR Green I (supplied as 10,000X final concentration by Invitrogen) was added to each well. The plates were mixed for 25 sec with gentle shaking and incubated overnight at room temperature in the dark. The following morning, fluorescence intensity at 485(14) nm excitation and 535(25) nm emission wavelengths was measured on an EnVision (Perkin Elmer) plate reader. Plate reads were normalized relative to the control inhibitor (0.29 uM artemisinin) and vehicle (DMSO) wells present on each plate and then corrected by an algorithm using vehicle-only control plates at the beginning and end of the compound plate stack.

References
Shah, S. (2010). The Fever: how malaria has ruled humankind for 500,000 years. New York, Sarah Crichton Books.
CDC web site, http://www.cdc.gov/malaria/about/biology/index.html

Mu, J., Myers, R.A., Jiang, H., Liu, S., Ricklefs, S., Waisberg, M., Chotivanich, K., Wilairatana, P., Krudsood, S., White, N.J., et Plasmodium falciparum genome-wide scans for positive selection, recombination hot spots and resistance to antimalarial drugs Nature Genetics 2010, 42, pp 268-271

Bennett TN, Paguio, M., Gligorijevic, B., Seudieu, C., Kosar, A. D., Davidson, E., and Roepe, P. D. Novel, rapid, and inexpensive cell-based quantification of antimalarial drug efficacy Antimicrobial Agents Chemother. 2004, 48(5): pp 1807-10

Yuan J, Johnson RL, Huang RL, Wichterman J, Jiang HY, Hayton K, Fidock DA, Wellems TE, Inglese J, Austin CP, Su XZ. Genetic mapping of targets mediating differential chemical phenotypes in Plasmodium falciparum. Nature Chem Biol 2009, 5(10), pp 765-771.

Cleveland, P.H. and Koutz, P.J. Nanoliter Dispensing for uHTS Using Pin Tools, ASSAY and Drug Devel Technol 2005, 3(2), pp, 213-225

Comment: Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.

External ID: VSVM-OFFLINE

Protocol: Two uL of HEK293 cell suspension are dispensed at 1000 cells/well into solid white 1536-well plates (Grenier) using a Multidrop Combi (Thermo Scientific). After addition of 23 nL compound by a pin tool (Kalypsys), the plate is incubated 1 h at 37 degrees C and then 3 uL of virus 1:100 dilution VSV-MARV is added. After 28 hr, 4 uL of assay reagent is added and the plates are read using a ViewLux (Perkin Elmer). Assays are performed in sub-saturating amounts of virus (MOI <0.5), therefore luciferase signals reflect the amount (titer) of virus able to infect the cells in presence of the compound.

Comment: Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.

External ID: VSVL-OFFLINE

Protocol: For screening, 1000 cells in 2 ul/well were dispensed into white solid 1536-well plates (Greiner) using a solenoid-based dispenser. Following transfer of 23nl compound or DMSO vehicle by a pin tool, 3 ul/well of VSV-LV was added. The plates were centrifuged 1 min at 1000 RPM and then incubated 16 hr at 37 C and 5% CO2. After addition of 4 ul/well SteadyLite (PerkinElmer) detection reagent, the plates were incubated 10 min at ambient temperature and luminescence was measured on a ViewLux (Perkin Elmer) plate reader.

Keywords: NIH Roadmap, MLPCN, MLI, MLSMR, qHTS, NCGC, Lassa virus, luciferase, cell assay, infection

Comment: Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.