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: UNMCMD_HOXA9_MYELOIDDIFFERENTIATION_PRIMARY_VALIDATIONSET

Protocol: This assay will be used to identify small molecules that promote the differentiation of acute myeloid leukemia cells. Cells will be arrested in differentiation by the expression of HoxA9 from an estrogen receptor-HoxA9 construct. The screen will be conducted in the presence of estrogen so HoxA9 will be continuously expressed. The cells also contain a GFP construct regulated by the lysozyme promoter. Differentiated cells will express GFP. Small molecules that promote differentiation will be identified by green fluorescence. In order to eliminate compounds that are green fluorescent in nature or compounds that activate the expression of GFP, a MAC1-APC antibody will be used to detect true differentiation. True hits will be identified as those cells that are both green and red fluorescent.

Cells are cultured in RPMI supplemented with 10% fetal bovine serum, penicillin/streptomycin,L-glutamine, 5% conditioned media containing stem cell factor (SCF; approx 100ng/milliL), and beta-estradiol (0.5 microM). The conditioned media is generated by a Chinese Hamster Ovary (CHO) cell line which constitutively expresses and secretes SCF into the supernatant. On day 0, cells and inert polystyrene beads (5 micron; Spherotech CPX-50-10), and Pluronic (final 0.04%, Sigma P5556) suspended in media are dispensed into 384-well plates using an automated dispenser. Slow, continuous stirring using a sterile magnetic stir-bar keeps the cells dispersed during the dispensing process. The compounds (dissolved in DMSO) are pinned into the cells. The plates are incubated for four days under standard conditions (37 degrees-C, humidified, 5% CO2). On day 4, the MAC1-APC antibody will be added. After a twenty minute incubation with the antibody, the plates are analyzed using high-throughput flow cytometry via the HyperCyt autosampler (IntelliCyt, USA) connected to a Cyan flow cytometer. Cell viability and green fluorescence is stable for 24 hours after the day 4 time point. Gating on the inert bead population provides a measure of sampling quality, as the same number of beads are seeded into each well on day 0. The percent of GFP and APC positive live cells is used to determine whether the test compound has a differentiating effect.

Calculations:

The data are exported into a Microsoft Excel spreadsheet template, and the PERCENT_RESPONSE is calculated for each well as follows:
PERCENT_RESPONSE = 100*(Sample%X+Y+ - DMSO%X+Y+)/(PCntrl%X+Y+ - DMSO%X+Y+)
where %X+Y+ is the percentage of the population in the double positive region (both GFP and APC positive) for either wells with Sample, DMSO control or Positive Control (5 microM fulvestrant).

Compounds were deemed active if PERCENT_RESPONSE is equal to or greater than 50%. And the PUBCHEM_ACTIVITY SCORE equal to the PERCENT_RESPONSE.

Comment: 1. Fenaux, P., et al., Effect of all transretinoic acid in newly diagnosed acute promyelocytic leukemia. Results of a multicenter randomized trial. European APL 91 Group. Blood, 1993. 82(11): p. 3241-9.
2. Lawrence, H.J., et al., The role of HOX homeobox genes in normal and leukemic hematopoiesis. Stem Cells, 1996. 14(3): p. 281-91.
3. Borrow, J., et al., The t(7;11)(p15;p15) translocation in acute myeloid leukaemia fuses the genes for nucleoporin NUP98 and class I homeoprotein HOXA9. Nat Genet, 1996. 12(2): p. 159-67.
4. Drabkin, H.A., et al., Quantitative HOX expression in chromosomally defined subsets of acute myelogenous leukemia. Leukemia, 2002. 16(2): p. 186-95.
5. Andreeff, M., et al., HOX expression patterns identify a common signature for favorable AML. Leukemia, 2008. 22(11): p. 2041-7.
6. Tedeschi, F.A. and F.E. Zalazar, HOXA9 gene expression in the chronic myeloid leukemia progression. Leuk Res, 2006. 30(11): p. 1453-6.
7. Faber, J., et al., HOXA9 is required for survival in human MLL-rearranged acute leukemias. Blood, 2009. 113(11): p. 2375-85.
8. Stegmaier, K., et al., Gene expression-based high-throughput screening(GE-HTS) and application to leukemia differentiation. Nat Genet, 2004. 36(3): p. 257-63.

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: 2134-01_Inhibitor_SinglePoint_HTS_Activity_Set2

Protocol:
1. Clone20-Shn3FFL cells (Lot#1-810) are cultured and expanded in OB media
to obtain sufficient cell numbers to plate across the ~2,100 384-well plates required
to screen in duplicate the 330,000 compounds in the MLSMR collection.
2. Cells are plated at a concentration of 3000 cells per well in 30ul of OB media using a Combi Multidrop (Thermo)
in white opaque 384-well plates (Corning 3570/8867BC).
3. Once plated, cells are incubated overnight at 37 degrees C 5% CO2 95% humidity prior to the addition of
compounds.
4. Compounds are then be added to plates at a volume of 100nl of compound using a steel pin tool (V&P Scientific).
5. Following the addition of compounds, cells will be incubated for an additional 24
hours at 37 degrees C 5%CO2 95% humidity.
6. After 24 hour incubation period, cells and luciferase reagents are allowed to
equilibrate to room temperature for 10 minutes.
7. For firefly luciferase assay, 20ul of Cell Titer Glo luciferase (Promega) is added
to each well with a Combi multidrop and then plates are incubated for 15 minutes at room temperature
on orbital shaker.
8. Plates are read on an Envision multi-mode plate reader (Perkin Elmer) to
obtain luminescence readings (0.1 s/well)

Comment: PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.
All well activities were then multiplied by -1 to create a positive activity readout value range, to match Pubchem convention.


PATTERN CORRECTION: The plate pattern correction algorithm 'Runwise Pattern (Multiplicative)' in Genedata (v7.0.3) was applied to the normalized plate data.

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the mean of the normalized and corrected sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 75.

PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 100.

PUBCHEM_ACTIVITY_OUTCOME:
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T

Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T

Samples passing AT only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T

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: HMS979

Protocol: Cation-adjusted Mueller Hinton II broth supplemented with 0.005% Tween-80 was inoculated with a single colony of S. aureus RN4220. Following overnight incubation, the culture was diluted 1:100 into fresh medium, and incubation was continued until the bacterial suspension reached an optical density at 600 nm (OD600) of 0.6, corresponding to a bacterial density of 5 x 10E8 colony forming units (CFU)/mL. An aliquot of this culture was diluted 1:400 with fresh medium and stored on ice until use. Assay plates were prepared for compound transfer in quadruplicate to enable screening of compounds at two temperatures in duplicate. The plate layout was as follows: wells in columns 1-22 were loaded with culture medium at 25 microL/well; wells in column 23 contained the screening negative control (medium only); wells in column 24 contained the screening positive control (medium containing 25 microg/mL chloramphenicol). 300 nL of experimental compounds were transferred by stainless steel pin array from library plate to assay plates.

25 microL of the bacterial suspension was added to each assay well. Assay plates were incubated at 42 degrees C in humidified chambers (humidity > 85%). After 20 h, the OD600 was measured using a plate reader in absorbance mode.

Comment: Data analysis description:

Absorbance values at 42 degrees C and 30 degrees C for each replicate were normalized to positive and negative control plate average absorbance, and replicate normalized values were averaged to calculate an average relative absorbance at both temperatures. A substance was considered a temperature-dependent positive at 42 degrees C if average relative absorbance <= 50 and the difference between relative absorbance at 30 degrees C and 42 degrees C >= 20 (relative absorbance 30 degrees C - relative absorbance 42 degrees C >= 20). A substance was considered a temperature-independent positive if relative absorbance at both 30 degrees C and 42 degrees C < 10.

Activity scores were calculated using average relative absorbance at both temperatures. Average relative absorbance <= 0 was scored as 100 for activity; average relative absorbance >= 100 was scored as 0 for activityy. Average relative absorbance between 0 and 100 was subtracted from 100 to generate activity scores for intermediate values (i.e. average relative absorbance = 40 corresponds to an activity score of 60).

Note that since this is treated as a panel assay, the query for active compounds includes many that were considered active at both temperatures. The final Pubchem activity score is the activity score at 42 degrees C, and compounds scored as active (PUBCHEM_ACTIVITY_OUTCOME = 2) include both temperature-dependent and temperature-independent active substances.

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: MH080376 Biochemical HTS for Inhibitors of the Proprotein Convertase Furin.

Protocol: Biochemical Furin HTS Assay protocol

Reaction Buffer Concentrations (final concentrations): 50 mM Hepes pH 7.5, 1 mM CaCl2, 1 mM beta-mercaptoethanol , 0.2 mg/ml BSA.

Substrate Stock Solution: 10 mM pERTKR-AMC in DMSO; stored in aliquots at -20 oC.

rhFurin714 prep (5.2 units/uL)Stored in aliquots at -80oC.

Furin Assay Protocol

The assay involves three liquid transfer steps of 5 uL each of 30 uM compound in 1% DMSO (10 uM final), 1 unit/5 uL rhFurin714 in 3X reaction buffer and 5 #L of 30 uM pERTKR-AMC substrate (10 uM final).

The assay plates used are Greiner 384-well, flat-bottom, low volume, black polystyrene plates (VWR catalog # 784076).

1. Add 5 uL of compound/controls to each well.
2. Add 5 uL of rhFurin714 in 3X buffer (1.0 units/well final).
3. Add 5 uL of 30 uM pERTKR-AMC fluorigenic substrate (10 uM/well final).
4. Incubate the plates for 1 hr at room temperature.
5. Stop the reaction by adding 5 uL of 1M Acetic acid.
6. Measure the amount of fluorigenic AMC released on the SpectraMax M5 using Ex = 345nm; Em = 440nm; cut-off 420 nm.

Comment: Active Criteria, Secondary Assay Plan, Hit and Lead Criteria.

Furin HTS Activity scoring rules:

The Furin inhibitor HTS run at the PMLSC utilized % inhibition calculated from maximum (n=32) and minimum (n=24) plate controls, with a hit criteria of >/= 25% inhibition to identify active compounds.

Furin Inhibitor scoring rules:

PUBCHEM_ACTIVITY_OUTCOME

1 - Substance is considered inactive when the % inhibition is < 25 %
2 - Substance is considered active when % activation is >/= 25 %
3 - Substance activity outcome is inconclusive

PUBCHEM_ACTIVITY_SCORE

0-40 scoring range is reserved for primary HTS data
a) if the % inhibition is >/= 25 %, the score is 40.
b) if the % inhibition is < 25 %, the score is 0.

Definition of Hit Criteria:
It is anticipated that all Furin inhibitor HTS actives will be confirmed in duplicate at the primary HTS concentration of 30 uM.
Furin inhibitor actives confirmed in the primary HTS format will then be run in 10-pt IC50 concentration response curves.
Confirmed hits will be subjected to structural confirmation by LCMS.

Secondary assay testing paradigm: Confirmed concentration dependent Furin inhibitors will be tested in the HeLa pcFur1.6 cell based ELISA assay.

External ID: UNMCMD_ABCB6_1o_ValidationSet

Protocol: Primary screening assay is based on measuring the accumulation of porphyrin in the mitochondria by a flow cytometer with excitation at 488nm and emission at 575 nm. Potential inhibitors of ABCB6 will be identified in the Primary Assay by a decrease in fluorescent signal.

1) Cell cultures of erythroleukemia K562 cell lines stably expressing ABCB6 will be grown at 37 degreesC, 5% CO2 and collected, resuspended in 5% Serum PBS to a cell density of 4-5 x 10^7 cells/ml. Aliquots of 10 microL volume will be transferred to 384-well plates containing 100 nanoLvolume of the test compounds or DMSO in 5 microL of 5% serum PBS.
2) Test compounds, stored as DMSO stocks, will be diluted in 5% serum PBS.
3) DMSO concentration will be 0.5%, which is well tolerated by the assays. Positive control are cells exposed to 70 microM benzenthonium chloride.
4) Microplates will be incubated for 1hr at 37 degreesC.
5) HyperCyt(R) sampling will consist of 1-2 microL taken from a 15 microL well volume at room temperature.
6) Data file analyses will use HyperView software programs to automatically detect the time-resolved data clusters (wells), and determine the median channel fluorescence. Data will be automatically exported to a Microsoft Excel spreadsheet to calculate the Zprime.

Calculations:

Percent response for this assay is calculated with the following equation:
PERCENT_RESPONSE = 100*(1-(Sample-PCntrl)/(NCntrl-PCntrl))
where Sample, PCntrl, and NCntrl are the median channel fluorescence for the Sample compound, positive control well (benzenthonium chloride), and negative control well (DMSO).

Compounds are deemed active if their PERCENT_RESPONSE is greater than the overall average plus 3 standard deviation for this collection of compounds (i.e., 51%). Note that in the column entitled PUBCHEM_ASSAYDATA_COMMENT, there are compounds with annotation of "PotentiallyFluorescent" if the raw median channel fluorescence measured is greater than twice the value measured for the negative control wells with DMSO.

Keywords: NIH Roadmap, NMMLSC, UNMCMD, ABCB6

Comment: N/A

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: UNMCMD_phenotypic_multiplex_HTS_for_antifungal efflux_pump_inhibitors_ValidationSet

Protocol: Yeast cultures (Saccharomyces cerevisiae strain AD1-8u background), expressing efflux pumps from Candida albicans, namely AD/CaCDR1; AD/CaCDR2 and AD/CaMDR1, and the control strain containing the empty pABC3 cassette without a heterologous efflux pump gene, AD/pABC3, (all supplied by the assay provider) are incubated at 30 degrees C with shaking (250 rpm) until an optical density (OD 600) of 1.5 to 2.0 is obtained. All cultures are adjusted to be at an equivalent OD of 0.6. The cells are collected by centrifugation and washed with PBS, then re-suspended as a tri-plex mixture in PBS with 10 microM Nile Red (Invitrogen USA). The mixture is placed at 4 degrees C to equilibrate with efflux pumps effectively turned off. Wells containing 5 microL of PBS with 2 percent glucose and 20 microM compound are plated during this time. Enniatin B is used as an on-plate positive control at 25 microM. Vector (AD/pABC3, stained in the same protocol as the tri-plex) is used as an off-plate control. The compound/buffer wells receive 5 microL of stained tri-plex mixture. In-well reagent summary: 5 microM Nile Red, 10 microM compound, 1 percent glucose. The assay plate is incubated inverted 30 degrees C for 1 hr. Accumulation of Nile Red is measured by sample delivery via HyperCyt (IntelliCyt, USA) to a Cyan (Beckman Coulter, USA) flow cytometer. Samples are excited with a 488 nm laser and a PE-Texas Red filter is used to detect the fluorescence signal from the Nile Red substrate.

Calculation:
The data are exported into a Microsoft Excel spreadsheet template, and the PERCENT_RESPONSE is calculated for each well as follows:

PERCENT_RESPONSE = 100((MFItest-MFIneg.control)/(MFIpos.control-MFIneg.control))
where MFI is median fluorescence intensity of cells with test compounds, positive or negative control wells.

RAW_MEDIAN = median fluorescence intensity of cells per well

Cutoff for determining compound as active is based on compound having response value larger than 4 standard deviations of the overall response values measured for the entire Validation compound set, i.e., 0.02 + 4*0.13 = 0.42.

The PUBCHEM_ACTIVITY_SCORE is determined by multiplying response value with 100. Meaning Active compounds have PUBCHEM_ACTIVITY_SCORE greater than 42.

Comment: N/A

External ID: MH083223 Targeting HIV-1 Nef with Small Molecules

Protocol: HIV Hck:Nef HTS Protocol
1. Recombinant Hck-YEEI protein, was purified to homogeneity from Sf-9 insect cells
2. Recombinant HIV-1 Nef (SF2 strain), was purified to homogeneity from E. coli
3. Z-Lyte assay reagents were purchased from (Invitrogen).

Several Hck and Nef protein preparations provided by the assay provider were utilized in the HTS campaign, and several Hck:Nef protein ratios were utilized 10 ng + 50 ng, 12.5 ng + 75 ng, 15 ng + 75 ng, and 20 ng + 100 ng in the 384-well HTS assay.

HTS Protocol:
1. Thaw compound plates, 2uL of 1 mM in 100% DMSO.
2. Spin compound plates down, 5 min 50 x g.
3. Dilute 2 uL 1mM compounds with 23ul of water on Flex Drop (80 uM, 8% DMSO).
4. Mix and Transfer 2.5ul of compound to assay plate on EP3.
5. Transfer 5ul of Max (2.5ul DMSO and 2.5ul Hck:Nef) and Min (2.5ul DMSO and 2.5ul Hck/1x kinase) controls (from pre-made control plates) to assay plate on EP3.
6. Add 2.5ul of Hck:Nef to assay plate(340 wells), spin down and incubate for 30 min at ambient temperature (40 uM, 4% DMSO).
7. Add 5 uL of ATP/Tyr complex to whole plate on MicroFlow, spin down plates and put on shaker to incubate at ambient temperature for 50 min (20 uM, 2% DMSO).
8. Add Development buffer to whole plate on MicroFlow, spin down plates and put on shaker to incubate at ambient temperature for 60 min.
9. Add Stop reagent to whole plate on MicroFlow, spin down.
10. Read Donor to Acceptor FRET Emission Ratio on M5 plate reader within 2 hrs. Excitation of the donor fluorophore at 400 nm, Donor to Acceptor FRET Ratio = ratio of Coumarin emission 445 nm to Fluorescein emission 520 nm.

Comment: Hck-Nef Assay HTS Activity scoring rules:
The 384-well format Hck-Nef inhibitor HTS run at the PMLSC utilized % inhibition calculated from maximum (n=32) and minimum (n=24) plate controls, with a hit criteria of >/= 50% inhibition to identify active compounds.
Max control: 2.5ul DMSO and 2.5ul Hck:Nef
Min control: 2.5ul DMSO and 2.5ul Hck/1x kinase (No Nef)
Hck-Nef Inhibitor scoring rules:
PUBCHEM_ACTIVITY_OUTCOME
1 - Substance is considered inactive when the % inhibition is < 50 %
2 - Substance is considered active when % inhibition is >/= 50 %
3 - Substance activity outcome is inconclusive
PUBCHEM_ACTIVITY_SCORE
0-40 scoring range is reserved for primary HTS data
a) if the % inhibition is >/= 50 %, the score is 40.
b) if the % inhibition is < 50 %, the score is 0.

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: HIV1-VIF_MS

Protocol: The assay protocol for screening and testing compounds included plating a mixture of GST-Vif "donor" protein and bio-A3G "acceptor" peptide with test compounds in white, non-binding surface 1536-well plates. LANCE reagents were purchased from PerkinElmer. Briefly, 15nM GST-Vif protein was added to the assay plate containing test compounds. Following a 30 minute incubation, 500 nM bio-A3G peptide was added. Following another 30 minute incubation, 2 nM Eu-labeled anti-GST and 50 nM streptavidin-Ulight detection reagents were added to the assay plate and incubated for 1 hour. After the final incubation, TR-FRET signals were measured using an Envision microplate reader with excitation at 340 nm and emission at 615 and 665 nm. The emission at 615 nm from Eu-labeled anti-GST induces emission at 665 nm from Ulight conjugated to streptavidin when the two molecules are in close proximity, resulting in a FRET signal. GST-Vif binding to the bio-A3G peptide produced a significant increase in the FRET signal over background levels measured in the presence of GST.

To evaluate the screening results each plate included negative and positive controls. Negative control wells included all assay components in the absence of an inhibitor and positive control wells included all assay components with GST-Vif 1-94 protein replaced by GST protein. Percent inhibition was determined as: 100*((Test Compound Signal - Median Negative Control Signal)/(Median Positive Control Signal - Median Negative Control Signal)).

Comment: In the Dose Response, compounds that showed >30% inhibition for at least one concentration were considered potentially active, however some compounds were tested in dose response on more than one occasion. If conflicting results were observed, the compound was deemed "inconclusive". Only compounds that consistently showed activity across all test dates were labeled "Active". Any compounds in the primary screen active range that were not available for testing in dose response were labeled "Inconslusive".
The following tiered system has been implemented at Southern Research Institute for use with the PubChem Score. Compounds in the primary screen are scored on a scale of 0-40 based on inhibitory activity where a score of 40 corresponds to 100% inhibition. In the confirmatory dose response screen, active compounds were scored on a scale of 41-80 based on the average IC50 result, inconclusive compounds were given the lowest confirmatory score of 41, and compounds that were tested and did not confirm as actives were given the score 0.

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: UNMCMD_DSG_VALIDATIONSET

Protocol: Protocol:
1) Protein G beads (1.06 million beads per 384 well plate) are coupled with the DSG3 antigen by overnight incubation with a cell lysate containing an Fc-DSG3 construct.
2) Coupled beads are used at 3000 beads per well
3) scFv-GFP reagent is diluted in Assay buffer (PBS with 1mM CaCl2, 0.05% BSA, 0.01% Na Azide), added to 382 well assay plates, and incubated with a 20 microM solution of test compounds for 60 minutes at RT.
4) Vehicle control (2%DMSO) and Blocking control (1/40 dilution of soluble DSG3 antigen) are similarly incubated with scFv-anti-DSG3-GFP
5) Pre-coupled beads are added to each well and plates are incubated for 60 minutes with rotation
6) scFv-anti-DSG3-GFP binding to beads is detected using flow cytometry and reported as the Median Channel Fluorescence

Calculations:
Z and Z' values were calculated individually for all plates, most plates passed a Z'>0.3.
An average response value was computed for each plate. Compounds were considered active if the associated well fluorescence was greater than 3SD below the Average Median Fluorescence of the individual plate.

dif = PLATE_CUTOFF - RESPONSE

If diff < 0
Then PUBCHEM_ACTIVITY_SCORE = 0
Else If diff > 100
Then PUBCHEM_ACTIVITY_SCORE = 100
Else
PUBCHEM_ACTIVITY_SCORE = diff

If (PUBCHEM_ACTIVITY_SCORE > 0) AND (RESPONSE > 0)
THEN PUBCHEM_ACTIVITY_OUTCOME = 2 (or ACTIVE)
If (PUBCHEM_ACTIVITY_SCORE > 0) AND (RESPONSE = 0)
THEN PUBCHEM_ACTIVITY_OUTCOME = 3 (or INCONCLUSIVE)
Else
PUBCHEM_ACTIVITY_OUTCOME = 1 (or INACTIVE)

Comment: N/A

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: 2132-01_Agonist_SinglePoint_HTS_Activity

Protocol:
Vibrio cholerae quorum-sensing modulator bioassay
Reporter strain:
1.BH1578 (V. cholerae AcqsA AluxQ carrying pBB1 cosmid, which contains the V. harveyi luxCDABE luciferase operon)
Materials:
LB Medium: Dissolve in 10 g/L Tryptone, 5 g/L Yeast Extract, and 10 g/L NaCl in distilled water, autoclave, store at room temperature
Tetracycline (10 mg/mL): Dissolve 10 mg tetracycline in 1 mL 100% ethanol, store at -20 degrees C, protect from light
LB/tet: add 1 mL tetracycline (10 mg/mL) to 1L of LB medium. Final concentration of tetracycline is 10 microg/mL. Prepare it fresh on a daily basis.
CAI-1 stock: Dissolve CAI-1 in DMSO to 50 mM (10.7 mg/mL), store at -20 degrees C
Procedures:
1.Grow up the BH1578 reporter strain in 100 mL LB/Tet at 30 oC for >16 hours with shaking (200 rpm). The final OD600 of each culture should be > 3.0
2.Dilute the culture to an OD600 of 0.9 with LB/Tet, mix well. (Note: avoid biofilm aggregates in the culture, a low speed centrifugation (200 rpm for 1 min) should remove most aggregates)
3.Add 20 uL of LB-Tet per 384 well assay plate with the Thermo Combi fluid dispenser. Add 150 nL of compound per well.
4.Dispense 10 microL of diluted culture into each well of a 384 well plate (Black with clear bottom Greiner 781096 plates). Compounds are screened at 20 microM final concentration. 1 uM CAI-1 was used as the positive control.
5.Incubate the plates at 30 degrees C for 6 hours.
5.Measure bioluminescence (USLum(384)) and OD600 in a Perkin-Elmer Envison Multilabel Reader

Comment: PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.

EXPECTED OUTCOME: Active compounds result in increasing readout signal.
NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Stimulators Minus Neutral Controls' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of 100.
Experimental wells values were scaled to this range.
PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the mean of the normalized sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 30.

PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 50.

PUBCHEM_ACTIVITY_OUTCOME:
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T

Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T

Samples passing AT only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T

tSamples passing PAR_T only were assigned an outcome of 2 (active) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T

NOTE: Raw signal values for inactive compounds were outside the linear detection range of the plate reader; therefore, no REPRODUCIBILITY_COSINE_TRANSFORM was calculated for inactive compounds.

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: MH083154

Protocol: Materials
MATERIALS
MEF-GFP-LC3 cells (provided by Dr. Xiao-Ming Yin, University of Pittsburgh)
384 well flat bottom microplates, collagen-coated (Falcon Biocoat)
DMEM growth medium (Gibco), supplemented with 10% Fetal bovine serum (HyClone) and antibiotics
Thapsigargin (Molecular Probes T-7459), dissolved in DMSO at 1 mM and frozen in aliquots at -20oC.
DMSO (Sigma 15,493-8)
Formaldehyde, 37 wt. % solution in water, A.C.S. reagent (Sigma 252549)
Test compounds plated into 384 well microplates (Greiner polypropylene).
Hanks balanced salt solution (HBSS, Fisher SH3026802)
Hoechst 33342 (Molecular Probes/Invitrogen H-1399)

PROTOCOL
Day 1. Cell seeding.
Plate 5,000 MEF-GFP-LC3 cells/40 [micro]L complete growth medium into the wells of collagen-coated 384 well microplates and incubate overnight at 37oC, 5% CO2.
Day 2. Compound treatment.
Thaw compound plates containing 2 [micro]L of 1 mM drug in DMSO.
Reconstitute wells A3:P22 of library compound plates in 38 [micro]L complete growth medium. Intermediate concentrations are 50 [micro]M compound and 5% DMSO
Load appropriate control wells on reconstituted library compound plates with 40 [micro]L of positive and negative controls (5% DMSO) and 5 [micro]M thapsigargin in 5% DMSO (Biomek2000, Perkin Elmer Multiprobe).
Transfer 10 [micro]L of treatment solution from reconstituted library compound plates to assay plates. (Perkin Elmer Janus MDT) and incubate for 18h at 37oC, 5% CO2. The final assay concentration will be 10 [micro]M compound and 1% DMSO.

Day 3. Fixation, staining, and analysis.
Prepare fixative (10.7% formaldehyde and 26.7 [micro]g/ml Hoechst 33342 in HBSS).
Dispense 30 [micro]L of fixative directly into all plates (Titertek MAP-C2).
Incubate for 30 min at room temp. under dimmed light.
Aspirate off fixative and wash plates three times with 50 [micro]L PBS (MAP-C2).
Seal plates and analyze on the ArrayScan Vti (Thermo Fisher Cellomics) for autophagosomal GFP-LC3 accumulation.
Acquire images in two channels (DAPI/FITC) using a 10x objective and an XF100 filter set on the ArrayScan VTi.

ANALYSIS
Analyze images by the Compartmental Analysis Bioapplication, configured to detect Hoechst 33342 stained nuclei, cytosolic GFP expression, and autophagosomes as GFP-expressing punctate objects located in the cytosol. First, nuclear and cytosolic areas were separated to avoid nuclear punctate objects to be included. Punctate structures were then detected in the cytosol by thresholding over local background. A selection threshold was then set using DMSO-treated cells to calculate the percentage of cells positive for autophagosome formation.

Comment: HCS parameters reported
1. %HIGH_RingSpotCountCh2: The percentage of cells containing GFP spots (Channel 2). The percentage of responders is computed on a plate by plate basis.
2. Z-score_%HIGH_RingSpotCountCh2: Z-score of the percentage of cells containing GFP spots (Channel 2).
3. HCS_cmpd_conc: Primary HCS compound concentration in [micro]M
4. MEAN_RingAvgIntenCh2. The average GFP intensity in the cytosol (Channel 2). The average intensity is reported on a per cell basis.
5. MEAN_RingSpotTotalAreaCh2. The total (integrated) intensity of GFP spots per cell (Channel 2). The total spot intensity is reported on a per cell basis.
6. SelectedObjectCountPerValidField. The number of nuclei per imaging field.
7. % Toxicity. % toxicity = 1-(SelectedObjectCountPerValidField in sample well / average SelectedObjectCountPerValidField of all MIN controls on the plate)*100
8. Assay Date : Date the HTS assay was performed

The high-content cell-based screen for modulators of autophagy conducted by the PMLSC utilized a Z-score statistical scoring method to identify active compounds (Brideau et al., 2003)
Brideau, C., Gunter, B., Pikounis, B., and Liaw, A. (2003). Improved statistical methods for hit selection in high-throughput screening. Journal of Biomolecular Screening 8, 634-647. 14711389

Target activity score (Z-score_%HIGH_RingSpotCountCh2)
The Z-score for a compound was computed on a plate by plate basis. The Z-score for the percentage of cells containing autophagosomes in Channel 2 (Xi) was defined as Xi = (Xi - Xm)/Sm, where Xm is the mean of all the percentage of cells containing autophagosomes values in wells A3:P22 on the microplate, and Sm is the standard deviation of these values. A cut-off Z-score of greater than 3 was selected as the definition as the active criterion.

Fluorescent outliers. (MEAN_RingAvgIntenCh2)
The average GFP intensity per cell was calculated on a per cell basis. A cut-off score of 1500 was selected as the definition for a fluorescence outlier.

Cytotoxicity outliers (% toxicity)
% toxicity was computed on a plate by plate basis. % toxicity was defined as 1-(cell density in sample well / average cell density of all MIN controls on the plate)*100. A cut-off score of more than 80% was selected as the definition for a cytotoxic compound.


Definition of an active compound
Z-score_%HIGH_RingSpotCountCh2 > 3, MEAN_RingAvgIntenCh2 > 1500, and % toxicity < 80%

PUBCHEM_ACTIVITY_OUTCOME
1 inactive. Substance is considered inactive when the Z-score_%HIGH_RingSpotCountCh2 > 3 at 10 [micro]M.
2 active. Substance is considered inactive when the Z-score_%HIGH_RingSpotCountCh2 > 3, MEAN_RingAvgIntenCh2 < 1500, and % toxicity < 80% at 10 [micro]M

PUBCHEM_ACTIVITY_SCORE
0-40 range is reserved for primary HTS data
a)If the substance is considered active the score is 40
b)If the substance is inactive the score is 0

Secondary assay testing paradigm:
Confirmation of inhibition of autophagosome formation in 2 independent tests at 10 [micro]M.
Concentration Response IC50 < 10 [micro]M in the autophagy inducer HCS assay.
Structural Verification
Indications of Specificity & Selectivity ~ PubChem X-target Query
Evidence of SAR.

Confirmed concentration dependent actives would be tested for A) autophagosome formation in a different cell line stably expressing LC3-GFP and B) for long lived protein degradation.

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: UNMCMD_RNA_Aptamer_Validation_Screen_GRK2_In

Protocol: GRK2 protein is biotinylated using biotinamidohexanoic acid N-hydroxysuccinimide ester (Sigma). The RNA aptamer is fluorescently labeled on the 3'end with carboxyfluorescein (synthesized and labeled byIDT). Streptavidin-coated beads (Spherotech) are incubated with biotinylated GRK2 (bGRK2) at a final concentration of 2 nM for 30 minutes. The BioTek Microflow liquid dispenser is used to dispense 4 microL of assay buffer to all but column 1 of a 384-well assay plate. The positive (blocked) control containing 50X unlabeled RNA aptamer in assay buffer is dispensed to column 1 by a Microflow liquid dispenser (BiotTek, USA). Compounds (10 microM in-well concentration) are transferred to assay wells via 100 nanoL pintool transfer on the Biomek FX liquid dispenser (Beckman Coulter, USA). A total of 3 microL of bead suspension is dispensed into assay wells using the Nanoquot liquid dispenser (BioTek, USA). Plates are incubated at RT for 30 min. 3 microL FAM-C13.28 aptamer (final concentration 2 nanoM, supplied by the assay provider) is added to assay wells using the Microflow liquid dispenser. The reaction is incubated for one hour at RT. In this flow cytometry-based HTS [Kuckuck, et al. 2001] a CyAn flow cytometer (Dako / Beckman Coulter) interfaced with a HyperCyt (IntelliCyt, USA) auto-sampler is used to measure the median fluorescence intensity associated with bead-bound bGRK2.

Calculation:
All plates passed the Z' test (Z'>.30) a compound was considered active if the PERCENT_RESPONSE > 30. The Z' mean for all the plates was 0.80 with a standard deviation of 0.05.

The 30% cutoff corresponds to about three times the standard deviation of PERCENT_RESPONSE from 'non-fluorescent' test compounds. Negative PERCENT_RESPONSE is primarily due to test compounds with innate fluorescence.

PUBCHEM_ACTIVITY_SCORE = PERCENT_RESPONSE
PUBCHEM_ACTIVITY_OUTCOME = 2 (or ACTIVE) if PUBCHEM_ACTIVITY_SCORE > 30, otherwise the PUBCHEM_ACTIVITY_OUTCOME = 1 (or INACTIVE).

Comment: N/A

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: UNMCMD_Botulinum_LightChainF_Protease_HTS_ValidationSet

Protocol: This assay will be used to identify small molecule inhibitors of Botulinum toxin type A and F light chain proteases (BoNTALC and BoNTFLC) and inhibitors of Bacillus anthracis Lethal Factor protease (LF) in a multiplex format. Proteases that are not inhibited will cleave their substrates and produce a loss of signal effect. If the proteases are inhibited by a small molecule cleavage will not occur, thus the signal should stay the same. Full length protease substrates are used in order to detect inhibitors that act at either the protease catalytic site or at sites of protease interaction with substrate distal to the cleavage site.

Protease inhibition assays were performed as previously described (5), but with modifications. Biotinylated GFP protease substrates for LF, BoNTALC, BoNTFLC, and a protease-resistant substrate (pinpointGFP) were prepared and loaded on color-coded streptavidin microspheres (Spherotech Blue Array Particle kit, 5.1 microm diameter) as previously described (5-7). Additions to wells were in sequence as follows: 1st, 4 microL protease buffer (50 mM HEPES, 100 mM NaCl, 1 mg/ml bovine serum albumin, 0.025% Tween-20, pH 7.4); 2nd, 2 microL of a mixture of 1.5 microM LF, 5 nM BoNTALC and 375 nM BoNTFLC in protease buffer; 3rd, 100 nL of test compounds (1 mM in DMSO); and 4th, 4 microL containing 2x10;5/ml of each set of microspheres. Plates were sealed and incubated at 24 degreesC overnight (16-18 h), rotating continuously from inverted to upright position until analyzed in 1536 well plate format with the HyperCyt Cluster Cytometer platform the following day.

The assay response range was defined by replicate control wells containing protease buffer alone (PCntrl, positive control) or the protease mixture alone (NCntrl, negative control). Additional positive controls included Ebselen, VAMP peptide and IN-2-LF (selective inhibitors of BoNTALC, BoNTFLC and LF, respectively), which were added separately and together as a mixture to validate protease inhibition. In each well the median fluorescence intensity (MFI) was determined for each set of substrate-bearing microspheres. A fifth streptavidin microsphere set included in each well had no GFP moiety attached and was used to quantify the contribution of innate test compound fluorescence to the assay readout. All MFI values for substrate-bearing microspheres were corrected by subtraction of the MFI value for the substrate-free microspheres in the same well.

Test compound inhibition of substrate cleavage by protease was then calculated as 100 x (MFI:Test - Mean MFI:NCntrl)/(Mean MFI:PCntrl - Mean MFI:NCntrl) in which MFI:Test represents corrected MFI in the
presence of test compound, and Mean MFI:PCntrl and MFI:NCntrl represent means for replicate MFI determinations in control wells.

PubChem Score equals percent inhibition.

Comment: Development of the HyperCyt Cluster Cytometer Platform for processing of 1536 well plates by high throughput flow cytometry was supported by NIH/NIMH Grant 1R01HG005066 to Bruce Edwards.

External ID: UNMCMD_Botulinum_LightChainA_Protease_HTS_ValidationSet

Protocol:
This assay will be used to identify small molecule inhibitors of Botulinum toxin type A and F light chain proteases (BoNTALC and BoNTFLC) and inhibitors of Bacillus anthracis Lethal Factor protease (LF) in a multiplex format. Proteases that are not inhibited will cleave their substrates and produce a loss of signal effect. If the proteases are inhibited by a small molecule cleavage will not occur, thus the signal should stay the same. Full length protease substrates are used in order to detect inhibitors that act at either the protease catalytic site or at sites of protease interaction with substrate distal to the cleavage site.

Protease inhibition assays were performed as previously described (5), but with modifications. Biotinylated GFP protease substrates for LF, BoNTALC, BoNTFLC, and a protease-resistant substrate (pinpointGFP) were prepared and loaded on color-coded streptavidin microspheres (Spherotech Blue Array Particle kit, 5.1 microm diameter) as previously described (5-7). Additions to wells were in sequence as follows: 1st, 4 microL protease buffer (50 mM HEPES, 100 mM NaCl, 1 mg/ml bovine serum albumin, 0.025% Tween-20, pH 7.4); 2nd, 2 microL of a mixture of 1.5 microM LF, 5 nM BoNTALC and 375 nM BoNTFLC in protease buffer; 3rd, 100 nanoL of test compounds (1 mM in DMSO); and 4th, 4 microL containing 2x10;5/ml of each set of microspheres. Plates were sealed and incubated at 24 degreesC overnight (16-18 h), rotating continuously from inverted to upright position until analyzed in 1536 well plate format with the HyperCyt Cluster Cytometer platform the following day.

The assay response range was defined by replicate control wells containing protease buffer alone (PCntrl, positive control) or the protease mixture alone (NCntrl, negative control). Additional positive controls included Ebselen, VAMP peptide and IN-2-LF (selective inhibitors of BoNTALC, BoNTFLC and LF, respectively), which were added separately and together as a mixture to validate protease inhibition. In each well the median fluorescence intensity (MFI) was determined for each set of substrate-bearing microspheres. A fifth streptavidin microsphere set included in each well had no GFP moiety attached and was used to quantify the contribution of innate test compound fluorescence to the assay readout. All MFI values for substrate-bearing microspheres were corrected by subtraction of the MFI value for the substrate-free microspheres in the same well.

Test compound inhibition of substrate cleavage by protease was then calculated as 100 x (MFI:Test - Mean MFI:NCntrl)/(Mean MFI:PCntrl - Mean MFI:NCntrl) in which MFI:Test represents corrected MFI in the
presence of test compound, and Mean MFI:PCntrl and MFI:NCntrl represent means for replicate MFI determinations in control wells.

PubChem Score equals the percent inhibition.

Comment: Development of the HyperCyt Cluster Cytometer Platform for processing of 1536 well plates by high throughput flow cytometry was supported by NIH/NIMH Grant 1R01HG005066 to Bruce Edwards.

External ID: MH081226: Primary HTS for Inhibitors of a Novel Necrotic Cell Death Pathway # Jurkat FADD-/- Model.

Protocol: Necroptosis Assay Protocol TNF-alpha Induced Cell Death in Jurkat FADD-/- Cells.

Based on the assay development experiments performed by the PMLSC the following assay conditions were selected for the 384-well TNF-alpha Jurkat FADD-/- necroptosis screen:
1. 20,000 Jurkat FADD-/- cells/well were seeded in 40 uL of complete media in white opaque 384-well microtiter plates.
2. Plate controls, compounds (10 uM final in well) and 40 ng/mL TNF-alpha were added in a single addition and cells were incubated for an additional 24 hrs at 37#C and 5% CO2.
3. 25 uL of Cell Titer Glo reagent was added to each well and the resulting luminescence measurement of cellular ATP levels was read on the Envision after 15 minutes at ambient temperature.

Materials:
1. Complete Media: RPMI1640 with L-glutamine, Penicillin-Streptomycin, 10% Newborn calf serum
2. Jurkat FADD-/- Cells: 20,000 cells/well in 40 μL complete media
3. TNF-alpha: 50 ug/mL stock in DMSO; final concentration = 40 ng/mL
4. Cell Titer Glo: use at 25 μL/well

Protocol:
1. Jurkat FADD-/- cells are seeded at 20,000 cells/well in 40 uL of complete media into 384-well white opaque-bottom polypropylene plates (Greiner Bio-one Cat #781080).
2.Compounds (10 uM final concentration) are added to the respective wells in a total volume of 10 uL/well.
3. Maximum and minimum controls (DMSO at 0.18% final concentration and TNF-alpha at 40 ng/mL final concentration respectively) are added to assay plates at 20 uL/well
4. TNF-alpha (40 ng/mL final concentration) is added at 10 uL/well to respective wells with compounds.
5. Cell plates are incubated overnight at 37#C and 5% CO2.
6. 25 uL Cell Titer Glo is added to each well and the luminescence signal is read on the Envision after 15 minutes.

Plate Controls:
Max: 0.18% DMSO in complete media
Min: 40 ng/mL TNF-alpha0.08% DMSO) + 0.1% DMSO in complete media (final DMSO concentration = 0.18%)

Comment: The TNF-alpha induced Jurkat FADD-/- necroptosis Assay HTS run at the PMLSC utilized % activation calculated from maximum (n=32) and minimum (n=24) plate controls, with a hit criteria of >/= 50% activation to identify active compounds.

TNF-alpha Jurkat FADD-/- necroptosis Assay Activity scoring rules:

PUBCHEM_ACTIVITY_OUTCOME

1 - Substance is considered inactive when the % activation is < 50%
2 - Substance is considered active when % activation is >/= 50%
3 - Substance activity outcome is inconclusive

PUBCHEM_ACTIVITY_SCORE

0-40 scoring range is reserved for primary HTS data
a) if the % activation is >/= 50%, the score is 40.
b) if the % activation is < 50 %, the score is 0.

Definition of a Hit:
Rapid HTS screen ≥ 50% inhibition of death at 10 uM.
Confirmation of ≥ 50% inhibition of TNF-alpha induced Jurkat FADD-/- cell death in 2 independent tests.
Concentration Response AC50 < 20 uM in the Jurkat FADD-/- cell death model.
Structural Verification
Indications of Specificity & Selectivity ~ PubChem X-target Query
Evidence of SAR.

External ID: HMS1262

Protocol: NEC is stored at -80 degrees at a concentration of 15mg/ml in single use aliquots.

On the day of the screen, 20ul of purified NEC is aliquoted using a Multidrop Combi reagent dispenser into 384 well plates (Corning 3824). 100nl of compound dissolved in DMSO was transferred to each well of the assay plated via pin transfer. The plates (NEC + compound) are incubated at room temperature for 3 hours. Acceptor and donor reagents (CisBio 620/665 pair) are combined then added to each well at 5 microL volumes at a concentration of 8 nM and 80nM respectively. The plates are spun at 1k rpm for 1 min and incubated overnight at 4 degrees, then for one hour the subsequent day at room temperature.

Flourescent measurements are read on the Envision 1 plate reader at ICCB-L. The raw data consists of two fluorescence readings - at 665 nm and 620 nm for the acceptor and donor respectively.

Comment: Data analysis:
The raw data consists of two fluorescence readings - at 665 and 620 nm for the acceptor and donor respectively. The data is processed as a ratio of the emission from the acceptor over the donor (homogeneous time resolved fluorescence ratio). Normalized percent inhibition (NPI) for all experimental wells is calculated based on plate averages for negative and positive control HTRF ratio. Positives are scored as any ratio with a 50% or greater inhibition as compared with the positive control (i.e. NEC + Untagged UL50). To be considered a hit, both replicates need to score as positive. Activity scores are derived from NPI, with 100 = 100% inhibition (> 100% set to 100) and 0 = no inhibition (< 0% set to 0). Note that some compounds with NPI <50% (activity scores < 50) are classified as potential hits based on additional criteria (typically by selecting wells with low ratios compared to other experimental wells on the plate).

External ID: UNMCMD_Anthrax_LethalFactor_Protease_HTS_ValidationSet

Protocol: This assay will be used to identify small molecule inhibitors of Botulinum toxin type A and F light chain proteases (BoNTALC and BoNTFLC) and inhibitors of Bacillus anthracis Lethal Factor protease (LF) in a multiplex format. Proteases that are not inhibited will cleave their substrates and produce a loss of signal effect. If the proteases are inhibited by a small molecule cleavage will not occur, thus the signal should stay the same. Full length protease substrates are used in order to detect inhibitors that act at either the protease catalytic site or at sites of protease interaction with substrate distal to the cleavage site.

Protease inhibition assays were performed as previously described (5), but with modifications. Biotinylated GFP protease substrates for LF, BoNTALC, BoNTFLC, and a protease-resistant substrate (pinpointGFP) were prepared and loaded on color-coded streptavidin microspheres (Spherotech Blue Array Particle kit, 5.1 microm diameter) as previously described (5-7). Additions to wells were in sequence as follows: 1st, 4 microL protease buffer (50 mM HEPES, 100 mM NaCl, 1 mg/ml bovine serum albumin, 0.025% Tween-20, pH 7.4); 2nd, 2 microL of a mixture of 1.5 microM LF, 5 nM BoNTALC and 375 nM BoNTFLC in protease buffer; 3rd, 100 nL of test compounds (1 mM in DMSO); and 4th, 4 microL containing 2x10;5/ml of each set of microspheres. Plates were sealed and incubated at 24 degreesC overnight (16-18 h), rotating continuously from inverted to upright position until analyzed in 1536 well plate format with the HyperCyt Cluster Cytometer platform the following day.

The assay response range was defined by replicate control wells containing protease buffer alone (PCntrl, positive control) or the protease mixture alone (NCntrl, negative control). Additional positive controls included Ebselen, VAMP peptide and IN-2-LF (selective inhibitors of BoNTALC, BoNTFLC and LF, respectively), which were added separately and together as a mixture to validate protease inhibition. In each well the median fluorescence intensity (MFI) was determined for each set of substrate-bearing microspheres. A fifth streptavidin microsphere set included in each well had no GFP moiety attached and was used to quantify the contribution of innate test compound fluorescence to the assay readout. All MFI values for substrate-bearing microspheres were corrected by subtraction of the MFI value for the substrate-free microspheres in the same well.

Test compound inhibition of substrate cleavage by protease was then calculated as 100 x (MFI:Test - Mean MFI:NCntrl)/(Mean MFI:PCntrl - Mean MFI:NCntrl) in which MFI:Test represents corrected MFI in the
presence of test compound, and Mean MFI:PCntrl and MFI:NCntrl represent means for replicate MFI determinations in control wells.

Comment: Development of the HyperCyt Cluster Cytometer Platform for processing of 1536 well plates by high throughput flow cytometry was supported by NIH/NIMH Grant 1R01HG005066 to Bruce Edwards.

External ID: PAD4_INH_FP_384_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds from the NIH Validation Collection that act as inhibitors of PAD4. In this assay, PAD4 is labeled in an active site-directed manner by the fluorescent ABPP probe rhodamine-conjugated F-amidine (RFA) in the presence of test compounds. The reaction is excited with linear polarized light and the intensity of the emitted light is measured as the polarization value (mP). As designed, test compounds that act as PAD4 inhibitors will prevent PAD4-probe interactions, thereby increasing the proportion of free (unbound) fluorescent probe in the well, leading to low fluorescence polarization in the well. Compounds were tested in singlicate at a final nominal concentration of 5 uM.

Protocol Summary:

Prior to the start of the assay, 10.0 ul of Assay Buffer (50 mM HEPES pH 7.6, 150 mM NaCl, 10 mM CaCl2, 0.01% Pluronic F-127, and 1 mM TCEP) containing 2 uM of PAD4 protein was dispensed into 384 microtiter plates. Next, 50 nL of test compound in DMSO or DMSO alone (0.5% final concentration) was added to the appropriate wells and incubated for 30 minutes at 25 C.

The assay was started by dispensing 1.1 ul of 750 nM RFA probe in Assay Buffer to all wells. Plates were centrifuged and after 5 hours of incubation at 37 C, fluorescence polarization was read on an Envision 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 30 seconds for each polarization plane (parallel and perpendicular). The well fluorescence polarization value (mP) was obtained via the PerkinElmer Viewlux software.

The percent inhibition for each compound was calculated as follows:

Percent inhibition = ( Test_Compound_mP - median_Negative_Control_mP ) / ( median_Positive_Control_mP - median_Negative_Control_mP )* 100

Where:

Negative_Control is defined as wells containing PAD4 and DMSO,
Test_Compound is defined as wells containing PAD4 in the presence of test compound,
Positive_Control is defined as wells containing no PAD4 protein.

Compounds with greater than 30% inhibition were 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 to 37, and 27 to 0 for inactive compounds .

List of Reagents:

Recombinant PAD4 (supplied by Assay Provider)
RFA probe (supplied by Assay Provider)
Tris HCl (Sigma, part T3038)
HEPES (Fisher, BP-410)
CaCl2 (Sigma, C3881)
TCEP (Pierce, part 20490)
NaCl (Sigma, part S6546)
Pluronic acid (Invitrogen, part P6866)
384-well plates (Greiner, part 781076)

Comment: 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: 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: 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: HMS750

Protocol: The stalled elongation complex used as a substrate in the screening assay was generated by pre-incubating 600 nM 3Dpol with 12 nM 5' fluorescein labeled 8-6 PETE RNA and 240 nM ATP for 30 minutes at room temperature in 50 mM HEPES, pH 7.0, 40 mM NaCl, 1.5 mM magnesium acetate, 60 microM ZnCl2, 4 mM DTT, and 0.1% Igepal detergent. This solution was dispensed into 384-well microplates (Corning 3710) at a volume of 25 microL/well. Experimental compounds were added by robotic pin transfer at 100 nL/well.

After an incubation time of ~60 min, the total fluorescence and fluorescence polarization signals from the labeled RNA were measured with Perkin Elmer EnVision microplate readers. This first reading provided data indicating whether compounds interfered with RNA binding to the stalled elongation complex. Polymerase elongation activity was then tested by adding 5 microL of a solution containing GTP, CTP, and UTP at 1.2 microM each, resulting in a final concentration of 200 nM for each of the four NTPs and 16.7 microg/ml for the compounds. This allowed for elongation to the end of the RNA template, and assay data were obtained with a second reading done after an incubation period of ~60 min, which is four times longer than the ~15 min needed for elongation under non-inhibited conditions.

Comment: Potential inhibitors were identified using a correlation plot combining standard Z-scores with an elongation efficiency measurement. A Z-score for each compound was calculated by the normal method of Z=(FPcompound-FPplate_mean)/StdDevplate_mean. An elongation efficiency value (FP %Elongation) was then calculated as E=(FPcompound-FPpositive)/(FPnegative-FPpositive), which reflects how the FP signal obtained in the presence of a compound compared to those of the unelongated positive controls (FPpositive) and fully elongated negative controls (FPnegative). Compounds that gave a FP signal greater than the starting material but less than the fully elongated controls thus had E values between 0% and 100%, while compounds that caused the RNA to dissociate from 3Dpol had negative E values due to the FP value associated with free RNA being lower than the unelongated FPpositive value. Finally, the elongation reaction also results in an increase in the total fluorescence intensity due to deprotonation of the fluorescein as it approaches the positively charged polymerase surface, providing an additional assessment of elongation. Compounds that resulted in total fluorescence %Elongation higher than that of the fully elongated control samples were rejected from the analysis. Positives were defined as having experimental FP Z-scores < -0.5 and FP %Elongation < 90%. Activity scores were calculated based on FP %Elongation. FP %Elongation <= 0 was scored as 100 for activity; FP %Elongation >= 100 was scored as 0 for activity. FP %Elongation values between 0 and 100 were subtracted from 100 to generate activity scores.

External ID: ES_Opera

Protocol: To perform high content microscopy screening, HeLa cells with stable expression of F508del-CFTR were cultured in 96-well PE ViewPlates (Perkin Elmer, Waltham, MA) for one day prior to well by well addition of two thousand known bioactive compounds from the NIH Roadmap Molecular Libraries Small Molecule Repository. Ten mM DMSO stocks of compounds were diluted 500-fold in growth medium, then added in equal volume to assay plates to yield a final screening concentration of 10 M.
After a 24h treatment period, cells were fixed, permeabilized, and labeled with 3G11 antibody, which recognizes intracellular CFTR epitopes in NBD1, then visualized with AlexaFluor488 anti-mouse conjugate and DRAQ5 fluorescent DNA label (Axxora, Farmingdale, NY). Nine microscopic fields per sample well were then imaged using the Evotec Operatrade mark QEHS automated confocal microscope equipped with a 20x magnification Olympus objective lens (Perkin Elmer). Automated analysis of the resulting images was performed using Acapellatrade mark (Perkin Elmer) software as follows. High contrast labeling of cellular nuclei by DRAQ5 enabled rapid image segmentation by intensity threshold. Corresponding cytoplasm and cell margins associated with each nucleus were subsequently defined by image threshold of lower contrast cytoplasmic / RNA staining of DRAQ5. Further segmentation of images into regions of interest for analysis included plasma membrane (a 2 pixel wide region corresponding to cell margin) and perinuclear region (a 5 pixel wide region encircling the outside of the nuclear mask). Three criteria were used to track expression and subcellular localization of: 1) F508del-CFTR signal in the region of interest, designated as the plasma membrane (PM); 2) F508del-CFTR signal in the perinuclear region of the cell, as a proxy for the ER; and 3) fluorescence signal across the entire cell (Total) to monitor total F508del-CFTR protein. Each measurement was expressed as the mean fluorescent intensity for all cells imaged in 9 microscopic fields per sample.

Comment:

External ID: JHICC_RGS_Inh_HTS

Protocol: Assay overview:

To screen for compounds that inhibit 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 show increase in the Fluo4 fluorescence in induced RGS4 expressed cells are considered agonist hits. M3 receptor and other endogenous receptor inhibitors 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, 15 ug/ml Blasticidin, 400 ug/ml G418, 200 ug/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 37 degrees C and 5% CO2.
4. Remove medium and add 20 ul /well of 2 uM Fluo4-AM solution to cells.
5. Incubate 30 minutes at 37 degrees C 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. Add 4 ul of 7x EC20 (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 prime 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 more than 3 times the standard deviation (SD) of the B scores of integrated ratios of all library compounds above the mean (B score ratio>3*SD+mean), AND the ratio of initial fluorescence intensity is within 5 times the standard deviation plus or minus the mean of the ratios of the library compounds, the compound is designated in the Outcome as active (value=2) as an inhibitor of RGS4. Otherwise, it is designated as inactive (value=1).
16. Score assignment: An active test compound is assigned a score between 5 and 100 by calculation of Int(((Log(ABS(B score ratio))-0.6)/1.25)*100), 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 Catalog number 10010049)
3. Medium: DMEM (Sigma, Catalog number D5796)
4. Fetal Bovine Serum (Gemini, Catalog number 100-106)
5. Hygromycin (Mediatech, Catalog number 30-240-CR)
6. 100x Penicillin-Streptomycin (Mediatech, Catalog number 30-001-CI)
7. Cell/stripper (Mediatech, Catalog number 25-056-Cl)
8. G418: (Invitrogen, Catalog number 11811-031)
9. Blasticidin (Sigma, Catalog number R21001)
10. Doxycycline hyclate (Sigma, Catalog number D9891)
11. HEPES (Sigma, Catalog number H4034)
12. Fluo-4 (Invitrogen, Catalog number F14202)
13. Pluronic F-127*20% in DMSO (Invitrogen, Catalog number P-3000MP)
14. Atropine (Sigma, Catalog number A0132)
15. Carbachol (Sigma, Catalog number C4382)
16. Triple-layer flask (VWR, Catalog number 62407-082)
17. BD Biocoat 384-well plates (BD, Catalog number (35)4663 and Lot number 7346273)

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: FLUC100

Protocol: NCGC Assay Protocol Summary:

Reagents: 50mM Tris acetate, pH 7.5; 10mM Mg acetate; 10uM D-luciferin (Sigma #L9504); 10uM ATP; 0.01% Tween-20; 0.05% BSA; 10nM P. pyralis luciferase (Sigma #L9506)
Control compounds used were two known firefly luciferase inhibitors (compounds (2) and (5) in Auld et al., 2010), and DMSO.

Assay Summary:
Three microliters containing firefly luciferase substrates in buffer (final concentrations: 50mM Tris acetate, pH 7.5, 10mM Mg acetate, 0.01% Tween-20, 0.05% BSA, 10uM D-luciferin, and 10uM ATP) are dispensed into each well of a Greiner white, solid-bottom 1536-well format plate using a flying reagent dispenser (FRD). These assay plates were then treated with 23nL of compound or DMSO using a Kalypsys pin tool, which allows for delivery of a 6-point interplate titration of each compound to the assay plate (quantitative HTS), with a final compound concentrations ranging from approximately 60muM to 7pM. One microliter of firefly luciferase in 500mM Tris-acetate buffer was then delivered by FRD to each well for a final enzyme concentration of 10nM. Luciferase activity was then measured using a ViewLux CCD imager (PerkinElmer), with an average exposure time of 2-30 seconds (2X binning, medium/high gain).

Keywords: NIH Roadmap, MLPCN, MLSMR, qHTS, miR-21, firefly luciferase, FLuc, miRNA.

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.