External ID: HAPV101

Protocol: NCGC Assay Protocol Summary:

The CellTiter-Glo# Luminescent Cell Viability Assay (Promega) is a homogeneous method to determine the number of viable cells in culture based on quantitation of the ATP present, which signals the presence of metabolically active cells. The homogeneous assay procedure involves adding a single reagent directly to cells cultured in serum-supplemented medium. Addition and mixing of the reagent results in cell lysis and generation of a luminescent signal proportional to the amount of ATP present. The amount of ATP is directly proportional to the number of cells present.

- Chemicals were dissolved with dimethyl sulfoxide (DMSO) into 12 different concentrations ranging from 0.26 nM to 46.0 uM and were aliquoted to 1536-well plate format via pin tool (Kalypsys, San Diego, CA). Negative control was established using vehicle, while positive control was established using staurosporine.
- Cells were grown to a concentration of at least 106 cells/ml, volume of at least 100 ml, and viability of 85% before treatment. For treatment, cells were filtered, diluted with fresh media to 3-4E5cells/ml, and plated to a tissue-culture treated 1,536 well white/solid bottom assay plates (Grenier Bio-One North Americal, NC) at 2000 cells/5uL/well using a flying reagent dispenser (Aurora discovery, Carlsbad, CA).
- ViewLux plate reader (PerkinElmer, Shelton, CT) was used to detect luminescent intensity in each well 40 hours post chemical treatment.

Comment: This is a profiling assay where compounds were screened against a panel of 81 immortalized human lymphoblast cell lines. Ranking of compounds within each cell line is described below.

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".
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: HAPC101

Protocol: NCGC Assay Protocol Summary:

The Caspase-Glo 3/7 Assay (Promega) provides a homogeneous luminescent assay that measures caspase-3/7 activities. The assay provides a proluminescent caspase-3/7 DEVD-aminoluciferin substrate and a proprietary thermostable luciferase in a reagent optimized for caspase-3/7 activity, luciferase activity and cell lysis. Adding the single Caspase-Glo 3/7 Reagent in an "add-mix-measure" format results in cell lysis, followed by caspase cleavage of the substrate. This liberates free aminoluciferin, which is consumed by the luciferase, generating a "glow-type" luminescent signal. The signal is proportional to caspase-3/7 activity. The stabilized luciferase and proprietary buffer system improve assay performance across a wide range of assay conditions, and the assay is less likely to be affected by compound interference unlike fluorescent- or colorimetric-based assays. The Caspase-Glo 3/7 Assay is designed for use with multiwell plate formats using either purified enzyme or cells in culture.
- Chemicals were dissolved with dimethyl sulfoxide (DMSO) into 12 different concentrations ranging from 0.26 nM to 46.0 uM and were aliquoted to 1536-well plate format via pin tool (Kalypsys, San Diego, CA). Negative control was established using vehicle, while positive control was established using staurosporine.
- Cells were grown to a concentration of at least 106 cells/ml, volume of at least 100 ml, and viability of 85% before treatment. For treatment, cells were filtered, diluted with fresh media to 3-4*105cells/ml, and plated to a tissue-culture treated 1,536 well white/solid bottom assay plates (Grenier Bio-One North Americal, NC) at 2000 cells/5uL/well using a flying reagent dispenser (Aurora discovery, Carlsbad, CA).
- ViewLux plate reader (PerkinElmer, Shelton, CT) was used to detect luminescent intensity in each well 40 hours post chemical treatment.

Comment: This is a profiling assay where compounds were screened against a panel of 81 immortalized human lymphoblast cell lines. Ranking of compounds within each cell line is described below.

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, active compounds are expected to have the activator phenotype and apparent activators are ranked higher than compounds that showed apparent inhibition.
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: CYP273

Protocol: Tox21 Assay Protocol Summary:

Two ul of enzyme-substrate mix was dispensed into medium binding white/solid 1536-well plates (Greiner Bio-One North America Inc., Monroe, NC) using a BioRaptr Flying Reagent Dispenser (FRD, Beckman Coulter, Brea, CA). Compounds dissolved in DMSO and positive control (furafyllline) were transferred to the assay plates at 23 nl using a Pintool station (Wako, San Diego, CA). The assay plates were incubated at room temperature for 10 min. Then 2 ul of NADPH regeneration solution was added to each well of the assay plates using an FRD and incubated at room temperature for 1 h. The reaction was stopped by adding 4 ul of detection reagent using an FRD and after 20 min incubation at room temperature the luminescence signal was measured using a ViewLux plate reader (Perkin Elmer, Shelton, CT). Data were expressed as relative luminescence units.

Comment: Disclaimer:

Although all reasonable efforts have been made to ensure the accuracy and reliability of the data, caution should be exercised when interpreting the results as artifacts are possible from nonspecific effects such as assay signal interference. The curve fitting and activity calls presented here are based on the NCATS analysis methods. Alternative analysis methods and interpretations of the data are available at EPA (http://actor.epa.gov) and NTP (http://tools.niehs.nih.gov/cebs3/ui/).

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: HERG01

Protocol: NCGC Assay Protocol Summary:

HERG assay (FluxORTM thallium flux assay) was initially developed by Invitrogen/Molecular Probes, and then miniaturized into 1536-well plate in a homogeneous format by NCGC. This assay measures the activity of potassium channel using thallium dye (FluxOR) flux as surrogate measurement for potassium into the cells with a FDSS-7000 kinetic plate reader (Hamamatsu Corp., Hamamatsu City, Japan). The hERG ion channel is transduced into mammalian cells (U2OS) using a baculovirus (Bacmam) construct harboring the hERG K+ ion channel. So far we screened LOPAC1280 library (Sigma), the NTP collection of 1408 compounds, and the NCGC Pharmaceutical Collection (NPC), in which many well defined HERG blockers are present. The rank order potencies of many of these compounds are similar to that of other HERG assays (membrane potential, Rb+ flux, patch clamp, etc). This quick and homogeneous assay is also found to be sensitive, specific, and robust.

Using the FluxORTM thallium flux assay, the activity of potassium channel using thallium dye (FluxOR) flux as surrogate measurement for potassium into the cells was measured in the U2OS cell line transduced with hERG K+ ion channel using a baculovirus (Bacmam) using Opti-MEM medium (Invitrogen) containing 2% fetal calf serum (FCS, HyCone) following loading buffer addition, compound treatment for around 10 minutes and finally adding stimulation buffer. The assay was performed in black clear Kalypsys 1536-well plates. In the screen, Astemizole was used as positive controls. Library compounds were measured for their ability to cause hERG channel blockage in the cell line, as reflected by a decrease in fluorescence intensity, in a concentration-dependent manner. Data were normalized to the controls for basal activity (DMSO only) and 100% inhibition (5uM Astemizole). AC50 values were determined from concentration-response data modeled with the standard Hill equation.

qHTS protocol for hERG-U2OS cellular assay

[Step] [Parameter] [Value] [Description]

1. Day 1: Replace medium in 70-80% confluent T225 flask with 2.5 mL of hERG-BacMam virus plus 12.5 mL of phosphate buffered saline (PBS) (corresponding roughly to a multiplicity of infection ratio of 100 virus particles/cell)
2. Incubation: 4 hrs @ room Temperature in Dark
3. Reagent; Remove virus; wash once with 25 ml DPBS
4. Reagent; 35 ml culture medium
5. Incubation; 37oC overnight
6. Day2: Reagent; 3 uL; 2000 U2OS cells/well
7. Time; 4 hr; 37oC incubation
8. Loading buffer; 1 uL; 0.7X;
9. Incubation: 1hr @ RT in Dark.
10. Compounds; 23 nL; 0.59 nM to 92 uM
11. Controls; 23 nL; Astemizole 1.4 nM to 92 uM
12. Time; 10min; 37oC incubation
13. Read fluorescence Intensity on FDSS for 10 Sec with 1 sec interval
14. Reagent; 1 uL; stimulation buffer
15. Read fluorescence Intensity on FDSS for 2 min with 1 sec interval
16. Detection; Fluorescence Intensity; FDSS

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: APP-Toga-CHIKV-nsP2-f2-f9

Protocol: PROTOCOL TABLE (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; DESCRIPTION.
1; Control / Compound; 20 nL; Echo 655 acoustic dispenser, Greiner 1536-well solid bottom black plate.
2; Enzyme; 4 uL; BioRAPTR FRD liquid dispenser (Beckman Coulter).
3; Incubation; 15 min; room temperature.
4; Reagent; 4 uL; 2.5 uM Peptide 2 substrate.
5; Incubation; 1 hr; room temperature.
6; Detection; Fluorescence; WiewLux microplate reader (PerkinElmer), 525 nm excitation, 598/25 nm emission.

NOTES (numbers refer to sequence numbers above).
1.Briefly, 20 nL DMSO, positive control ZnAc (20nM final concentration), and test compounds were transferred into a 1,536-well solid bottom black plate (789176-F, Greiner One) via Echo 655 acoustic dispenser (Beckman Coulter). For primary screens, compounds were tested at 7 concentrations, 1:3 dilution points ranging from 25 uM to 34 nM. Follow-up confirmatory screens were carried out at 11 concentrations, 1:3 dilution points from 25 uM to 0.42 nM.
2.Four uL nsP2pro enzyme mix (150 nM final concentration) in 10 mM Tris-HCl pH 8.0 with 0.01% Tween 20 assay buffer was dispensed into the plate using a BioRAPTR FRD liquid dispenser (Beckman Coulter).
3.The plate was incubated at room temperature (protected from light) for 15 min
4.Four microliter of peptide 2 substrate (2.5 uM final concentration) in assay buffer was added to the plate.
5.After 1 hour, plates were immediately read on a ViewLux high-throughput CCD imager (Exposure = 10 sec, Gain = High, Speed = Slow, Binning = 2X). The above assay was also incorporated in the NCATS HTS facility41, which allowed for robotic liquid and compound dispensing, microplate handling, and fluorescence reading.

REFERENCE:
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.

Comment: Disclaimer:
Although all reasonable efforts have been made to ensure the accuracy and reliability of the data, caution should be exercised when interpreting the results as artifacts are possible from nonspecific effects such as assay signal interference. The curve fitting and activity calls presented here are based on the NCATS analysis methods [1].

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. For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2.For all inactive compounds, with a ratio 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 a ratio activity curve class = -1.1, score = 80+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -1.2 && abs(ratio.max_response) > 6*10, score = 60+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -2.1 || ( ratio.curve_class==-2.2 && abs(ratio.max_response) > 6*10), score = 40+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -1.2 || ratio.curve_class == -2.2, score = 20+abs((log_ac50+4.5)*inf_activity/20). Inconclusive compounds, with a donor curve class other than 4, have PUBCHEM_ACTIVITY_SCORE of 10. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39.

Reference:
1. Inglese J, Auld DS, Jadhav A, et al. Quantitative high-throughput screening: a titration-based approach that efficiently identifies biological activities in large chemical libraries. Proc Natl Acad Sci U S A. 2006;103(31):11473-11478.

External ID: APP-Toga-CHIKV-nsP2-f3_f16

Protocol: PROTOCOL TABLE (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; DESCRIPTION.
1a; Reagent; 3 uL; Papain in assay buffer.
1b; Reagent; 3uL; Assay buffer
2; Compound; 17 nL; Echo 655 acoustic dispenser (Beckman Coulter).
3; Incubation; 15 min; room temperature in the dark.
4: Reagent; 1 uL; Z-FR-AMC in assay buffer.
5; Incubation; 15 sec, 271g; centrifugation.
6; Detection; Fluorescence; time 0, Viewlux microplate reader (PerkinElmer) 355 nm excitation, 460 nm emission
7; Incubation; 10 min; room temperature
8; Detection; Fluorescence; time 10; ViewLux microplate reader (PerkinElmer), 355 nm excitation, 460 nm emission.

NOTES (numbers refer to sequence numbers above).
1a. Medium-binding black solid-bottom Greiner plates (cat. 789176-F). Dispense 3 uL of 1.33X papain in assay buffer (100 mM sodium acetate, pH 5.5, 5 mM dl-cysteine, 0.01% Tween-20 v/v) to columns 1, 2, 5-48 via Wako/Kalypsys dispenser. Papain (cat. P4762, Sigma) was dispensed to produce 5 nM final concentration. Enzyme solution kept on ice during experiment. Prepare papain as 50% glycerol (v/v) at final concentration of 10 mg/mL (427 uM, 100 U/mL). Sources: microplates (Greiner 789176-F), papain (Sigma P4762), sodium acetate (Bioworld 40120009-2), cysteine (Sigma 861677), Tween-20 (Sigma P1379), water (GIBCO 10977-015; Gibco).
1b. Dispense 3 uL of assay buffer (100 mM sodium acetate, pH 5.5, 5 mM dl-cysteine, 0.01% Tween-20 v/v) to columns 3 and 4 via Wako/Kalypsys dispenser. Assay buffer solution kept on ice during experiment.
2. Compound addition by single pin transfer (17 nL) using Wako pintool. Compound solutions in source plate: 169 nM to 10 mM DMSO stock solutions. Final concentrations: 0.664 nM to 39.2 uM, respectively. Final DMSO concentration: 0.5% (v/v).
3. RT incubation. Kalypsys lids used as microplate coverings. Protected from light.
4. Dispense 1 uL of 4x substrate mixture in assay buffer (100 mM sodium acetate, pH 5.5, 5 mM dl-cysteine, 0.01% Tween-20 v/v) to columns 1-48 via Wako/Kalypsys dispenser. Z-FR-AMC substrate was dispensed to produce 2 uM final concentration. Substrate solution kept on ice during experiment. Prepare Z-FR-AMC as 32.1 mM stock solution in DMSO. Source: Z-FR-AMC (HCl salt) (Bachem I-1160).
5. Centrifugation step to remove bubbles.
6. ViewLux microplate reader (PerkinElmer, Waltham, MA). Optics: excitation filter 340 (30) nm, emission filter 450 (20) nm. Energy = 2000. Exposure time = 2 sec.
7. RT incubation. Incubation time 10 min.
8. ViewLux microplate reader. Optics: excitation filter 340 (30) nm, emission filter 450 (20) nm. Energy = 2000. Exposure time = 2 sec.

REFERENCE:
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.

Comment: Disclaimer:
Although all reasonable efforts have been made to ensure the accuracy and reliability of the data, caution should be exercised when interpreting the results as artifacts are possible from nonspecific effects such as assay signal interference. The curve fitting and activity calls presented here are based on the NCATS analysis methods [1].

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. For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, with a ratio 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 a ratio activity curve class = -1.1, score = 80+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -1.2 && abs(ratio.max_response) > 6*10, score = 60+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -2.1 || ( ratio.curve_class==-2.2 && abs(ratio.max_response) > 6*10), score = 40+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -1.2 || ratio.curve_class == -2.2, score = 20+abs((log_ac50+4.5)*inf_activity/20). Inconclusive compounds, with a donor curve class other than 4, have PUBCHEM_ACTIVITY_SCORE of 10. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39.

Reference:
1. Inglese J, Auld DS, Jadhav A, et al. Quantitative high-throughput screening: a titration-based approach that efficiently identifies biological activities in large chemical libraries. Proc Natl Acad Sci U S A. 2006;103(31):11473-11478.

External ID: APP-Toga-CHIKV-nsp2-p

Protocol: PROTOCOL TABLE (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; DESCRIPTION.
1; Control / Compound; 20 nL; Echo 655 acoustic dispenser, Greiner 1536-well solid bottom black plate.
2; Enzyme; 4 uL; BioRAPTR FRD liquid dispenser (Beckman Coulter).
3; Incubation; 15 min; room temperature.
4; Reagent; 4 uL; 2.5 uM Peptide 2 substrate.
5; Incubation; 1 hr; room temperature.
6; Detection; Fluorescence; WiewLux microplate reader (PerkinElmer), 525 nm excitation, 598/25 nm emission.

NOTES (numbers refer to sequence numbers above).
1. Briefly, 20 nL DMSO, positive control ZnAc (20nM final concentration), and test compounds were transferred into a 1,536-well solid bottom black plate (789176-F, Greiner One) via Echo 655 acoustic dispenser (Beckman Coulter). For primary screens, compounds were tested at 7 concentrations, 1:3 dilution points ranging from 25 uM to 34 nM. Follow-up confirmatory screens were carried out at 11 concentrations, 1:3 dilution points from 25 uM to 0.42 nM.
2. Four uL nsP2pro enzyme mix (150 nM final concentration) in 10 mM Tris-HCl pH 8.0 with 0.01% Tween 20 assay buffer was dispensed into the plate using a BioRAPTR FRD liquid dispenser (Beckman Coulter).
3. The plate was incubated at room temperature (protected from light) for 15 min
4. Four microliter of peptide 2 substrate (2.5 uM final concentration) in assay buffer was added to the plate.
5. After 1 hour, plates were immediately read on a ViewLux high-throughput CCD imager (Exposure = 10 sec, Gain = High, Speed = Slow, Binning = 2X). The above assay was also incorporated in the NCATS HTS facility41, which allowed for robotic liquid and compound dispensing, microplate handling, and fluorescence reading..

REFERENCE:
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.

Comment: Disclaimer:
Although all reasonable efforts have been made to ensure the accuracy and reliability of the data, caution should be exercised when interpreting the results as artifacts are possible from nonspecific effects such as assay signal interference. The curve fitting and activity calls presented here are based on the NCATS analysis methods [1].

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. For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, with a ratio 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 a ratio activity curve class = -1.1, score = 80+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -1.2 && abs(ratio.max_response) > 6*10, score = 60+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -2.1 || ( ratio.curve_class==-2.2 && abs(ratio.max_response) > 6*10), score = 40+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -1.2 || ratio.curve_class == -2.2, score = 20+abs((log_ac50+4.5)*inf_activity/20). Inconclusive compounds, with a donor curve class other than 4, have PUBCHEM_ACTIVITY_SCORE of 10. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39.

Reference:
1. Inglese J, Auld DS, Jadhav A, et al. Quantitative high-throughput screening: a titration-based approach that efficiently identifies biological activities in large chemical libraries. Proc Natl Acad Sci U S A. 2006;103(31):11473-11478.

External ID: APP-Toga-CHIKV-nsP2-f

Protocol: PROTOCOL TABLE (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; DESCRIPTION.
1; Enzyme; 2 uL; 1,536-well solid-bottom, black plate (Greiner Bio One).
2; Compounds; 20 nL; Echo 655 acoustic dispenser (Beckman Coulter).
3; Incubation; 15 min; room temperature in the dark.
4: Reagent; 2 uL; Ac-DE-[Lys(5-TAMRA)]-EE-[Abu-psi(COO)Ala]-S-[Lys(ECLIPSE-7)]-amide.
5; Detection; Fluorescence; time 0, ViewLux microplate reader (PerkinElmer) 525 nm excitation, 598/25 nm emission
6; Incubation; 15 min; room temperature
7; Detection; Fluorescence; time 15; ViewLux microplate reader (PerkinElmer), 525 nm excitation, 598/25 nm emission.

NOTES (numbers refer to sequence numbers above).
1. Two microliter of NS3-4A HCV enzyme solution (5 nM final concentration) in assay buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, with 0.01% Tween 20) were dispensed into a 1,536-well solid-bottom black plate (Greiner Bio One) using the Kalypsys Pintool Station.
2. Twenty nanoliter of compounds, positive control Aprotinin (50 uM final concentration), or neutral control DMSO were transferred to the assay plates. Compounds in counter-screen assays were tested at 11 concentrations, 1:3 dilution points from 25 uM to 0.42 nM.
3. The assay plates were incubated in the dark for 15 min.
4. After incubation, 2 uL of 0.625 uM substrate (Ac-DE-[Lys(5-TAMRA)]-EE-[Abu-psi(COO)Ala]-S-[Lys(ECLIPSE-7)]-amide) in assay buffer (for NS3-4A assay) was added using the Kalypsys Pintool Station.
5-7. Plates were immediately read at time 0 then covered with stainless steel gasket lids and incubated for 15 min at room temperature in the dark. Fluorescence intensity in the assay plates were again read after incubation using the ViewLux imager equipped with Tamara fluorescence optics. The change in fluorescence intensity over the 15-min reaction period was normalized against DMSO and inhibitor controls as described below.

REFERENCE:
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.

Comment: Disclaimer:
Although all reasonable efforts have been made to ensure the accuracy and reliability of the data, caution should be exercised when interpreting the results as artifacts are possible from nonspecific effects such as assay signal interference. The curve fitting and activity calls presented here are based on the NCATS analysis methods [1].

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. For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, with a ratio 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 a ratio activity curve class = -1.1, score = 80+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -1.2 && abs(ratio.max_response) > 6*10, score = 60+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -2.1 || ( ratio.curve_class==-2.2 && abs(ratio.max_response) > 6*10), score = 40+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -1.2 || ratio.curve_class == -2.2, score = 20+abs((log_ac50+4.5)*inf_activity/20). Inconclusive compounds, with a donor curve class other than 4, have PUBCHEM_ACTIVITY_SCORE of 10. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39.

Reference:
1. Inglese J, Auld DS, Jadhav A, et al. Quantitative high-throughput screening: a titration-based approach that efficiently identifies biological activities in large chemical libraries. Proc Natl Acad Sci U S A. 2006;103(31):11473-11478.

External ID: APP-Toga-CHIKV-nsP2-full-length

Protocol: PROTOCOL TABLE (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; DESCRIPTION.
1; Control / Compound; 20 nL; Echo 655 acoustic dispenser, Greiner 1536-well solid bottom black plate.
2; Enzyme; 4 uL; BioRAPTR FRD liquid dispenser (Beckman Coulter).
3; Incubation; 15 min; room temperature.
4; Reagent; 4 uL; 2.5 uM Peptide 2 substrate.
5; Incubation; 1 hr; room temperature.
6; Detection; Fluorescence; WiewLux microplate reader (PerkinElmer), 525 nm excitation, 598/25 nm emission.

NOTES (numbers refer to sequence numbers above).
1. Briefly, 20 nL DMSO, positive control ZnAc (20nM final concentration), and test compounds were transferred into a 1,536-well solid bottom black plate (789176-F, Greiner One) via Echo 655 acoustic dispenser (Beckman Coulter). For primary screens, compounds were tested at 7 concentrations, 1:3 dilution points ranging from 25 uM to 34 nM. Follow-up confirmatory screens were carried out at 11 concentrations, 1:3 dilution points from 25 uM to 0.42 nM.
2. Four uL NSP2 full length enzyme mix (150 nM final concentration) in 10 mM Tris-HCl pH 8.0 with 0.01% Tween 20 assay buffer was dispensed into the plate using a BioRAPTR FRD liquid dispenser (Beckman Coulter).
3. The plate was incubated at room temperature (protected from light) for 15 min
4. Four microliter of peptide 2 substrate (2.5 uM final concentration) in assay buffer was added to the plate.
5. After 1 hour, plates were immediately read on a ViewLux high-throughput CCD imager (Exposure = 10 sec, Gain = High, Speed = Slow, Binning = 2X). The above assay was also incorporated in the NCATS HTS facility41, which allowed for robotic liquid and compound dispensing, microplate handling, and fluorescence reading.

REFERENCE:
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.

Comment: Disclaimer:
Although all reasonable efforts have been made to ensure the accuracy and reliability of the data, caution should be exercised when interpreting the results as artifacts are possible from nonspecific effects such as assay signal interference. The curve fitting and activity calls presented here are based on the NCATS analysis methods [1].

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. For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, with a ratio 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 a ratio activity curve class = -1.1, score = 80+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -1.2 && abs(ratio.max_response) > 6*10, score = 60+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -2.1 || ( ratio.curve_class==-2.2 && abs(ratio.max_response) > 6*10), score = 40+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -1.2 || ratio.curve_class == -2.2, score = 20+abs((log_ac50+4.5)*inf_activity/20). Inconclusive compounds, with a donor curve class other than 4, have PUBCHEM_ACTIVITY_SCORE of 10. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39.

Reference:
1. Inglese J, Auld DS, Jadhav A, et al. Quantitative high-throughput screening: a titration-based approach that efficiently identifies biological activities in large chemical libraries. Proc Natl Acad Sci U S A. 2006;103(31):11473-11478.

External ID: APP-Toga-CHIKV-nsP2-f18-cell-based

Protocol: PROTOCOL TABLE (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; DESCRIPTION.
1a; Cell Transfection; Lipofectamine 2000.
1b; Cell; 4 uL; Aurora 1536-well, white, tissue culture plate.
2; Incubation; 4 hr; 37C, 5% CO2, 95% humidity.
3; Compound; 20 nL; Echo 650 Liquid Handler.
4; Incubation; overnight; 37C, 55 CO2, 95% humidity.
5; Reagent; 4 uL; Nano-Glo Luciferase assay system.
6; Centrifugation; 2 sec.
7; Detection; Luminescence; ViewLux plate reader (PerkinElmer).

NOTES (numbers refer to sequence numbers above).
1a. For the 1,536-well assay, cells were transfected in T75 flasks using a reverse transfection procedure, where 9 mL of complexes containing 45 uL Lipofectamine 2000 (ThermoFisher Scientific), 20 ug nsP2 plasmid, and 0.2 ug Nluc reporter plasmid, were combined with 10 mL of HEK293T cell suspension (1x10^6 cells/mL, 10 million cells total). Complexes containing Transfection Carrier DNA (Promega) and Nluc reporter plasmid (also at 1:100 ratio) were used as a high-signal control. After 24h, cells were harvested by trypsinization and resuspended at a density of 1x10^6 cells/ml.
1b. Cells were dispensed (4 uL cell/well) into 1,536-well white plates (Aurora, cyclic olefin polymer, cat# EWB041000A) using a Multidrop Combi.
2-4. After 4 hours, compounds (20 nL) were subsequently pinned using an Echo 650 Series Liquid Handlers and incubated overnight at 37C.
5. Nano-Glo (Promega) reagent was added (4 uL /well) according to the manufacturer's recommendations.
6-7. The Plates were centrifuged and analyzed for luminescence intensity using a ViewLux reader.

REFERENCE:
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.