External ID: JHICC_RGS_Act_HTS

Protocol: Assay overview:

To screen for compounds that activate the RGS4 protein, a HEK293 cell line which stably expresses M3R and inducibly expresses RGS4 is employed. RGS4 function is monitored by calcium flux with a commercially available Fluo4-AM dye. Compounds that do not show increase in the Fluo4 fluorescence in induced RGS4 expressed cells are considered as activator/potentiator hits. M3 receptor and other endogenous receptor activators/potentiators will be excluded through later counter-screening against non-induced parental cells.

Protocol for RGS4 Primary Screen:
1. Cell culture: Cells (HEK293-FlpIn-TREx/M3R/RGS4) are routinely cultured in DMEM (high glucose, w/ glutamine), 10%FBS, 1%Pen/Strep, 15ug/ml Blasticidin, 400ug/ml G418, 200ug/ml Hygromycin.
2. Cell plating: Add 50 ul/well of 200,000 cells/ml re-suspended in DMEM/high glucose medium with 10% FBS, 1% Pen/Strep. Include 10 ng/ml Doxycyclin (DOX) to induce RGS4 expression.
3. Incubate overnight at 37C and 5% CO2.
4. Remove medium and add 20 ul /well of 2uM Fluo4-AM solution to cells.
5. Incubate 30 minutes at 37C in incubator.
6. Prepare 6x compound plates and control plates on Cybi-Well system: test compounds are prepared using assay buffer (HBSS-HEPES pH 7.4).
7. Remove Fluo4-AM dye solution and add 20 ul/well of assay buffer to cells.
8. Incubate 30 minutes at room temperature (RT).
9. Add 6x compounds in cell plates and incubate 20 minutes at RT.
9. Load cell plates on Hamamatsu FDSS 6000 kinetic imaging plate reader
10. Measure fluorescence for 10 seconds at 1Hz to establish baseline
11. After 100 seconds, add 4 ul of ECmax (carbachol) into the cell plates and record fluorescence for 100 seconds.
12. Calculate ratio readout as F(max-min)/F0 and integrated ratio readout.
13. Calculate the average and standard deviation for negative and positive controls in each plate, as well as Z and Z' factors [26]
14. Calculate B scores [27] for test compounds using integrated ratios calculated in Step 12
15. Outcome assignment: If the B score of the test compound is less than 4 times the standard deviation (SD) of the B scores of integrated ratios of all library compounds above the mean (B score Activator Ratio16. Score assignment: An active test compound is assigned a score between 5 and 100 by calculation of Int(((Log(ABS(B score ratio))-0.82)/0.44)*100), and they are normalized to the smallest and largest LOG(B score ratio), B score ratio, as in the result definition. The inactive test compounds are assigned a score of 0.


List of reagents

1. HEK293-FlpIn-TREx/M3R/RGS4 cell lines (provided by assay provider)
2. PBS: pH7.4 (Invitrogen Cat#10010049)
3. Medium: DMEM (Sigma, Cat#D5796)
4. Fetal Bovine Serum (Gemini, Cat# 100-106)
5. Hygromycin (Mediatech, Cat#30-240-CR)
6. 100x Penicillin-Streptomycin (Mediatech, Cat#30-001-CI)
7. Cell/stripper (Mediatech, Cat#25-056-Cl)
8. G418: (Invitrogen, Cat#11811-031)
9. Blasticidin (Sigma, Cat#R21001)
10. Doxycycline hyclate (Sigma, Cat#D9891)
11. HEPES (Sigma, Cat#H4034)
12. Fluo-4 (Invitrogen, Cat #F14202)
13. Pluronic F-127*20% in DMSO (Invitrogen, Cat#P-3000MP)
14. Atropine (Sigma, Cat#A0132)
15. Carbachol (Sigma, Cat# C4382)
16. Triple-layer flask (VWR, Cat #62407-082)
17. BD Biocoat 384-well plates (BD, Cat# (35)4663 and Lot #7346273)

Comment: To screen for compounds that activate the RGS4 protein, a HEK293 cell line which stably expresses M3R and inducibly expresses RGS4 is employed. RGS4 function is monitored by calcium flux with a commercially available Fluo4-AM dye. Compounds that do not show increase in the Fluo4 fluorescence in induced RGS4 expressed cells are considered as activator/potentiator hits. M3 receptor and other endogenous receptor activators/potentiators will be excluded through later counter-screening against non-induced parental cells.

External ID: EYA2477

Protocol: The Eya2 enzyme was kindly provided by the laboratory of Dr. Rui Zhao. The 3-O-methyl-fluorescein phosphate (OMFP) substrate and all buffer reagents were purchased from Sigma Aldrich.
qHTS Assay Setup:
1. Buffer- 50 mM HEPES pH 7.5, 50 mM NaCl, 5 uM MgCl2, 0.05% BSA, 1 mM DTT.
2. OMFP Stock- 10 mM solution prepared in 100% DMSO and stored in single use aliquots at -20C.

Assay Procedure-
1. Dispense 1.5 ul per well of Eya2 at 0.2 uM in black medium binding 1536 well Greiner plate
2. Transfer 23 nl of compound and control compound (EGTA) to plate
3. Incubate at ambient for 10 minutes
4. Dispense 1.5 ul per well of OMFP at 50 uM
5. Incubate at ambient for 10 minutes
6. Read on ViewLux using Excitation = 485 nm, Emission = 525 nm

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: BCCG-A405-UPR-XBP1-PrimaryAgonist-Assay

Protocol: UPR CHO-XBP1 1536-Well Assay Protocol
A. Brief Description of the Assay:
The purpose of this assay is to detect activators of the IRE1-XBP1 (adaptive) arm of the Unfolded Protein Response pathway.
B. Materials:
CHO-XBP1 Cell Line
F12 nutrient mix HAMs (Invitrogen, Cat# 11765047)
Fetal Bovine Serum, heat-inactivated (Hyclone, Cat# SH30396)
Penicillin/Streptomycin, liquid (Invitrogen, Cat# 15140122)
L-glutamine (100X ) (Invitrogen, Cat# 25030081)
MEM Non-Essential Amino Acids Solution 10 mM (100X) (Invitrogen, Cat# 11140050)
Trypsin-EDTA 0.25% (Invitrogen, Cat# 25200-056)
DPBS (Hyclone, Cat# 30028.02)
T225 TC Flask (Nunc, Cat# 159934)
Cell strainer, 40 um (BD, Cat# 352340)
Aurora 1536 well white solid bottom TC plate (Aurora Biotechnology 00029846)
Tunicamycin (Sigma-Aldrich, Cat# T7765)
Steady-Glo Luciferase Assay System (1L) (Promega, Cat# E2550)

C. Plate Map:
Positive control (P) in columns 1 and 2, 10ug/mL Tunicamycin
Negative control (N) in columns 3 and 4, No Tunicamycin
Test compound (T) in columns 5 - 48, No Tunicamycin

D. Procedures:
Day1 Cell Seeding
1. Prepare cell suspensions as described in section F. Cell culture.
2. Set up Kalypsys dispenser as described in section G. Kalypsys dispenser setting.
3. Plate 1000 cells/well in 5 uL of assay media into columns 1-48 of a 1536-well assay plate, using straight tip dispense on a Kalypsys dispenser.
4. Centrifuge plates at 500 rpm for 1 minute on Eppendorf centrifuge 5810. Use Kalypsys metal lids.
5. Incubate overnight at 37 degrees, 100% relative humidity, 5% CO2 for 16-18 hours.

Day2 Compound Addition
6. Using LabCyte Echo, transfer 50 nL from a 2 mM Echo qualified plate containing test compounds into assay plate Col. 5 - 48 (final concentration of test compounds is 20 uM, 1% DMSO), 50 nL of 1 mg/mL Tunicamycin in DMSO to positive control wells in Columns 1 and 2, and 50 nL of DMSO to negative control wells in Columns 3 and 4.
7. Centrifuge plates at 1000 rpm for 1 minute on a Vspin centrifuge.
8. Incubate plates in incubator (37 degrees, 100% relative humidity, 5% CO2) for 6 hours.
9. Set up Kalypsys dispenser and Perkin Elmer Envision as described in section G. Kalypsys dispenser setting and H. Envision setting.
10. Following 6 hours incubation, remove lid and incubate plate for 10 min in at room temp.
11. Add 3 uL of Steady-Glo to each wells (Columns 1 - 48) using a Kalypsys dispenser.
12. Centrifuge plates at 2000 rpm for 2 minutes on a Vspin centrifuge.
13. Lid plate and incubate for 10 min at room temp.
14. Read plates using Envision using a luminescence protocol.
E. Recipe:
Growth Media
F12 nutrient mix HAMs supplemented with 10% hi-FBS, 1X Penicillin/Streptomycin, 1X L-glutamine, and 1X MEM-NEAA
Assay Media
Filtered Growth Media
Trypsin
Dilute 0.25% Trypsin/EDTA to 0.05% Trypsin/EDTA using DPBS
Positive Control
Tunicamycin at 10 ug/mL final.
(Note: Tunicamycin is reconstituted in DMSO to a concentration of 10mg/ml).

F. Cell Culture:
Prepare Growth Media/Assay Media as described in section E. Recipe.
Procedure to expand and maintain cells:
CHO-CHOP cells are seeded into T225 flasks at 3.75 x 105 cells. Cells are passaged twice a week (Monday or Tuesday, and Thursday or Friday depending on cell growth). Confluency should be maintained at <75%. After 3 days incubation, ~2.5X10^7 cells are expected per T225 flask. Incubate cells in 5% CO2.
1. Put media in water bath and leave for 30 minutes. Also leave Trypsin at room temp for 15 minutes. Keep DPBS at room temp (no need to warm up DPBS at 37 degrees).
2. Aspirate off old media from T225 flask.
3. Wash the flasks with 20 mL DPBS per T225 flask. Leave cells in DPBS for about 30 seconds.
4. Add 6.5 mL 0.05% Trypsin solution into the flask. Rock the flask gently so that the solution covers all over the surface.
5. Allow the cells to detach by incubating at room temperature for about 4 minutes.
6. Wash the flask with 25 mL fresh growth media.
7. Collect the cell suspension in a 50 mL sterile conical tube.
8. Centrifuge at 900 rpm for 5 minutes. Once pelleted, remove the supernatant and resuspend the cell pellet carefully in 1 mL fresh growth media with p1000 pipette.
9. Add 19 mL of growth media and mix gently.
10. Filter the cell suspension with cell strainer.
11. Count the cells and prepare stock flasks with
3.00 x 10^5 cells per T225 flask for 4 days incubation.
3.75 x 10^5 cells per T225 flask for 3 days incubation.
Procedure to prepare cells for the assay:
Follow steps 1 - 3 above
4. Add 5 mL Trypsin into the flask. Rock the flask gently so that the solution covers all over the surface.
5. Allow the cells to detach by incubating at room temperature for about 4 minutes.
6. Wash the flask with 25 mL fresh growth media.
7. Collect the cell suspension in a 50 mL sterile conical tube.
8. Centrifuge at 500 rpm for 10 minutes. Once pelleted, remove the supernatant and resuspend the cell pellet carefully in 1 mL fresh assay with p1000 pipette.
9. Add 19 mL of assay media and mix gently.
10. Filter the cell suspension with cell strainer.
11. Count the cells and adjust cell density to 1.0x10^5 cells/mL in media.

Comment: Compounds that test with activity greater than or equal to 30% activation at 20 uM concentration relative to the positive control are defined as actives in the primary assay.
To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the assay is described below.

Activity Scoring
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % activity in the assay demonstrated by a compound at 20 uM concentration:
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
Scoring for Single concentration confirmation screening is not applicable to this assay.
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.
2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay

External ID: JHICC_MrgX1_AlloAgonist_Primary

Protocol: Protocol:
1) Seed cells (MRGX1-HEK293) 15,000/50ul/well on BD PDK-pre-coated 384-well plate, overnight.
2) Dump the medium thoroughly and add 20 microl of Fluo4 NW solution.
3) 37 degrees C and 5% CO2 incubation in the dark for 0.5 hr and Room temperature incubation in the dark for 0.5 hr.
4) Mount the cell plate to FDSS, with compound plate from plate loading (1 by 1, 4 microl, 6x this step, 10x final); 2nd plate on Stage II (10 plate/2nd addition plate, 6 microl, 5x this step, 6.67x final) and 3rd plate on Stage III (10 plate/3rd addition plate, 6 microl, 6x this step, 6x final).
5) Read for the first 2 additions for 10 plates (~5 min/plate; total 55 min)
6) After that, read the 3rd addition of all 10 plates (~3 min/plate)
7) Change tips and repeat step 5 to step 7.
8) Calculate ratio readout as F(max-min)/F0 for the 2nd addition
9) Calculate the average and standard deviation for negative and positive controls in each plate, as well as Z and Z' factors [6].
10) Calculate B scores [7] for test compounds using ratios calculated in Step 9.
11) Outcome assignment: If the B score of the test compound is more than 3 times the standard deviation (SD) of the B scores of ratios of the library compounds (>=3*SD), AND the B score of initial fluorescence intensity is within 5 times the standard deviation of the B scores of the library compounds, AND not as a MrgX1 agonist in 1st addition, the compound is designated in the Outcome as active as an allosteric agonist hit of the MrgX1 target. Otherwise, it is designated as inactive.
12) Score assignment: An active test compound is assigned a score between 5 and 100 by calculation of with normalized ratioBScore, ratioBScore as in the result definition. Among the active compounds in the assay, the activity score range is 100-5. All inactive test compounds are assigned to the score 0.

List of reagents
(1) Cells (MrgX1 stably expressed HEK293 cells) from Dr. Xinzhong Dong.
(2) PBS: pH7.4 (Gibco, Cat#10010)
(3) Medium: DMEM (Mediatech 10-014-CV)
(4) Fetal Bovine Serum (Gibco, Cat# 26140)
(5) 200 mM L-Glutamine(Gibco, Cat#25030)
(6) Penicillin-Streptomycin(Mediatech, Cat#30-001-CI)
(7) Trypsin-EDTA: 0.25% Trypsin (Gibco, Cat#25300)
(8) G418 (100X): 50mg/mL(filtered) (Gibco, Cat#11811-031)
(9) HEPES (Sigma, Cat#H4034)
(10) BD Biocoat 384-well plates (BD, Cat#(35)4663 and Lot #7346273)
(11) Fluo-4 NW Calcium Assay Kit (Invitrogen Cat# F36205)
(12) BAM8-22 (Tocris, Cat#: 1763)

Comment: Possible artifacts of this assay can include, but are not limited to: non-intended chemicals or dust in or on wells of the microtiter plate, compounds that non-specifically modulate the cell host or the targeted activity, and compounds that quench or emit light or fluorescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR.

External ID: JHICC_MrgX1_Antagonist_Primary

Protocol: Protocol:
1) Seed cells (MRGX1-HEK293) 15,000/50ul/well on BD PDK-pre-coated 384-well plate, overnight.
2) Dump the medium thoroughly and add 20 microl of Fluo4 NW solution.
3) 37 degrees C and 5% CO2 incubation in the dark for 0.5 hr and Room temperature incubation in the dark for 0.5 hr.
4) Mount the cell plate to FDSS, with compound plate from plate loading (1 by 1, 4 microl, 6x this step, 10x final); 2nd plate on Stage II (10 plate/2nd addition plate, 6 microl, 5x this step, 6.67x final) and 3rd plate on Stage III (10 plate/3rd addition plate, 6 microl, 6x this step, 6x final).
5) Read for the first 2 additions for 10 plates (~5 min/plate; total 55 min)
6) After that, read the 3rd addition of all 10 plates (~3 min/plate)
7) Change tips and repeat step 5 to step 7.
8) Calculate ratio readout as F(max-min)/F0 for the 3rd addition
9) Calculate the average and standard deviation for negative and positive controls in each plate, as well as Z and Z' factors [12].
10) Calculate B scores [13] for test compounds using ratios calculated in Step 8.
11) Outcome assignment: If the B score of the test compound is more than 3 times the standard deviation (SD) of the B scores of ratios of the library compounds (<=3*SD), AND the B score of initial fluorescence intensity is within 5 times the standard deviation of the B scores of the library compounds, AND not as a MrgX1 agonist in 1st addition, the compound is designated in the Outcome as active as an antagonist hit of the MrgX1 target. Otherwise, it is designated as inactive.
12) Score assignment: An active test compound is assigned a score between 5 and 100 by calculation of with normalized ratioBScore, ratioBScore as in the result definition. Among the active compounds in the assay, the activity score range is 100-5. All inactive test compounds are assigned to the score 0.

List of reagents
(1) Cells (MrgX1 stably expressed HEK293 cells) from Dr. Xinzhong Dong.
(2) PBS: pH7.4 (Gibco, Cat#10010)
(3) Medium: DMEM (Mediatech 10-014-CV)
(4) Fetal Bovine Serum (Gibco, Cat# 26140)
(5) 200 mM L-Glutamine(Gibco, Cat#25030)
(6) Penicillin-Streptomycin(Mediatech, Cat#30-001-CI)
(7) Trypsin-EDTA: 0.25% Trypsin (Gibco, Cat#25300)
(8) G418 (100X): 50mg/mL(filtered) (Gibco, Cat#11811-031)
(9) HEPES (Sigma, Cat#H4034)
(10) BD Biocoat 384-well plates (BD, Cat#(35)4663 and Lot #7346273)
(11) Fluo-4 NW Calcium Assay Kit (Invitrogen Cat# F36205)
(12) BAM8-22 (Tocris, Cat#: 1763)

Comment: Possible artifacts of this assay can include, but are not limited to: non-intended chemicals or dust in or on wells of the microtiter plate, compounds that non-specifically modulate the cell host or the targeted activity, and compounds that quench or emit light or fluorescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR.

External ID: SBCCG-A413-tim10-1-Primary-Antagonist-Assay

Protocol: Assay Materials:
Yeast Strain: ySHDSTim10ts
Growth Media: YPD broth (TEKNOVA)
SD Assay Media: 1.2 g/L Yeast Nitrogen Base w/o amino acids, 5 g/L Ammonium Sulfate, 20 g/L Dextrose, 13.8 g/L Succinate, supplemented with 1 X Amino Acid Mix (2 g/L) (Sunrise Science Products, San Diego, CA)
SD Minimal Media: 1.2 g/L Yeast Nitrogen Base w/o amino acids, 5 g/L Ammonium Sulfate, 13.8 g/L Succinate, (Sunrise Science Products, San Diego, CA)
Assay Plate: Corning 1536 Well White Plate (Catalogue #: 3725)
Detection Reagent: Bactiter-GLO (Promega)

I. Compound Addition:

1. Using LabCyte Echo, transfer 40 nL from a 2 mM Echo qualified plate containing test compounds into assay plate columns 3 - 48 (final concentration of test compounds is 20 uM, 1 % DMSO). Transfer 40 nL of DMSO to positive and negative control wells in columns 1 - 4.
2. Centrifuge plates at 1000 rpm for 1 min.

II. Set up of tim10-1 assay:

The day before the screen, frozen culture was thawed at room temperature and resuspended in growth media at a cell density of 5x10^5/ml in approx. 100 ml. The culture was grown overnight at 25 oC with shaking (225 rpm).

3. In the morning of the Set-Up day prepare sufficient amount of SD Assay Media for negative control and compound wells and SD Minimal Media for positive control wells in order to obtain enough yeast cell culture for plating the desired number of 1536 well plates with 4 ul yeast cell suspension / well.
4. Count the yeast cells from the overnight Growth Media culture.
5. Dilute yeast to a final concentration of 2000 yeast/well in SD Media.
6. Pellet at 2400 rpm for 5 min at RT. Aspirate off supernatant.
7. Add 25 ml of sterile Water. Re-suspend cells by gently shaking. Pellet again at the same conditions, and wash the yeast cells in 25 ml sterile water a second time.
8. Re-suspend in SD Assay Media for negative control and compound wells.
9. For positive control wells, use SD Minimal Media with no yeast.
10. Add 4 ul yeast cells per well using combi and cover each plate with plastic lid.
11. Spin the plates at 2000 rpm for 1 min, incubate at 25 oC, inverted in a stack of 4, wrapped in saran wrap for 22-24 hours.

IV. Reading plates:
12. After 22-24 hours of incubation, add 3 ul of substrate solution (should be at RT) to all the wells of each plate using combi.
13. Plates are spun again at 2000 rpm, and left at RT for 15 min.
14. Read plates using a Perkin Elmer ViewLux using a luminescence protocol.

Comment: Compounds with %Activity >= 40% are defined as actives in this assay.

To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the assay is described below.

Activity Scoring
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary and single-concentration confirmation screening data.
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30.
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.

2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay

3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay

External ID: SBCCG-A539-APOBEC3A-Inhibitor-Primary-Assay

Protocol: Assay Materials:

Protein dilution buffer: Tris (pH 7.4) at 15 mM, NaCl at 150 mM, Triton X-100 at 0.5%, glycerol at 10%
Oligo dilution buffer: TE at 20 mM
Oligo: 5' d FAM-AAATATCCCAAAGAGAGA-TAMRA 3': BioSearch Technologies
UDG: New England Biolabs
A3A-MycHis: University of Minnesota
1N NaOH
2M Tris (pH 7.9)
Assay plate: Corning 1536 Well Black Solid Bottom Plate (Catalogue # 3724)

I. Compound Addition:
1- Using LabCyte Echo, transfer 40 nL from 2 mM compound source plate into assay plate Columns 5-48 (final concentration of test compounds is 20 uM, 1.0% DMSO), and 40 nL of DMSO to control wells in Columns 1-4.

II. Preparing reagents:
2- Prepare protein dilution buffer
3- Prepare oligo dilution buffer
4- Prepare intermediate A3A at 0.4 ng/ul. The final concentration of A3A in the plate is 0.2 ng/ul.
5- Prepare Oligo/UDG intermediate with 1 uM for Oligo and 0.002U/ul for UDG. The final concentration in the plate is 0.5 uM for Oligo and 0.001U/ul for UDG.
III. Reagent Addition
6- Add 2 ul of protein dilution buffer in columns 1 & 2
7- Add 2 ul of A3A in columns 3 to 48
8- Add 2 ul of Oligo/UDG to all columns
9- Cover the plate with Kalypsis metal lid; incubate the plate at room temperature for 45 minutes.
10- Add 1 ul of 1N NaOH to all columns
11- Cover the plate with Kalypsis metal lid; incubate the plate at room temperature for 30 minutes.
12- Add 1 ul of 2M Tris (pH 7.9) to all columns
13- Cover the plate with Kalypsis metal lid; incubate the plate at room temperature overnight.
IV. Reading plates:
14- Read the plate on PerkinElmer-EnVision plate reader (485/535)

Comment: Compounds that demonstrated an activity of >= 40% at 20 uM concentration and <= 30% at 20 uM in "uHTS identification of APOBEC3G DNA Deaminase Inhibitors via a fluorescence-based single-stranded DNA deaminase assay" (AID TBD)are defined as actives in this assay.

To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented.

Activity Scoring
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % activity in the assay:
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
Scoring for Single concentration confirmation screening is not applicable to this assay.
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.

2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay

3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay

External ID: BCCG-A235-SUMO-HTRF-Assay

Protocol: Assay materials:
1) E1, E2, GST-SUMO and His6-RanGap-1 proteins were obtained from the assay provider's laboratory.
2) Assay Buffer: 50mM Tris-HCl pH 7.4, 0.3mM DTT, 10 mM MgCl2, 0.005% Tween-20
3) Anti-GST XL665 and Anti-His Europium-labeled antibodies were purchased from Cis-Bio (Cat # 61GSTXLB and 61HISXLB)
4) Corning 1536-well, white plates (Cat #3725)

uHTS Procedure
1) Dispense 2ul of assay buffer into columns 1 and 2, and 2ul of Mixture 1, containing 37.5 nM E1 and 100 nM His-RanGap-1 in the assay buffer, into columns 3-48 of the 1536-well assay plate.
2) Using a HighRes biosolutions pintool dispense 70nl of 2mM compounds in DMSO to columns 5-48
3) Dispense 70nl of DMSO to columns 1-4.
4) Using Thermo Scientific MultiDrop Combi, dispense 2 uL of Mixture 2, containing 20 mM ATP, 12.5 nM E2 and 30 nM GST-SUMO in assay buffer, to all wells.
5) Incubate for 30 min. at room temperature.
6) Add 1ul of 500 mM KF
7) Read plate on a BMG Labtech PheraStar in a Homogeneous Time-Resolved
Fluorescence mode (Ex: 337 nm; Em: 620/665 nm). The ratio of fluorescence 665 over 620 was utilized as a readout of the assay.
8) Data analysis was performed using CBIS software (ChemInnovations, Inc).
9) The value of fluorescence at 620 nm for each sample was normalized to the value of the average value of fluorescence at 620 nm in the plate negative control wells to calculate F_ratio parameter.

Procedure for dose-response confirmation assay

1) Using Thermo Scientific MultiDrop Combi, dispense 2ul of assay buffer into columns 1 and 2, and 2ul of Mixture 1, containing 37.5 nM E1 and 100 nM His-RanGap-1 in the assay buffer, into columns 3-48 of the 1536-well assay plate.
3) Using Labcyte Echo550, dispense 70 nL of serially diluted compounds to columns 5-48 and DMSO to columns 1-4.
4) Using Thermo Scientific MultiDrop Combi, dispense 2 uL of Mixture 2, containing 20 mM ATP, 12.5 nM E2 and 30 nM GST-SUMO in assay buffer, to all wells.
5) Incubate lidded plate for 30 min. at room temp.
6) Add 1ul of 500 mM KF
7) Read plate on a BMG Labtech PheraStar in a Homogeneous Time-Resolved
Fluorescence mode (Ex: 337 nm; Em: 620/665 nm). The ratio of fluorescence 665 over 620 was utilized as a readout of the assay.
8) Data analysis was performed using CBIS software (ChemInnovations, Inc).

Comment: Compounds that demonstrated an inhibition of >= 50% and 0.5 <= F_ratio <= 1.5 at 35 uM concentration are defined as actives in primary HTS assay. Compounds with Inhibition >= 50% that demonstrate F_ratios outside these boundaries are optically interfering with the assay (quenching or fluorescent) and the results are marked as "inconclusive".

The compounds identified as primary screening actives proceed to single-concentration confirmation stage. These compounds were retested in duplicate at a single 35 uM concentration in confirmation screen. Compounds with an average of >= 33% inhibition are considered active of the confirmation assay.

Substance SID26670119 was reordered for the confirmation assay. A different batch of the compound, SID7970403, was received and used.

Compounds with an IC50 < 100 uM are considered active in the dose response assay.
To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the assay is described below.

Activity Scoring
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data-the score is correlated with % inhibition in the assay demonstrated by a compound at 35 uM concentration:
a. If primary % inhibition is less than 0%, then the assigned score is 0
b. If primary % inhibition is greater than 100%, then the assigned score is 40/(1+(F_ratio - 1)^2)
c. If primary % inhibition is between 0% and 100%, then the calculated score is (%inhibition)*0.4/(1+(F_ratio-1)^2)
d. For compounds retested at 35 uM, if primary % inhibition is greater than 100%, then the assigned score is 40
e. For compounds retested at 35 uM, if primary % inhibition is between 0% and 100%, then the calculated score is %inhibition)*0.4

2) Second tier (41-80 range) is reserved for dose-response confirmation data
a. Inactive compounds of the confirmatory stage are assigned a score value equal 41.
b. The score is linearly correlated with a compound's potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.
c. The Hill coefficient is taken as a measure of compound behavior in the assay via an additional scaling factor QC:
QC = 2.6*[exp(-0.5*nH^2) - exp(-1.5*nH^2)]

This empirical factor prorates the likelihood of target- or pathway-specific compound effect vs. its non-specific behavior in the assay. This factor is based on expectation that a compound with a single mode of action that achieved equilibrium in the eIF4H assay demonstrates the Hill coefficient value of 1. Compounds deviating from that behavior are penalized proportionally to the degree of their deviation.
d. Summary equation that takes into account all the items discussed above is
Score = 44 + 6*(pIC50-3)*QC,
Where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units. This equation results in the Score values above 50 for compounds that demonstrate high potency and predictable behavior. Compounds that are inactive in the assay or whose concentration-dependent behavior are likely to be an artifact of that assay will generally have lower Score values.

3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay

External ID: JHICC_CaCC_Inh_Primary

Protocol: 1. Cell culture: Cells are routinely cultured in DMEM (high glucose, w glutamine), 10% FBS, 1%Pen/Strep.
2. Cell plating: Add 50 ul/well of 8,000/well re-suspended in media on BD PDK-pre-coated 384-well plate.
3. Incubate overnight at 37C and 5% CO2
4. Remove medium and add 25 ul /well of HBSS-HEPES (1x) (pH 7.4) to cells
5. Prepare 7.5X compound plates and control plates on Cybi-Well system: test compounds are prepared using assay buffer; controls are assay buffer(IC0), activator control ionomycin and inhibitor control NFA (all with DMSO concentrations matched to that of test compounds)
6. Add 5 microl drugs/compounds and controls to cell plates by Cybi and incubate at RT for 20 min.
7. Load cell plates to Hamamatsu FDSS 6000 kinetic imaging plate reader
8. Prepare 8.5x trigger solution containing 4.25 uM ionomycin in 138mM iodide-HBSS solution (pH 7.4)
9. Measure fluorescence for 10 seconds at 1Hz to establish baseline
10. Add 5 ul of trigger solution into the cell plates on FDSS and continue measuring fluorescence for 50 seconds
11. Calculate ratio readout as F(max-min)/F0
12. Calculate the average and standard deviation for negative and positive controls in each plate, as well as Z and Z' factors [15].
13. Calculate B scores [16] for test compounds using ratios calculated in Step 11.
17. Outcome assignment: If the B score of the test compound is more than 3 times the standard deviation (SD) of the B scores of ratios of the library compounds (>=3*SD), AND the B score of initial fluorescence intensity is within 5 times the standard deviation of the B scores of the library compounds, the compound is designated in the Outcome as active as an inhibitor/blocker of the CaCC (TMEM16A) channels. Otherwise, it is designated as inactive.
18. Score assignment: An active test compound is assigned a score between 0 and 100 by calculation of with normalized ratioBScore, ratioBScore as in the result definition. Among the active compounds in the assay, the activity score range is 100-25. All inactive test compounds are assigned to the score 0.


List of reagents

(1)Cells (TMEM16A/YFP-H148Q/ I152L/HEK293) from JHICC.
(2)PBS: pH7.4 (Gibco, Cat#10010)
(3)Medium: DMEM (Cellgro, Cat# 10-013-CV )
(4)Fetal Bovine Serum (Gibco, Cat# 26140)
(5)200 mM L-Glutamine(Gibco, Cat#25030)
(6)Penicillin-Streptomycin(Mediatech, Cat#30-001-CI)
(7)Trypsin-EDTA: 0.25% Trypsin (Gibco, Cat#25300)
(8)G418 (100X): 50mg/mL(filtered) (Gibco, Cat#11811-031)
(9)HEPES (Sigma, Cat#H4034)
(10)BD Biocoat 384-well plates (BD, Cat#(35)4663 and Lot #7346273)
(11)Hygromycin (mediatech Cat# 30-240-CR)
(12)Niflumic acid (Sigma, Cat#:N0630)

Comment: Possible artifacts of this assay can include, but are not limited to: non-intended chemicals or dust in or on wells of the microtiter plate, compounds that non-specifically modulate the cell host or the targeted activity, and compounds that quench or emit light or fluorescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR.

External ID: JHICC_MrgX1_Agonist_Primary

Protocol: Protocol:
1) Seed cells (MRGX1-HEK293) 15,000/50ul/well on BD PDK-pre-coated 384-well plate, overnight.
2) Dump the medium thoroughly and add 20 microl of Fluo4 NW solution.
3) 37 degrees C and 5% CO2 incubation in the dark for 0.5 hr and Room temperature incubation in the dark for 0.5 hr.
4) Mount the cell plate to FDSS, with compound plate from plate loading (1 by 1, 4 microl, 6x this step, 10x final); 2nd plate on Stage II (10 plate/2nd addition plate, 6 microl, 5x this step, 6.67x final) and 3rd plate on Stage III (10 plate/3rd addition plate, 6 microl, 6x this step, 6x final).
5) Read for the first 2 additions for 10 plates (~5 min/plate; total 55 min)
6) After that, read the 3rd addition of all 10 plates (~3 min/plate)
7) Change tips and repeat step 5 to step 7.
8) Calculate ratio readout as F(max-min)/F0
9) Calculate the average and standard deviation for negative and positive controls in each plate, as well as Z and Z' factors [6].
10) Calculate B scores [7] for test compounds using ratios calculated in Step 9.
11) Outcome assignment: If the B score of the test compound is more than 3 times the standard deviation (SD) of the B scores of ratios of the library compounds (>=3*SD), AND the B score of initial fluorescence intensity is within 5 times the standard deviation of the B scores of the library compounds, the compound is designated in the Outcome as active as an agonist hit of the MrgX1 target. Otherwise, it is designated as inactive.
12) Score assignment: An active test compound is assigned a score between 5 and 100 by calculation of with normalized ratioBScore, ratioBScore as in the result definition. Among the active compounds in the assay, the activity score range is 100-5. All inactive test compounds are assigned to the score 0.

List of reagents
(1) Cells (MrgX1 stably expressed HEK293 cells) from Dr. Xinzhong Dong.
(2) PBS: pH7.4 (Gibco, Cat#10010)
(3) Medium: DMEM (Mediatech 10-014-CV)
(4) Fetal Bovine Serum (Gibco, Cat# 26140)
(5) 200 mM L-Glutamine(Gibco, Cat#25030)
(6) Penicillin-Streptomycin(Mediatech, Cat#30-001-CI)
(7) Trypsin-EDTA: 0.25% Trypsin (Gibco, Cat#25300)
(8) G418 (100X): 50mg/mL(filtered) (Gibco, Cat#11811-031)
(9) HEPES (Sigma, Cat#H4034)
(10) BD Biocoat 384-well plates (BD, Cat#(35)4663 and Lot #7346273)
(11) Fluo-4 NW Calcium Assay Kit (Invitrogen Cat# F36205)
(12) BAM8-22 (Tocris, Cat#: 1763)

Comment: Possible artifacts of this assay can include, but are not limited to: non-intended chemicals or dust in or on wells of the microtiter plate, compounds that non-specifically modulate the cell host or the targeted activity, and compounds that quench or emit light or fluorescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR.

External ID: SBCCG-A415-tim10-Primary-Antagonist-Assay

Protocol: Assay Materials:
Yeast Strain: ySHDSTim10 Isogeneic Control yeast strain (TIM10 strain)
Growth Media: YPD broth (TEKNOVA)
SD Assay Media: 1.2 g/L Yeast Nitrogen Base w/o amino acids, 5 g/L Ammonium Sulfate, 20 g/L Dextrose, 13.8 g/L Succinate, supplemented with 1 X Amino Acid Mix (2 g/L) (Sunrise Science Products, San Diego, CA)
SD Minimal Media: 1.2 g/L Yeast Nitrogen Base w/o amino acids, 5 g/L Ammonium Sulfate, 13.8 g/L Succinate, (Sunrise Science Products, San Diego, CA)
Assay Plate: Corning 1536 Well White Plate (Catalogue #: 3725)
Detection Reagent: Bactiter-GLO (Promega)

I. Compound Addition:

1. Using LabCyte Echo, transfer 20 nL from a 2 mM Echo qualified plate containing test compounds into assay plate columns 3 - 48 (final concentration of test compounds is 10 uM, 0.5 % DMSO). Transfer 20 nL of DMSO to positive and negative control wells in columns 1 - 4.
2. Centrifuge plates at 1000 rpm for 1 min.

II. Set up of tim10-1 assay:

The day before the screen, frozen culture was thawed at room temperature and resuspended in growth media at a cell density of 5x10^5/ml in approx. 100 ml. The culture was grown overnight at 25 oC with shaking (225 rpm).
3. In the morning of the Set-Up day prepare sufficient amount of SD Assay Media for negative control and compound wells and SD Minimal Media for positive control wells in order to obtain enough yeast cell culture for plating the desired number of 1536 well plates with 4 ul yeast cell suspension / well.
4. Count the yeast cells from the overnight Growth Media culture.
5. Dilute yeast to a final concentration of 2000 yeast/well in SD Media.
6. Pellet at 2400 rpm for 5 min at RT. Aspirate off supernatant.
7. Add 25 ml of sterile Water. Re-suspend cells by gently shaking. Pellet again at the same conditions, and wash the yeast cells in 25ml sterile water a second time.
8. Re-suspend in SD Assay Media for negative control and compound wells
9. For positive control wells, use SD Minimal Media with no yeast.
10. Add 4 ul yeast cells per well using combi and cover each plate with plastic lid.
11. Spin the plates at 2000 rpm for 1 min, incubate at 25 oC, inverted in a stack of 4, wrapped in saran wrap for 22-24 hours.

IV. Reading plates:
12. After 22-24 hours of incubation, add 3 ul of substrate solution (should be at RT) to all the wells of each plate using combi.
13. Plates are spun again at 2000 rpm, and left at RT for 15 min.
14. Read plates using a Perkin Elmer ViewLux using a luminescence protocol.

Comment: Compounds with %Activity >= 40% are defined as "active" in this assay.

To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the assay is described below.

Activity Scoring
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary and single-concentration confirmation screening data.
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30.
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.

2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay

3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay

External ID: BCCG-A237-eIF4H-FP-Assay

Protocol: HTS Assay Protocol

Assay materials:
1) His6-eIF4H (Obtained from Assay Provider)
2) 5'-fluorescein-labeled poly(U)10 (Purchased from Dharmacon)
3) Assay Buffer: 5 mM Hepes pH 7.5, 0.005 % Tween-20

Procedure:
1. Dispense 2 ul of assay buffer into columns 1 and 2 of a black, Corning (#2725) 1536 well assay plate.
2. Add 2 ul of eIF4H protein, 30 nM final concentration into columns 3-48.
3. Add 2 ul of 5'-fluorescein-labeled poly(A)10 2.5 nM final concentration into columns 1-48.
4. Using a HighRes biosolutions pintool dispense 70 nl of 2 mM compounds in DMSO to columns 5-48.
5. Using a HighRes biosolutions pintool dispense 70 nl of DMSO to columns 1-4.
6. Read plate on a BMG PHERAstar at 485/520/520nm in Fluorescence Polarization mode.
i. Positioning delay = 0.0
ii. Flashes/well = 10
7. Data analysis was performed using CBIS software (ChemInnovations, Inc).
8. Fluorescence intensity of each sample was normalized to the average fluorescence intensity value of the plate negative control wells to calculate F_ratio parameter.

Dose Response Protocol

Assay materials:
1) His6-eIF4H (Obtained from Assay Provider)
2) 5#-fluorescein-labeled poly(U)10 (Purchased from Dharmacon)
3) Assay Buffer: 5 mM Hepes pH 7.5, 0.005 % Tween-20

Procedure:
1. Using a Labcyte Echo, DMSO and test compounds are transferred to wells of a black, Corning 1536 well assay plate. DMSO only is transferred to columns 1-4 (Control wells), while varying volumes of test compounds are transferred to columns 5-48 to achieve the desired test concentrations. Compounds are transferred from a 2 mM stock to give the stated final concentration. Test compound wells in the assay plate are back-filled with DMSO to equalize final assay concentrations.
2. After compounds have been added, dispense 2 ul of assay buffer into columns 1 and 2 of a black, Corning (#2725) 1536 well assay plate.
3. Add 2 ul of eIF4H protein, 30 nM final concentration into columns 3-48.
4. Add 2 ul of 5#-fluorescein-labeled poly(A)10 2.5 nM final concentration into columns 1-48.
5. Read plate on a BMG PHERAstar at 485/520/520nm in Fluorescence Polarization mode.
i. Positioning delay = 0.0
ii. Flashes/well = 10
6. Data analysis was performed using CBIS software (ChemInnovations, Inc).
7. Fluorescence intensity of each sample was normalized to the average fluorescence intensity value of the plate negative control wells to calculate F_ratio parameter.

Comment: Compounds that demonstrated % activity of >= 50 % and 0.4 =< F_ratio <= 1.5 at 35 uM concentration are defined as actives of the primary screen in this assay. Compounds with activity >50% that demonstrate F_ratios outside these boundaries are optically compounds interfering with the assay (quenching or fluorescent) and the results are marked as "inconclusive".

Selected compounds from the primary screen were retested and those that confirmed in duplicate at 20uM concentration were considered "active". If the results of the duplicates were not consistent they are marked as "inconclusive."

Compounds were tested in a dose response assay and those with an IC50 < 100 uM are considered to be "active."

To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the assay is described below.

Activity Scoring
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data-the score is correlated with %Inhibition in the assay demonstrated by a compound at 35 uM concentration:
a. If primary %Inhibition is less than 0%, then the assigned score is 0
b. If primary %Inhibition is greater than 100%, then the assigned score is 40/(1+(F_ratio - 1)^2)
c. If primary %Inhibition is between 0% and 100%, then the calculated score is (%Inhibition)*0.4/(1+(F_ratio-1)^2)

2) Second tier (41-80 range) is reserved for dose-response confirmation data
a. Inactive compounds of the confirmatory stage are assigned a score value equal 41.
b. The score is linearly correlated with a compound's potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.
c. The Hill coefficient is taken as a measure of compound behavior in the assay via an additional scaling factor QC:
QC = 2.6*[exp(-0.5*nH^2) - exp(-1.5*nH^2)]

This empirical factor prorates the likelihood of target- or pathway-specific compound effect vs. its non-specific behavior in the assay. This factor is based on expectation that a compound with a single mode of action that achieved equilibrium in the eIF4H assay demonstrates the Hill coefficient value of 1. Compounds deviating from that behavior are penalized proportionally to the degree of their deviation.
d. Summary equation that takes into account all the items discussed above is
Score = 44 + 6*(pIC50-3)*QC,
Where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units. This equation results in the Score values above 50 for compounds that demonstrate high potency and predictable behavior. Compounds that are inactive in the assay or whose concentration-dependent behavior are likely to be an artifact of that assay will generally have lower Score values.

3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in to this assay

External ID: TRXR101

Protocol: Assay protocol: 2 uL of reagents (buffer in column 4 as negative control and 90 nM rTrxR1 in columns 1-3 and 5-48) were dispensed into Greiner black solid-bottom 1,536-well assay plates, followed by 1 uL of NADPH (400 uM final concentration) to each well. The plates were centrifuged at 1000 rpm for 15 seconds and subsequently incubated for 5 min at room temperature (~22 deg C) to allow for rTrxR1 reduction. Compounds (23 nL) were then transferred via Kalypsys pin tool equipped with 1536-pin array (10 nL slotted pins, V&P Scientific, San Diego, CA). In addition, a duplicate 2-fold serial dilution of the control compounds auranofin, a known gold-based TrxR1 inhibitor, and juglone (5-hydroxy-1,4-naphthoquinone), a natural TrxR1 substrate, were pin-transferred to columns 2 and 3, respectively. After incubation for 15 min at room temperature (~22 deg C), 1 uL of selenite (400 uM final concentration) were dispensed to each well. The plate was immediately transferred to a ViewLux high-throughput CCD imager (PerkinElmer), wherein kinetic measurements of NADPH fluorescence (Ex 340 nm, Em 450 nm) were acquired (8 minute kinetic read, see Table 1). Read 1 was utilized to assess the capacity of a compound to serve as an rTrxR1 substrate, i.e. a decrease in NADPH fluorescence compared to the no-compound background is an indication of a substrate behavior for that particular compound. For inhibitory activity of a compound, delta values, computed as the difference in fluorescence intensity between the first and last reads of an 8-minute time kinetic window, were used. All reagents were diluted in an assay buffer consisting of 50 mM Tris-HCl, pH 7.5, 2 mM EDTA, and 0.01% Tween-20.

Throughout the screen, reagent bottle and all liquid lines were made light-tight to minimize reagent degradation. All screening operations were performed on a fully integrated robotic system (Kalypsys, San Diego, CA) containing one RX-130 and two RX-90 anthropomorphic robotic arms (Staubli, Duncan, SC). Library plates were screened starting from the lowest and proceeding to the highest concentration, and a 'double-pinning' step of the highest concentration was required to access higher concentrations of compounds. Vehicle-only plates, with DMSO being pin-transferred to the columns 5-48, were inserted uniformly at the beginning and the end of each library in order to monitor and record any shifts in the background, which can be affected by reagent dispensers or loss in enzyme activity over time. Screening data were corrected, normalized, and concentration-effect relationships were derived by using publicly-available curve fitting algorithms developed in-house (http://ncgc.nih.gov/pub/openhts).

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: CHOK1_ANT_FLUO8_1536_1X%INH CSRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that non-selectively inhibit Gq signaling. In this assay, the parental CHO cell line (not transfected with any GPCRs) is used to monitor inhibition of Gq activity by test compound. Cells are incubated with test compounds, followed by measurement of intracellular calcium as monitored by the FLUO-8 fluorescent, cell permeable calcium indicator dye. As designed, compounds that act as Gq antagonists will decrease calcium mobilization, resulting in decreased relative fluorescence of the indicator dye, and thus decreased well fluorescence. These compounds are considered nonselective Gq inhibitors. Compounds are tested in singlicate at a nominal concentration of 5.6 uM.

Protocol Summary:

The CHO cell line was routinely cultured in T-175 sq cm flasks at 37 C and 95% relative humidity (RH). The growth media consisted of Ham's F-12 Nutrient Media (F-12) supplemented with 10% v/v heat-inactivated qualified fetal bovine serum, 25 mM HEPES, and 1X antibiotic mix (penicillin, streptomycin, and neomycin).

The day before the assay, 2000 cells in 3 uL of growth media were seeded into each well of 1536 well microtiter plates and allowed to incubate at 37 C, 5% CO2, and 95 % RH for 23 hours. Next, 2 uL of the fluorogenic Fluo-8 intracellular calcium indicator mixture with 1 mM trypan red plus (prepared according to the manufacturer's protocol) was added to each well. After incubation for 1 hour at 37 C, 5% CO2, and 95 % RH, 25 nL of test compound in DMSO, or DMSO alone were dispensed to the appropriate wells. The assay was started after an additional 30 minute incubation at room temperature, by performing a basal read of plate fluorescence (470-495 nm excitation and 515-575 nm emission) for 6 seconds on the FLIPR Tetra (Molecular Devices). Next, 15 nL of ATP in DMSO (EC84 average response), or DMSO alone were dispensed to the appropriate wells. Then a real time fluorescence measurement was immediately performed for the remaining 94 seconds of the assay.

A ratio for each well was calculated to normalize assay data, according to the following mathematical expression:

Ratio = I_Max / I_Min

Where:

I_Max represents the maximum measured fluorescence emission intensity over the 100 second read.
I_Min represents the minimum (basal) measured fluorescence emission intensity before compound was added.

Percent inhibition was calculated from the median ratio as follows:

% Inhibition = ( 1 - ( ( Ratio_Test_Compound - Median_Ratio_High_Control ) / ( Median_Ratio_Low_Control - Median_Ratio_High_Control ) ) ) * 100

Where:

Test_Compound is defined as wells containing test compound.
Low_Control is defined as wells containing ATP Challenge.
High_Control is defined as wells containing DMSO alone (no ATP).

A mathematical algorithm was used to determine nominally inhibiting compounds in the screen. Two values were calculated for each assay plate: (1) the average percent inhibition of test compound wells and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter for each plate, i.e. any compound that exhibited greater % inhibition than that particular plate's cutoff parameter was declared active.

PubChem Activity Outcome and Score:

The PubChem Activity Score range for active compounds is 100-22, and for inactive compounds 61-0.

List of Reagents:

CHO cells (ATCC, part CCL-61)
Fluo-8 No Wash Calcium Assay Kit (AAT Bioquest, part 36316)
Trypan red plus (ABD Bioquest, part 2456)
Ham's F-12 media (Invitrogen, part 11765-054)
Trypsin-EDTA solution (Invitrogen, part 25200-056)
Fetal Bovine Serum (Invitrogen, part 16140-071)
100X Penicillin-Streptomycin-Neomycin mix (Invitrogen, part 15640-055)
T-175 tissue culture flasks (Nunc, part 159910)
Agonist: ATP (Sigma-Aldrich, part A6559)
1536-well plates (Greiner, part 789072)

Comment: Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that quench or emit fluorescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR.

External ID: PROCASPASE3_ACT_EPIABS_1536_1X%ACT PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that activate procaspase 3 activity. This assay employs a procaspase 3 mutant enzyme, D9A/D28A/D175A (called PC-3 D3A) which is unable to autoproteolyze itself because its aspartic acid cleavage sites have been mutated to alanines. The mutant has a fully functional active site that can process the peptidic Ac-DEVD-pNA chromogenic substrate. Cleavage of substrate by procaspase 3 hydrolyzes the bond between the aspartic acid and p-nitroaniline, leading to release of yellow p-nitroaniline and an increase in well absorbance at 405 nm. In this assay, PC-3 D3A enzyme is pre-incubated with test compounds, followed by addition of substrate and measurement of well epi-absorbance. As designed, compounds that activate procaspase 3 activity will increase substrate hydrolysis, leading to an increase in well absorbance. Compounds are tested in singlicate at a nominal concentration of 8.5 uM.

Protocol Summary:

Prior to the start of the assay, 2.5 uL of zinc-free Assay Buffer (50 mM HEPES, 300 mM NaCl, pH 7.4, 0.01% Triton-X 100) containing 2 uM of PC-3 D3A protein were dispensed into 1536 microtiter plates. Next, 43 nL of test compound in DMSO or DMSO alone (0.8% final concentration) were added to the appropriate wells and incubated for 1 hour at 25 C.

The assay was started by dispensing 2.5 uL of 400 uM Ac-DEVD-pNA in Assay Buffer to all wells. Plates were centrifuged and after 2 hours of incubation at 25 C, epi-absorbance was read on the EnVision plate reader using a photometric filter set (excitation = 405 nm, emission = 450 nm) and a dichroic mirror with 425 nm cutoff. Fluorescence emission was read at 10 flashes per well at two time points (0 minutes and 120 minutes).

Prior to further calculations, the following formula was used to calculate absorbance:

Abs = ( -Log10( ( [ Raw2 ] / [ Mean Reference2 ] ) ) - ( -Log10 ( ( [ Raw1 ] ) / [ Mean Reference ] ) )

Where:

Raw1 is defined as the read at T0 minutes.
Raw2 is defined as the read at T120 minutes.
Mean Reference is defined as a mean of values from wells containing buffer only at T0.
Mean Reference2 is defined as a mean of values from wells containing buffer only at T120.

The percent activation for each compound was calculated as follows:

% Activation = ( ( Ratio_Test_Compound - Median_Ratio_Low_Control ) / ( Median_Ratio_High_Control - Median_Ratio_Low_Control ) ) * 100

Where:

Test_Compound is defined as wells containing test compound.
Low_Control is defined as wells containing DMSO.
High_Control is defined as wells containing DMSO and 5 uM PC-3 D3A.

A mathematical algorithm was used to determine nominally activating compounds in the primary screen. Two values were calculated: (1) the average percent activation of all compounds tested, and (2) three times their standard deviation.The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % activation than the cutoff parameter was declared active.

PubChem Activity Outcome and Score:

The reported PubChem Activity Score has been normalized to 100% observed primary activation. Negative % activation values are reported as activity score zero.

The PubChem Activity Score range for active compounds is 100-9, and for inactive compounds 9-0.

List of Reagents:

Recombinant PC-3 D3A (procaspase 3 enzyme) (Assay Provider)
Assay Buffer (Assay Provider)
Chromogenic Substrate (Ac-DEVD-pNA) (Assay Provider)
1536 SWSN plates (Corning, part 7254)

Comment: Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well absorbance. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.

External ID: PolI100

Protocol: Three microliters of reagents (buffer in column 3 and 4 as negative control and 10 nM Pol iota in columns 1, 2, and 5-48) will be dispensed into 1,536-well black solid-bottomed plate. Compounds (23 nL) will be transferred via Kalypsys pin tool equipped with 1536-pin array. The plates will then be incubated for 15 min at room temperature, and 1 muL substrate (50 nM final concentration) will be added to start the reaction and kinetically read twice at 0 min and 10 min on the Viewlux reader.

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: PolK100

Protocol: Three microliters of reagents (buffer in column 3 and 4 as negative control and 10 nM Pol kappa in columns 1, 2, and 5-48) were dispensed into a 1536-well black solid-bottom plate. Compounds (23 nL) were transferred via Kalypsys pin tool equipped with 1536-pin array. The plates were then incubated for 15 min at room temperature, and 1 uL substrate (50 nM final concentration) were then added to start the reaction and kinetically read twice at 0 min and 10 min on the Viewlux reader

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: SBCCG-A419-tim23-1-Primary-Antagonist-Assay

Protocol: Assay Materials:
Yeast Strain: ySHDSTim23ts (tim23-1 strain)
Growth Media: YPD broth (TEKNOVA)
SD Assay Media: 1.2 g/L Yeast Nitrogen Base w/o amino acids, 5 g/L Ammonium Sulfate, 20 g/L Dextrose, 13.8 g/L Succinate, supplemented with 1 X Amino Acid Mix (2 g/L) (Sunrise Science Products, San Diego, CA)
SD Minimal Media: 1.2 g/L Yeast Nitrogen Base w/o amino acids, 5 g/L Ammonium Sulfate, 13.8 g/L Succinate, (Sunrise Science Products, San Diego, CA)
Assay Plate: Corning 1536 Well White Plate (Catalogue #: 3725)
Detection Reagent: Bactiter-GLO (Promega)

I. Compound Addition:

1. Using LabCyte Echo, transfer 40 nL from a 2 mM Echo qualified plate containing test compounds into assay plate columns 3 - 48 (final concentration of test compounds is 20 uM, 1 % DMSO). Transfer 40 nL of DMSO to positive and negative control wells in columns 1 - 4.
2. Centrifuge plates at 1000 rpm for 1 min.

II. Set up of tim23-1 assay:

The day before the screen, frozen culture was thawed at room temperature and resuspended in growth media at a cell density of 5x10^5/ml in approx. 100 ml. The culture was grown overnight at 25 oC with shaking (225 rpm).
3. In the morning of the Set-Up day prepare sufficient amount of SD Assay Media for negative control and compound wells and SD Minimal Media for positive control wells in order to obtain enough yeast cell culture for plating the desired number of 1536 well plates with 4 ul yeast cell suspension / well.
4. Count the yeast cells from the overnight Growth Media culture.
5. Dilute yeast to a final concentration of 1000 yeast/well in SD Media.
6. Pellet at 2400 rpm for 5 min at RT. Aspirate off supernatant.
7. Add 25 ml of sterile Water. Re-suspend cells by gently shaking. Pellet again at the same conditions, and wash the yeast cells in 25 ml sterile water a second time.
8. Re-suspend in SD Assay Media for negative control and compound wells
9. For positive control wells, use SD Minimal Media with no yeast.
10. Add 4 ul yeast cells per well using combi and cover each plate with plastic lid.
11. Spin the plates at 2000 rpm for 1 min, incubate at 25 oC, inverted in a stack of 4, wrapped in saran wrap for 22-24 hours.

IV. Reading plates:

12. After 22-24 hours of incubation, add 3 ul of substrate solution (should be at RT) to all the wells of each plate using combi.
13. Plates are spun again at 2000 rpm, and left at RT for 15 min.
14. Read plates using a Perkin Elmer ViewLux using a luminescence protocol.

Comment: Compounds with %Activity >= 50% are defined as actives in this assay.

To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the assay is described below.

Activity Scoring
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary and single-concentration confirmation screening data.
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30.
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.

2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay

3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay

External ID: TRXR100

Protocol: Assay protocol: 2 uL of reagents (buffer in column 4 as negative control and 90 nM rTrxR1 in columns 1-3 and 5-48) were dispensed into Greiner black solid-bottom 1,536-well assay plates, followed by 1 uL of NADPH (400 uM final concentration) to each well. The plates were centrifuged at 1000 rpm for 15 seconds and subsequently incubated for 5 min at room temperature (~22 deg C) to allow for rTrxR1 reduction. Compounds (23 nL) were then transferred via Kalypsys pin tool equipped with 1536-pin array (10 nL slotted pins, V&P Scientific, San Diego, CA). In addition, a duplicate 2-fold serial dilution of the control compounds auranofin, a known gold-based TrxR1 inhibitor, and juglone (5-hydroxy-1,4-naphthoquinone), a natural TrxR1 substrate, were pin-transferred to columns 2 and 3, respectively. After incubation for 15 min at room temperature (~22 deg C), 1 uL of selenite (400 uM final concentration) were dispensed to each well. The plate was immediately transferred to a ViewLux high-throughput CCD imager (PerkinElmer), wherein kinetic measurements of NADPH fluorescence (Ex 340 nm, Em 450 nm) were acquired (8 minute kinetic read, see Table 1). Read 1 was utilized to assess the capacity of a compound to serve as an rTrxR1 substrate, i.e. a decrease in NADPH fluorescence compared to the no-compound background is an indication of a substrate behavior for that particular compound. For inhibitory activity of a compound, delta values, computed as the difference in fluorescence intensity between the first and last reads of an 8-minute time kinetic window, were used. All reagents were diluted in an assay buffer consisting of 50 mM Tris-HCl, pH 7.5, 2 mM EDTA, and 0.01% Tween-20.

Throughout the screen, reagent bottle and all liquid lines were made light-tight to minimize reagent degradation. All screening operations were performed on a fully integrated robotic system (Kalypsys, San Diego, CA) containing one RX-130 and two RX-90 anthropomorphic robotic arms (Staubli, Duncan, SC). Library plates were screened starting from the lowest and proceeding to the highest concentration, and a 'double-pinning' step of the highest concentration was required to access higher concentrations of compounds. Vehicle-only plates, with DMSO being pin-transferred to the columns 5-48, were inserted uniformly at the beginning and the end of each library in order to monitor and record any shifts in the background, which can be affected by reagent dispensers or loss in enzyme activity over time. Screening data were corrected, normalized, and concentration-effect relationships were derived by using publicly-available curve fitting algorithms developed in-house (http://ncgc.nih.gov/pub/openhts).

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: PROCASPASE7_ACT_EPIABS_1536_1X%ACT CSRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that increase the activity of procaspase-7, an enzyme with a high degree of structural similarity to caspase-3. This assay also serves as a counterscreen for procaspase-3 activators. This assay employs a procaspase-7 triple-mutant enzyme (D23A/D198A/D206A) (PC-7 D3A) which is unable to autoproteolyze itself because its aspartic acid cleavage sites have been mutated to alanines. The mutant has a fully functional active site that can process the peptidic Ac-DEVD-pNA chromogenic substrate. Cleavage of this substrate by procaspase-7 hydrolyzes the bond between the aspartic acid and p-nitroaniline, leading to release of yellow p-nitroaniline and an increase in well absorbance at 405 nm. In this assay, the enzyme is pre-incubated with test compounds, followed by addition of substrate and measurement of well epi-absorbance. As designed, compounds that activate procaspase-7 activity will increase substrate hydrolysis, leading to an increase in well absorbance. Compounds are tested in singlicate at a nominal concentration of 8.5 uM.

Protocol Summary:

Prior to the start of the assay, 2.5 uL of zinc-free Assay Buffer (50 mM HEPES, 300 mM NaCl, pH 7.4, 0.01% Triton-X 100) containing 2 uM of PC-7 D3A protein were dispensed into 1536 microtiter plates. Next, 43 nL of test compound in DMSO or DMSO alone (0.8% final concentration) were added to the appropriate wells and incubated for 1 hour at 25 C.

The assay was started by dispensing 2.5 uL of 400 uM Ac-DEVD-pNA in Assay Buffer to all wells. Plates were centrifuged and after 2 hours of incubation at 25 C, epi-absorbance was read on the EnVision plate reader using a photometric filter set (excitation = 405 nm, emission = 450 nm) and a dichroic mirror with the 425 nm cutoff. Fluorescence emission was read at 10 flashes per well at two time points (0 minutes and 120 minutes).

Prior to further calculations, the following formula was used to calculate absorbance:

Absorbance = ( -Log10 * ( [Raw2] / [Mean Reference2] ) ) - ( -Log10 * ( [Raw1] / [Mean Reference] ) )

Where:

Raw1 is defined as the read at T0 minutes.
Raw2 is defined as the read at T120 minutes.
Mean Reference is defined as a mean of values from wells containing buffer only at T0.
Mean Reference2 is defined as a mean of values from wells containing buffer only at T120.

The percent activation for each compound was calculated as follows:

% Activation = ( ( Ratio_Test_Compound - Median_Ratio_Low_Control ) / ( Median_Ratio_High_Control - Median_Ratio_Low_Control ) ) * 100

Where:

Test_Compound is defined as wells containing test compound.
Low_Control is defined as wells containing DMSO.
High_Control is defined as wells containing DMSO and 5 uM PC-7.

PubChem Activity Outcome and Score:

A mathematical algorithm was used to determine nominally activating compounds in the primary screen. Two values were calculated: (1) the average percent activation of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % activation than the cutoff parameter was declared active.

The reported PubChem Activity Score has been normalized to 100% observed primary activation. Negative % activation values are reported as activity score zero.

The PubChem Activity Score range for active compounds is 100-14, and for inactive compounds 14-0.

List of Reagents:

Recombinant Procaspase 7 enzyme (Assay Provider)
Assay Buffer (Assay Provider)
Chromogenic Substrate (Ac-DEVD-pNA) (Assay Provider)
1536 SWSN plates (Grainer, part 789175 )

Comment: Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well absorbance. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.

External ID: PPAFAH_INH_FP_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that act as inhibitors of the plasma platelet activating factor acetylhydrolase (pPAFAH), which has a catalytic cysteine residue and is sensitive to thiol alkylating agents such as N-ethylmaleimide. In this biochemical assay, recombinant pPAFAH protein is incubated with test compounds and a Rh-conjugated activity-based probe. The reaction is excited with linear polarized light and the intensity of the emitted light is measured as the polarization value. The assay is performed by incubating test compounds with pPAFAH for a defined period, followed by addition of the FP-rhodamine probe and measurement of fluorescence polarization at a specific time point. As designed, test compounds that act as pPAFAH inhibitors will prevent pPAFAH-probe interactions, thereby increasing the proportion of free (unbound) fluorescent probe in the well, leading to low fluorescence polarization. Compounds are tested in singlicate at a final nominal concentration of 3.39 uM.

Protocol Summary:

Prior to the start of the assay, 4.0 uL of Assay Buffer (0.01% Pluronic acid, 50 mM Tris HCl pH 8.0, 150 mM NaCl, 1 mM DTT) containing 12.5 nM of pPAFAH protein were dispensed into 1536 microtiter plates. Next, 17 nL of test compound in DMSO or DMSO alone (0.34% final concentration) were added to the appropriate wells and incubated for 30 minutes at 25 C.

The assay was started by dispensing 1.0 uL of 375 nM FP-Rh probe in Assay Buffer to all wells. Plates were centrifuged and after 15 minutes of incubation at 25 C, fluorescence polarization was read on a Viewlux microplate reader (PerkinElmer, Turku, Finland) using a BODIPY TMR FP filter set and a BODIPY dichroic mirror (excitation = 525 nm, emission = 598 nm). Fluorescence polarization was read for 15 seconds for each polarization plane (parallel and perpendicular).

Prior to further calculations, the following formula was used to calculate fluorescence polarization (FP):

FP = ( Raw1 - Raw2 ) / ( Raw1 + Raw2 )

Where:

Raw1 is defined as the S channel.
Raw2 is defined as the P channel.

The percent inhibition for each compound was calculated as follows:

100 * ( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control ) )

Where:

Low_Control is defined as wells containing pPAFAH and DMSO.
Test_Compound is defined as wells containing pPAFAH in the presence of test compound.
High_Control is defined as wells containing no pPAFAH protein.

A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.

PubChem Activity Outcome and Score:

The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.

The PubChem Activity Score range for active compounds is 100-14, and for inactive compounds 14-0.

List of Reagents:

Recombinant pPAFAH protein (supplied by Assay Provider-Brian Bahnson)
FP-Rh probe (supplied by Assay Provider-Benjamin Cravatt)
Tris HCl (Sigma, part T3038)
NaCl (Sigma, part S6546)
Pluronic Acid (Invitrogen, part P6866)
DTT (Invitrogen, part 15508-013)
1536-well plates (Corning, part 7261)

Comment: Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.

External ID: SMAD3201

Protocol: Suspensions of trypsinized HEPG2 CAGA-GFP cells were dispensed into white, tissue culture-treated, solid 1536-well plates at 5uL/well (1000 cells/well final concentration) in DMEM medium supplemented with 1% FBS. Plates were incubated at 37 degrees C for 2 hours, after which 23 nL of compounds or DMSO were delivered to each well using a pin tool. One uL of recombinant TGF-beta in DMEM (1% FBS) was then dispensed (500 pg/mL final concentration), and plates were incubated at 37 degrees C for 18 hours. Two uL of CellTiter Glo (Promega), a luminescence-based viability reagent, was dispensed, followed by a 10 minute room temperature incubation. The plates were then measured on a PerkinElmer ViewLux plate reader for luminescence (clear filter) using a 5 second exposure. The %Activity was determined from the corrected luminescence values. Wells containing media only (no cells) were used to normalize %Activity of identified toxic compounds; media-only wells corresponded to 100%Activity (complete cell-killing), while DMSO-dosed cell controls were used to normalize 0%Activity (no toxicity).

Concentration-response curves were fitted to the signals arising from the resulting luminescence. The concentration-effect curves were then classified based on curve quality (r2), response magnitude and degree of measured activity, and compounds were subsequently categorized based on their curve class. Toxic compounds showed concentration-dependent decreases in luminescence, concordant with a decrease in intracellular ATP concentration (CellTiter Glo's marker of viability), and thus a decrease in the number of viable cells. Inactive (non-toxic) compounds showed no effect on luminescence signal. Active (toxic) compounds showed concentration dependent decrease in luminescence.

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".

2. For all inactive (non-toxic) compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active (toxic) compounds, a score range was given for each curve class type given above. Active (toxic) 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: FEN1001

Protocol: Three uL of reagents (buffer in column 3 and 4 as negative control and 10 nM FEN1 in columns 1, 2, and 5-48) were dispensed into 1,536-well black solid-bottomed plate. Compounds (23 nL) were transferred via Kalypsys pin tool equipped with 1536-pin array (10 nL slotted pins, V&P Scientific, San Diego, CA). The plates were then incubated for 15 min at room temperature, and 1 uL substrate (50 nM final concentration) were added to start the reaction read twice at 0 min read and 15 min on ViewLux reader. Throughout the screen, reagent bottle and all liquid lines were chilled and made light-tight to minimize reagent degradation. All screening operations were performed on a fully integrated robotic system (Kalypsys, San Diego, CA) containing one RX-130 and two RX-90 anthropomorphic robotic arms (Staubli, Duncan, SC). Library plates were screened starting from the lowest and proceeding to the highest concentration, and a "double-dipping" step of the highest concentration was required to access higher concentrations of compounds. Vehicle-only plates, with DMSO being pin-transferred to the entire column 5-48 compound area, were inserted uniformly at the beginning and the end of each library in order to monitor for and record any shifts in the background, which can be affected by reagent dispensers or loss in enzyme activity overtime. Screening data was corrected, normalized, and concentration-effect relationships were derived by using publicly-available curve fitting algorithms developed in-house (http://ncgc.nih.gov/pub/openhts). A four parameter Hill equation was fitted to the concentration-response data by minimizing the residual error between the modeled and observed responses.

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.