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: ADAM17_INH_QFRET_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that inhibit ADAM17. This assay employs a fluorophore and quencher pair. F =EDANS fluorophore, Q = DABCYL quencher. When intact, EDANS emission at 460nm is quenched by DABCYL via fluorescence resonance energy transfer. Upon cleavage of the scissile bond (A~V) by ADAM protease, the distance between fluorophore and quencher increases resulting in fluorescence increase at 460nm. Compounds are tested in singlicate at a final nominal concentration of 6.95 micromolar.

Protocol Summary:

Prior to the start of the assay, 2.5 microliters 2X ADAM17 enzyme (20 nM in Assay Buffer: 50 mM HEPES, 0.01% Brij, pH 7.5) are dispensed into 1536 microtiter plates. Compounds are added to plate (final concentration TBD) and incubated for 30 minutes at 25 degrees Celsius. The assay is started by dispensing 2.5 microliter of2X DM2 substrate (20 uM in Assay Buffer) to all wells. Plates are centrifuged and after 3 hours of incubation at 25 degrees Celsius, fluorescence is measured (excitation = 359nm, emission = 460nm).

The % inhibition for each well was then calculated as follows:

%_Inhibition = ( RFU_Test_Compound - MedianRFU_Low_Control ) / ( MedianRFU_High_Control - MedianRFU_Low_Control ) * 100

Where:

Test_Compound is defined as wells containing test compound.
High_Control is defined as wells treated with 1 micromolar Marimastat
Low_Control is defined as wells containing DMSO.


Interval Cutoff:
A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Four values were calculated: (1) the average percent inhibition of all high controls tested plus three times the standard deviation of the high controls, (2) the average percent inhibition of all low controls tested minus three times the standard deviation of the low controls, (3) the average percent inhibition of all compounds tested between (1) and (2), and (4) three times their standard deviation. The sum of two of these values, (3) and (4), was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition/activity 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 activity score range for active compounds is 100-8, for inactive 8-0.

List of Reagents:

ADAM17 enzyme (R&D Systems, part # 930-ADB)
EDANS-DABCYL DM2 peptide substrate (Supplied by Assay Provider)
0.5M HEPES solution, pH7.5 (Teknova, part #101319-900)
Brij-35 (Sigma-Aldrich, part # P1254)
1536 well plate (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. 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, 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: USP8 FAST DUB HTS Primary

Protocol: Deubiquitinases were purified from E. Coli. Ub-Rho substrate was purchased from Boston BioChem (Cat #: U-555). Assay buffer consisted of 50 mM Tris pH 8, 50 mM NaCl, 0.002% Tween20, 5 mM DTT in MilliQ water. Stop buffer consisted of assay buffer + 0.2% trifluoroacetic acid.

Protocol for Inhibitors of USP8: Primary Screen
1.#Compound, 20 nL of screening compounds (final concentration of 20 uM) to columns 1-46 of a Greiner 1536 well Black, flat bottom, polystyrene microplate (Griener 782076) (columns 45-46: DMSO control)
2.#Reagent, 2.5 uL of assay buffer to columns 47-48 on all plates (No enzyme control)
3.#Reagent, 2.5 uL of USP8 in assay buffer (final concentration of 1 nM) to columns 1-46
4.#Reagent, 2.5 uL of Ub-Rho in assay buffer (final concentration of 50 nM) to all wells
5.#Time, Incubate at room temperature for 30 minutes
6.#Detection, Fluorescence, PheraStar, FITC Module

Comment: Normalization of raw data to positive and negative control wells were applied to the data using Helios, a high-throughput screening data analysis program developed at Novartis.(Gubler et al., 2018)

Compounds demonstrating greater than or equal to 30% inhibition of USP8 (USP8_EFFICACY >/= 30) were called as active, compounds demonstrating activity below this threshold were called as inactive.

External ID: USP17 FAST DUB HTS Primary

Protocol: Deubiquitinases were purified from E. Coli. Ub-Rho substrate was purchased from Boston BioChem (Cat #: U-555). Assay buffer consisted of 50 mM Tris pH 8, 50 mM NaCl, 0.002% Tween20, 5 mM DTT in MilliQ water. Stop buffer consisted of assay buffer + 0.2% trifluoroacetic acid.

Protocol for Inhibitors of USP17: Primary Screen
1. Compound, 50 nL of screening compounds (final concentration of 50 uM) to columns 1-46 of a Greiner 1536 well Black, flat bottom, polystyrene microplate (Griener 782076) (columns 45-46: DMSO control)
2. Reagent, 2.5 uL of assay buffer to columns 47-48 on all plates (No enzyme control)
3. Reagent, 2.5 uL of USP17 in assay buffer (final concentration of 1 nM) to columns 1-46
4. Reagent, 2.5 uL of Ub-Rho in assay buffer (final concentration of 40 nM) to all wells
5. Time, Incubate at room temperature for 3 hours
6. Reagent, 2.5 uL of stop buffer to all wells
7. Centrifuge plates (5s, 350 rpm)
8. Detection, Fluorescence, PheraStar, FITC Module

Comment: Normalization of raw data to positive and negative control wells were applied to the data using Helios, a high-throughput screening data analysis program developed at Novartis.(Gubler et al., 2018)

Compounds demonstrating greater than or equal to 30% inhibition of USP17 (USP17_EFFICACY >/= 30) were called as active, compounds demonstrating activity below this threshold were called as inactive.

External ID: USP7 FAST DUB HTS Primary

Protocol: Deubiquitinases were purified from E. Coli. Ub-Rho substrate was purchased from Boston BioChem (Cat #: U-555). Assay buffer consisted of 50 mM Tris pH 8, 50 mM NaCl, 0.002% Tween20, 5 mM DTT in MilliQ water. Stop buffer consisted of assay buffer + 0.2% trifluoroacetic acid.

Protocol for Inhibitors of USP7: Primary Screen
1. Reagent, 2.5 uL of USP7 in assay buffer (final concentration of 4 nM) to columns 1-46 of a Greiner 1536 well Black, flat bottom, polystyrene microplate (Griener 782076)
2. Compound, 20 nL of screening compounds (final concentration of 20 uM) to columns 1-46 (columns 45-46: DMSO control)
3. Reagent, 2.5 uL of assay buffer to columns 47-48 on all plates (No enzyme control)
4. Time, Incubate at room temperature for 60 minutes
5. Reagent, 2.5 uL of Ub-Rho in assay buffer (final concentration of 40 nM) to all wells
6. Time, Incubate at room temperature for 10 minutes
7. Detection, Fluorescence, PheraStar, FITC Module

Comment: Normalization of raw data to positive and negative control wells were applied to the data using Helios, a high-throughput screening data analysis program developed at Novartis.(Gubler et al., 2018)

Compounds demonstrating greater than or equal to 30% inhibition of USP7 (USP7_EFFICACY >/= 30) were called as active, compounds demonstrating activity below this threshold were called as inactive.

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

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

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

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

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

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

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

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

External ID: JHICC_MrgX1_AlloAgonist_Primary

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

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

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

External ID: JHICC_MrgX1_Antagonist_Primary

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

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

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

External ID: 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: Bursicon-induced LGR2 mediated cAMP production in LGR-2/CRE6x-Luciferase co-transfected HEK293 cells Inhibition - 7011-01_Antagonist_SinglePoint_HTS_Activity

Protocol: Protocol

Day 0:
Cell Culture
HEK 293 cells are plated in DMEM culture media (containing 10% Fetal Bovine Serum (FBS) and 1X penn/strep/glutamine) at a density of 5x106 cells/T175 flask and grown for 3 days at 37 degrees C in 5% CO2 incubator.

Day 3:
Transient Transfection
Cells are transiently transfected in DMEM transfection media (containing 1X penn/strep/glutamine without serum) using polyethyleneimine (PEI).
1)Mix 2.9ug/flask of cDNA encoding the wild-type Bursicon Receptor with 14.6ng/flask of cDNA encoding a CRE-luciferase reporter gene with a PEST/HCL sequence in 2mL of transfection media
2)Add 61uL/flask of PEI of 1mg/mL in 1.5mL of transfection media
3)Mix the 2mL cDNA solution with 1.5mL of PEI solution and incubate at RT for 20 mins
4)Aspirated the culture media from the cells in T175 flask
5)Add 25 mL of transfection media and 3.5mL of transfection mixture to T175 falsk.
6)Mix the media with transfection mixture well and transfect for 2 days at 37 degrees C in 5% CO2 incubator

Day 5:
Cell Plating
1)Typsinize the Cells with 5mL of 0.05% Trypsin and incubate at 37oC for 5 min.
2)Add 5mL of DMEM assay media (containing DMEM without phenol red, 10% NuSerum and 1x Pen/Strep/Glutamine) to inactive trypsin
3)Collect cells by centrifugation at 1000rpm for 5 min
4)Suspend the cells in DMEM assay media to a cell density of 4x105 cells/mL
5)Plate the cells to 1536-well Aurora Mako plate with 2000 cells/well/5uL using ViaFill
6)Incubate the cells at 37 degrees C in 5% CO2 incubator on GS system for overnight

Day 6:
Compound Treatment, Agonist Stimulation and Detection
Assay Setup
1)Thaw aliquoted Bursicon conditioned media and diluted with DMEM assay media at a 1/20 dilution. Filter the solution through a 0.22uM filter
2)Add DMEM condition media, the diluted Bursicon conditioned media and SteadyGlo to the bottles and setup BioRAPTR

Run automation protocol on GS system
1)Take the assay plate with transfected cells from the incubator
2)Transfer 5nL/well of 10mM compound to assay plate
3)Incubate 30 mins in incubator
4)Add 1uL/well of DMEM assay media to PosCon wells and diluted Bursicon solution to the other wells using BioRAPTR (Beckman) based on the plate map
5)Incubate the assay plate for 4 h in incubator
6)Take the plate out from the incubator and equilibrate the assay plate at RT for 10 mins
7)Add 1uL/well of SteadyGlo to assay plate using BioRAPTR, incubate at RT for 10 mins
8)Read the plate on ViewLux (PerkinElmer) for Luminescence.

Culture Media
DMEM high glucose, no glutamine (Invitrogen, 11960)
Pen/Strep/Glutamine (Invitrogen 10378-016)
Fetal Bovine Serum (Atlanta Biological, S10350)

Transfection Media
DMEM high glucose, no glutamine (Invitrogen, 11960)
Pen/Strep/Glutamine (Invitrogen 10378-016)

Assay Media
DMEM no phenol red (Invitrogen, 21063-029)
Pen/Strep/Glutamine (Invitrogen 10378-016)
NuSerum (BD biosciences, 24883)

0.05% Trypsin-EDTA (Invitrogen, 25300-062)
Aurora 1536-well Mako plate (Aurora/Brooks, 00028778)

Comment:

External ID: HMS1149-MLP

Protocol: A 558 bp region upstream of hspX was PCR amplified and cloned upstream of the GFP-mut2 (Cormack et al., 1996). This construct was cloned into a modified version of the replicating plasmid pSE100 and transformed into CDC1551 to generate CDC1551(hspX'::GFP).

The M. tuberculosis CDC 1551 (hspX'::GFP) fluorescent biosensor was grown in pH 7.0 Middlebrook 7H9 medium to mid- to late-log phase. Black-walled, clear-bottom, tissue-culture treated 384-well microtiter plates (Corning #3712) were filled with 25 uL of buffered pH 7.0 7H9 medium using a Matrix Combi. 100 nL of compound was then transferred to each well via stainless steel pin array. Plates were manually sealed with DMSO-resistant aluminum seals and frozen at -80C until they were ready to be screened. The cultures were re-suspended in pH 7.0 7H9 and 25 uL was dispensed into each well of the 384-well assay plates already containing the perturbator utilizing a Cy-Bio Selma liquid handler robot to an OD595 of 0.05. The plates were then placed in a Ziploc bag with a moistened paper towel (to limit evaporation) and incubated for 6 days at 37C. Fluorescence and optical density (OD) readings were made on an EnSpire plate reader (Perkin Elmer, Inc.) at excitation and emission wavelengths of 488 and 509 nm as a top read, with the OD being taken at 595 nm as a bottom read.

The ~328,633 compound library was arrayed into black-walled, clear-bottom tissue-culture treated 384-well microtiter plates (Corning #3712) by pin transfer (100 nL), containing 25 uL of buffered pH 7.0 7H9 medium. In a previous screen utilizing the same compound library and screening method, four random compound plates were screened in duplicate to observe variation in hit robustness across plates and method. The result was that the duplicate plates did not vary in a statistically significant manner and due to the high volume of compounds to be screened (~328,633), replicates were not utilized.

Negative control: DMSO, 0.1% final concentration in buffered pH 7.0 7H9 in column 2 or column 23 of each plate

Positive control: 0.3 uM Rifampicin final concentration in buffered pH 7.0 7H9 in column 1 or column 24 of each plate

Comment: Analysis methods:
To calculate normalized fluorescence percent inhibition, plate average negative control fluorescence intensity (based on all plates within a screening run) was subtracted from well fluorescence intensity, divided by the difference between plate negative and positive control average fluorescence intensity (based on all plates within a screening run), and multiplied by 100. To calculate normalized growth inhibition, plate average negative control absorbance (based on all plates within a screening run) was subtracted from well absorbance, divided by the difference between plate negative and positive control average absorbance (based on all plates within a screening run), and multiplied by 100. Normalized fluorescence percent inhibition was divided by normalized growth inhibition to calculate a ratio. Wells were considered active if growth was not greatly decreased and showed either a 3-fold selectivity fluorescence to growth inhibition ratio (with 35-45% fluorescence inhibition) or a 1.5-fold selectivity fluorescence to growth inhibition ratio (with > 45% fluorescence inhibition). These compounds may be directly or indirectly inhibiting DosRS signaling. Wells were considered general inhibitors of M. tuberculosis growth if both fluorescence and growth inhibition > 50%. Activity scores were scaled from 100 to 0 and derived from normalized fluorescence percent inhibition, with 100 (or > 100) = 100% inhibition and 0 (or < 0) = no inhibition.

External ID: 7124-01_Inhibitor_SinglePoint_HTS_Activity

Protocol:
Protocol:
1) Plate 10 uL BL2-NFkB-luc cells at 12.5K cells/well, using a Multidrop Combi reagent dispenser (1,250 cells/uL).
2) Add 25 nL compound or positive control, by pin transfer; IKK inhibitor VII will be used at 50 uM.
3) Incubate at 37 masculineC for 1 hour (allowing compounds to enter cells for action).
4) Add 5 uL 192 ng/mL tCD40L per well for a final concentration of 64 ng/ml using a Thermo Multidrop Combi.
5) Incubate at 37 masculineC for 4 hrs, then leave plate at RT for 30 minutes.
6) Add 15 uL SteadyGlo luciferase substrate (Promega) per well using a Thermo Multidrop Combi.
7) Incubate at RT for 5min, then read Luminescence using LJL Analyst HT plate 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 decreasing readout signal.

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v10.0.2):
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.10.0.2) 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 -50.

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 55.

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 1 (inactive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T

Samples passing PAR_T only were assigned an outcome of 1 (inactive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T

External ID: ABHD4_INH_FP_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this biochemical assay is to identify compounds that act as inhibitors of alpha/beta hydrolase domain containing 4 (ABHD4). In this assay, ABHD4 protein is incubated with test compounds and fluorophosphonate-rhodamine (FP-Rh) activity-based probe. 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 ABHD4 inhibitors will prevent ABHD4-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 nominal test concentration of 8.92 micromolar.

Protocol Summary:

Prior to the start of the assay, 4 microliters of assay buffer (50 mM HEPES pH 7.0, 150 mM NaCl and 0.01% Pluronic F-127) were dispensed into column 2 only. Next, 4 microliters of assay buffer containing 46.9 ug/mL of ABHD4 mutant protein (S159A) were dispensed into columns 1 and 3 of 1536 microtiter plates and 4 microliters of assay buffer containing 46.9 ug/mL of ABHD4 wild type protein were dispensed into columns 4 thru 48. Then, 45 nL of test compound in DMSO or DMSO alone (0.89% final concentration) were added to the appropriate wells and incubated for 45 minutes at 25 degrees Celsius.

The assay was started by dispensing 1.0 microliter of 188 nM FP-Rh in assay buffer to all wells. Plates were centrifuged and after 60 minutes of incubation at 25 degrees Celsius, 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 = 525nm, emission = 598nm) for 30 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:

Test_Compound is defined as wells containing ABHD4 wild type protein in the presence of test compound and FP-Rh.
High_Control is defined as wells containing ABHD4 mutant protein, DMSO and FP-Rh.
Low_Control is defined as the median of the wells containing test compounds.

PubChem Activity Outcome and Score:


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.

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

The activity score range for active compounds is 100-6, for inactive 6-0.

List of Reagents:

mABHD4 wild type protein (supplied by Assay Provider)
mABHD4 (S159A) mutant protein (supplied by Assay Provider).
FP-Rh probe (supplied by Assay Provider)
HEPES (Sigma, part 83264)
NaCl (Sigma, part S6546)
Pluronic F-127 (Invitrogen, part P6866)
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, 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: SIAE_INH_FP_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this biochemical assay is to identify compounds that act as inhibitors of sialic acid acetylesterase (SIAE). In this assay, SIAE protein is incubated with test compounds and fluorophosphonate-rhodamine (FP-Rh) activity-based probe. 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 SIAE inhibitors will prevent SIAE-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 nominal test concentration of 9.66 micromolar.

Protocol Summary:

Prior to the start of the assay, 3 microliters of assay buffer (1X DPBS and 0.01% Pluronic F-127) were dispensed into column 1 thru column 3 of 1536 microtiter plates. Next, 3 microliters of assay buffer containing 0.73uM of SIAE protein were dispensed into columns 4 thru 48. Then, 39 nL of test compound in DMSO or DMSO alone (0.97% final concentration) were added to the appropriate wells and incubated for 45 minutes at 25 degrees Celsius.

The assay was started by dispensing 1.0 microliter of 300 nM FP-Rh in assay buffer to all wells. Plates were centrifuged and after 120 minutes of incubation at 25 degrees Celsius, 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 = 525nm, emission = 598nm) for 25 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:

Test_Compound is defined as wells containing SIAE protein in the presence of test compound and FP-Rh.
High_Control is defined as wells containing DMSO, FP-Rh but, no SIAE protein.
Low_Control is defined as the median of the wells containing test compounds.

PubChem Activity Outcome and Score:

A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Four values were calculated: (1) the average percent inhibition of all high controls tested plus three times the standard deviation of the high controls, (2) the average percent inhibition of all low controls tested minus three times the standard deviation of the low controls, (3) the average percent inhibition of all compounds tested between (1) and (2), and (4) three times their standard deviation. The sum of two of these values, (3) and (4), was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition/activity than the cutoff parameter was declared active.

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

The activity score range for active compounds is 100-2, for inactive 2-0.

List of Reagents:

SIAE protein (supplied by Assay Provider)
FP-Rh probe (supplied by Assay Provider)
DPBS (Mediatech, part 20-031-CV)
Pluronic F-127 (Invitrogen, part P6866)
1536-well plates (Greiner, part 789176)

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, 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: HMS1126

Protocol: 384-well assay plates (Corning 3820) were filled with Mix 1 at 10 microL per well. Mix 1 contained 133.3 microM potassium phosphate pH 7.5, 133.3 microM manganese sulfate, 5% Cayman enzyme mix, and 2 microM BmaI1. 300 nL of each experimental compound were pin-transferred to assay wells. 3.33 microL of 40 mM acetovanillone in DMSO was added to positive control wells before the reaction was started.

The primary assay was initiated with 3.33 microL Mix 2. Mix 2 contained 100 microM octanoyl-ACP, 800 microM 10-acetyl-3,7-dihydroxyphenoxazine (ADHP), and 140 microM S-adenosylmethionine (SAM). The secondary assay was initiated by addition of 3.33 microL of Mix 4. Mix 4 contained 133.3 microM potassium phosphate pH 7.5, 133.3 microM manganese sulfate, and 4% Cayman enzyme mix. The assay was allowed to proceed for 40 to 60 minutes, at which time it was stopped by the addition of 3.33 microL of 40 mM acetovanillone to all wells except the positive control wells.

Plates were read on a PerkinElmer EnVision (525/25 excitation filter, 590/20 emission filter, 555 mirror. Gain 165, excitation light 5%, 10 flashes).

Library plates were screened using a primary assay and a counterscreen assay, with all assay plates for a given library plate prepared on the same day. For every compound library plate, there were four daughter plates. Primary plates A & B were prepared as described; counterscreen plates CNTL-A and CNTL-B contained the same components except for 5'-Deoxy-5'-(methylthio)adenosine instead of BmaI1, acyl-ACP, and SAM. Counterscreen plates also contained 3% enzyme mixture instead of 3.5%.

Positive control: All wells of column 24 contained the final enzymatic reaction mix, with acetovanillone added before the assay was started, and no experimental compound.
Negative control: All wells in column 23 contained only the final enzymatic reaction mix (no experimental compound).

Comment: For each non-excluded experimental well, a robust Z-score was calculated based on the fluorescence intensity median and normalized MAD for each replicate in the primary assay and the counterscreen assay. The absolute value of each experimental screen well Z-score was subtracted from the absolute value of plate average positive control Z-score. Wells with a robust Z-score < 2 units from the plate average positive control Z-score for both primary screen replicates, and a robust Z-score < 3 for at least one counterscreen replicate, were considered positive.

Activity scores were calculated for both the primary screen and the counterscreen using normalized percent of control values. For each well (both replicates), the positive control plate average fluorescence intensity was subtracted from the well fluorescence intensity, divided by the difference between plate average negative and positive control fluorescence intensity, subtracted from 1, and multiplied by 100. Some plates lacked a positive control; the positive control average of all plates was used for these plates. Replicate normalized percent of control values were averaged for the primary screen and counterscreen separately to determine activity scores for each. Values less than 0 were set to 0 and values greater than 100 were set to 100.

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: UHRF1-CPGDNA_INH_TRFRET_1536_1X%INH CSRUN for MBD2-CPGDNA INH

Protocol: Assay Overview:

The purpose of this assay is to determine whether compounds identified as inhibitors of MBD2 are nonselective, as determined by inhibition of methylated DNA-binding activity of the SRA domain polypeptide of ubiquitin-like with PHD and ring finger domains 1 (UHRF1). The UHRF1 gene encodes a member of a subfamily of RING-finger type E3 ubiquitin ligases. It contains a Set-and RING Associated (SRA) domain that binds to partially methylated DNA sequences during DNA synthesis. UHRF1 exhibits a different mode of DNA binding than MBD2, binding so-called hemi-methylated cytosines in order to promote their fully methylation. This assay is performed in a similar manner as the MBD2 screen, except that it employs recombinant UHRF1 SRA domain protein (UHRF1_SRA) and hemi-methylated DNA as its substrate. This assay monitors the ability of compounds to inhibit binding of cloned recombinant UHRF1_SRA to a FAM-labeled 14 bp hairpin oligonucleotide containing a hemimethylated CpG. In this biochemical assay, test compounds are incubated with UHRF1_SRA and a 5-carboxyfluorescene (FAM)-labeled 14bp hairpin DNA oligonucleotide containing one hemimethylated CpG dinucleotide in the presence of Tb-labeled anti-His antibody. An inhibitor of the UHRF1_SRA:FAM hemimethylated CpG interaction will inhibit binding between UHRF1 and FAM hemimethylated oligo, leading to reduced energy transfer and reduced well FRET (fluorescein emission / Tb emission). Compounds are tested in singlicate at a final nominal concentration of 4.4 micromolar.

Protocol Summary:

The assay was started by dispensing 5 microliters of Control Mix in assay buffer (4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 125 mM NaCl, 10 mM Tris-HCl (pH 7.4), and 0.2% Tween-20) containing 125 nanomolar of UHRF1 protein, 5 micromolar of FAM labeled methylated CpG DNA oligo and 5 nanomolar of Terbium labeled anti-his antibody into columns 1 thru columns 2 of 1536 microtiter plates. Next, 5 microliters of Experimental Mix containing 125 nanomolar of UHRF1 protein, 100 nanomolar FAM labeled Hemimethylated CpG DNA oligo and 5 nanomolar Terbium labeled anti-his antibody in assay buffer were dispensed into columns 3 thru 48. Then, the plates were centrifuged and pinned with 22 nL of test compound in DMSO, Mitoxantrone (1.3 micromolar final concentration) in DMSO or DMSO alone (0.44% final concentration). The plates were incubated for 60 minutes at 25 degrees Celsius and TR-FRET was measured on a Viewlux microplate reader (Perkin Elmer, Turku, Finland). After excitation at 340 nm, well fluorescence was monitored at 495 nm (Tb) and 525 nm (FAM). For each well, a fluorescence ratio was calculated according to the following mathematical expression:
Ratio = I525nm / I495nm

Where:

I525nm represents the measured fluorescence emission at 525 nm.
I495nm represents the measured fluorescence emission at 495 nm.

The percent inhibition for each compound was calculated using as follows:

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

Where:

Test_Compound is defined as wells containing the Experimental Mix in the presence of test compound.
High_Control is defined as wells containing the Experimental Mix, Mitoxantrone and DMSO.
Low_Control is defined as the median of the wells containing test compounds.

PubChem Activity Outcome and Score:

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.

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 100-19, and for inactive compounds 19-0.

List of Reagents:

UHRF1_SRA protein (supplied by Assay Provider)
FAM labeled Hemimethylated CpG DNA Oligo (IDT-custom order)
FAM labeled Methylated CpG DNA Oligo (IDT-custom order)
Lanthascreen Tb Anti-His Antibody (Invitrogen, part PV5895)
MgCl2 (Fisher, part BP214)
NaCl (Sigma, part S6546)
DTT (Fisher, part BP172)
EDTA (Sigma, part E7889)
Tris (Amresco, part 0497)
Tween 20 (Sigma, part P9416)
Glycerol (Sigma, part G7893)
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, 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-A1016-RevErbaLBD-Primary-Assay

Protocol: This assay is to identify modulators of Rev-erb alpha protein binding to DNA

A. Materials:
REV-alpha_beta purified protein = Rastinejad lab
FITC-DNA = Rastinejad lab
Tris = Biorad (cat #161-0719)
DTT = Akron Biotechnology (cat #AK2948-0005)
5M NaCl = Sigma-Aldrich (cat #56546-1L)
Glycerol 99.5% = Acros (cat #327255000)
Tween 20 = Sigma-Aldrich (cat #P1379)
Corning high base black plates = Corning (cat #3724)
Molecular grade water = Cellgro (cat #46-000-CM)

B. Plate Map:
Negative (low) control in columns 1 and 2 is 6nM DNA and 35nM Protein, DMSO
Positive (high) control in columns 3 and 4, Protein at 750nM and 6nM DNA, DMSO
Test compound in columns 5 - 48, Protein at 35nM + 6nM DNA + test compound

C. Procedures:
Step#Description
1#Prepare 2X Rev-erb alpha protein stock and 2X DNA stock
2#Using LabCyte Echo, transfer xnL from a 2 mM Echo qualified plate containing test compounds into assay plate Col. 5 - 48. Add same volume of DMSO in col 1-4.
3#Spin plates at 1000 rpm for 1 minute in centrifuge.
4#Using the bioraptr, add 3 uL/well of (35nM protein control) to columns 1 and 2 and test compound wells.
5#Using the bioraptr, add 3 uL/well of Mix 2 (750nM protein) to col. 3-4 for the positive control
6#Using the bioraptr, add 3uL/well of Mix 3 (DNA) to col. 1-48.
7#Spin plates at 1000 rpm for 1 minute in centrifuge.
8#Incubate plates in the dark at room temperature for 90 minutes.
9#Set up Perkin Elmer EnVision as described in section Instrument setting.
10#Read plates on EnVision using FP Dual enhanced mirror, FP 480 excitation filter, FP-P-pol 535 and FP-S-pol 535 emissin filters

Comment: Actives were selected based on, % response = 45% or greater

External ID: HGDAF12_AG_LUMI_1536_1X%ACT PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that act as agonists of the nuclear receptor daf-12. In this assay, CV-1cells are co-transfected with a GAL4-responsive reporter plasmid (UAS-tk-luc) and expression vector encoding GAL4-hgDAF12. The ability of compounds to increase transcriptional activity is assessed by measuring luciferase expression from the reporter gene plasmid. Compounds were tested in singlicate at a final nominal concentration of 6.8 micromolar.

Protocol Summary:

HEK293 cells were routinely cultured in T-175 flasks containing 25 mL of DMEM media supplemented with 10% v/v fetal bovine serum and 1% v/v antibiotic-antimycotic mix at 37 C, 5% CO2 and 95% relative humidity (RH). The day prior to run the assay, the HEK293 cells were harvested using 5 mL of TrypLE reagents and seeded in fresh media at a density of 10 million cells per T175 flask. The following day, cells were transfected with 1 mL of serum-free OptiMEM containing 8 ug of the hgDAF12-expressing vector, 20 ug of the MH1000-tk-luc reporter plasmid and 80 uL of transfection reagent. Twenty four hours post transfection, cells were harvested using 5 mL of preheated TrypLE and resuspended at a concentration of 1 million cells per mL in phenol-red free DMEM supplemented as above. Sodium butyrate, a compound shown to increase the luminscent signal in this assay, was used as a positive control.

The assay was started by dispensing 5 uL of cell suspension into each well of white, solid-bottom 1536-well plates using a flying reagent dispenser (Aurora) and placed in the incubator for 3 hours. Cells were then treated with 34 nL/well of either test compounds, DMSO (Low Control, final concentration 0.68%) or 2 mM of Sodium Butyrate (High Control). Plates were incubated for 24 hours at 37 C, 5% CO2 and 95%RH and then removed from the incubator and equilibrated to room temperature for 10 minutes. Luciferase activity was detected by addition of 5 uL of One-Glo reagent to each well. After a 15 minute incubation time, light emission was measured with the ViewLux reader (PerkinElmer).

The percent activation of each test compound was calculated as follows:

%_Activation = 100 * ( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median__Low_Control ) )

Where:

High_Control is defined as wells treated with 2 mM Sodium Butyrate
Low_Control is defined as wells treated with DMSO only.
Test_Compound is defined as wells treated with test compound.

PubChem Activity Outcome and Score:

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.

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 100-12, and for inactive compounds 12-0.

List of Reagents:

MH1000-tk-luc luciferase reporter plasmid (Assay Provider)
hgDAF12 expressing plasmid (Assay Provider)

List of Consumables:

Sodium Butyrate (Sigma, part B5887)
HEK293 cells (ATCC, part CRL-1573)
DMEM (Invitrogen, part 11965)
FBS (Hyclone, part SH30088.03)
Antibiotic-Antimycotic 100X Liquid Solution (Gibco, part 15240)
TransIT 293 (Mirus Corporation, part MIR-2700)
OptiMEM (Invitrogen, part 31985)
TrypLE Trypsin Replacement Enzyme (Invitrogen, part 12604)
One-Glo (Promega, part E6130)
1536-well plates (Greiner part 789173)

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 luciferase activity and hence well luminescence. 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.

In absence of a Protein Entry for the DAF-12 protein from H. glycines in NCBI's database, links to the target sequence and origin refer to the closest known orthologue from the prototypic parasitic helminth C.elegans.

External ID: TPD2_INH_EPIABS_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that act as inhibitors of the activity of tyrosyl DNA phosphodiesterase 2 (TDP2). TDP2 is a divalent cation-dependent enzyme that repairs TopII-associated DNA strand breaks. It is hypothesized that inhibitors of TDP2 may serve as useful adjuvants in combination with cancer drugs such as etoposide.

In this biochemical assay, recombinant human TDP2 protein is incubated at 37 degrees Celsius with the T5PNP substrate in the presence of Mg2+-containing assay buffer.T5PNP is a substrate for snake venom phosphodiesterase, as well as a substrate for TDP2. As a substrate for TDP2, T5PNP is used to mimic the TopII-DNA complex. TDP2 cleaves the phosphodiester bond in T5PNP, and the chromogenic p-nitrophenol group is released. As time increases the TDP2 enzyme will increasingly catalyze hydrolysis of the T5PNP substrate, resulting in increased release of p-nitrophenol and detection at 415nM wavelength. Compounds are tested in singlicate at a final nominal concentration of 12.8microM.

Protocol Summary:

Prior to the start of the assay, 2 ul of a solution containing T5PNP subtrate (final concentration 5mM) in assay buffer (50mM Tris-HCl pH7.5, 1mM DTT, 1mM MgCl2, 50mM KCl and 100ug/ml BSA) was dispended into a all wells of a 1536 well plate. Next, 39nL of test compound in DMSO or DMSO alone (1% final concentration) was added to the appropriate wells. The assay was started by dispensing 1 ul of a solution contanting TDP2 enzyme (120nM final concentration) in assay buffer to wells in columns 4-48 and 1ul of assay buffer alone to wells in columnes 1-3. Plates were centrifuged and incubated for 2hrs at 37 degrees Celsius at which time fluorescence intensity was measured (Ex. 405nm and Em. 405nm) using a EnVision microplate reader (Perkin Elmer).

Prior to further calculations, the following formula was used to calculate Epi Absorbance (EPIABS)

EPIABS = -log10( sample / background)

Where:

Sample is defined as the fluorescent intensity of wells containing test compounds or DMSO.
Background is defined as the fluorescent intensity of wells containing buffer and T5PNP only.

The % inhibition for each well was then calculated as follows:

%_Inhibition = ( EPIABS_Test_Compound - MedianEPIABS_Low_Control ) / ( MedianEPIABS_High_Control - MedianEPIABS_Low_Control ) * 100

Where:

Test_Compound is defined as wells containing test compound, TDP2 and T5PNP.
High_Control is defined as wells containing only Buffer and T5PNP.
Low_Control is defined as wells containing DMSO, TDP2 and T5PNP.

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 100-22, and for inactive compounds 22-0.

List of Reagents:

TDP2 (supplied by Assay Provider)
Tris Base(Sigma, 93349)
DTT (Sigma, 43815)
BSA (Sigma, A2153)
MgCl2 (Sigma,M2670)
KCl (Sigma, P9333)
T5PNP (Sigma, T4510)
1536 well plates (Corning 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. 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, 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: HMS937_MLP

Protocol: 10 microL of proteasome-containing buffer (0.15 nM proteasome in 50 mM Tris-HCl, pH 7.4, 1 mg/ml ovalbumin, 1 mM ATP-MgCl2, 1mM DTT) were added to each well of 384-well plates (Corning 3820), except for column 1, which was used for positive controls.

100 nL of each experimental compound were transferred to the assay plates by pin transfer, and then incubated for 30 minutes at room temperature.

10 microL of 1 microM Ub-AMC in buffer (50 mM Tris-HCl, pH 7.4, 1 mg/ml ovalbumin, 1 mM ATP-MgCl2, 1mM DTT) were added to all wells of the assay plates, and then incubated for 25 minutes at room temperature. The fluorescence intensity of each well was then read using a Perkin Elmer EnVision plate reader at 355 nm/460 nm (excitation/emission).

For positive controls, 10 microL of 1 microM Ub-AMC in buffer (50 mM Tris-HCl, pH 7.4, 1 mg/ml ovalbumin, 1 mM ATP-MgCl2, 1mM DTT) were added to all wells in column 1 on each assay plate, and incubated for 25 minutes at room temperature before fluorescence reading.

For negative controls, 10 microL of proteasome-containing buffer (0.15 nM proteasome in 50 mM Tris-HCl, pH 7.4, 1 mg/ml ovalbumin, 1 mM ATP-MgCl2, 1mM DTT) were added to all wells in column 2 on each assay plate, and then 10 microL of 1 microM Ub-AMC in buffer (50 mM Tris-HCl, pH 7.4, 1 mg/ml ovalbumin, 1 mM ATP-MgCl2, 1mM DTT) was added. After incubation at room temperature for 25 minutes, fluorescence intensity was read.

Comment: Activity scores were calculated based on percent (%) inhibition, which was in turn calculated from fluorescence intensity (FI) readings as follows: % Inhibition = (1-((Vi-Vp)/(Vn-Vp)))*100, where Vi is the FI of the individual well, Vp the average FI of the positive controls on that plate, and Vn the average FI of the negative controls on that plate. A final % inhibition value was calculated as the average of replicate results for each individual compound and used to determine the Activity Score. If the % inhibition was less than zero, the Activity Score was set to Zero. Compounds showing >20% inhibition were considered active.

External ID: PLCG1_INH_QFRET_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this biochemical assay is to identify compounds that act as inhibitors of the activity of phospholipase C isozymes, PLC-G1. In this assay, PLC-G1 isozyme is incubated with test compounds and fluorogenic reporter WH-15. As designed, test compounds that act as PLC-G1 inhibitors will prevent the hydrolysis of WH-15 fluorogenic reporter, thus preventing the release of IP3, a quinomethide derivative, and 6-aminoquinoline, which is highly fluorescent, leading to decreasing well fluorescence. Compounds are tested in singlicate at a nominal test concentration of 12.2 micromolar.

Protocol Summary:

Prior to the start of the assay, 2 microliters of PLC-G1 at a final concentration of 5pg/ul (in 50 mM HEPES pH 7.2, 70 mM KCl, 3mM CaCL2, 3mM EGTA, 2mM DTT, 0.04mg/mL acid-free BSA, with Cholate 0.5%) are dispensed into 1536 microtiter plates, 1 microliter of assay buffer is dispensed into columns 4-48 and 1 microliter of 0.2M EGTA is added to columns 1-3. Compounds are added to plate (final concentration 12.2uM) and incubated for 10 minutes at 25 degrees Celsius. The assay start by the addition of 2 microliter of WH-15 fluorogenic reporter at a final concentration 10uM in Assay Buffer to all wells. Plates were centrifuged and after 90 min of incubation at 25 degrees Celsius fluorescence is measured at 355nm excitation and 535nm emmision.

The percent inhibition for each compound was calculated as follows:

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

Where:

Test_Compound is defined as wells containing PLCG1 in the presence of test compound and WH15 fluoreogenic reporter.
High_Control is defined as wells containing PLCG1, WH15 fluoreogenic reporter and EGTA.
Low_Control is defined as the median of the wells containing test compounds.

A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Four values were calculated: (1) the average percent inhibition of all high controls tested plus three times the standard deviation of the high controls, (2) the average percent inhibition of all low controls tested minus three times the standard deviation of the low controls, (3) the average percent inhibition of all compounds tested between (1) and (2), and (4) three times their standard deviation. The sum of two of these values, (3) and (4), was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition/activity 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 activity score range for active compounds is 100-11, for inactive 11-0.

List of Reagents:

PLCG1 isozyme (Supplied by Assay Provider)
WH-15 fluorogenic reporter (Supplied by KXTBio)
HEPES (Fisher, BP310)
Sodium cholate hydrate (Sigma, C6445)
CaCl2 (Sigma, 06991)
EGTA (Fisher, O2783)
DTT (Fisher, BP172)
KCl (Sigma, P9541)
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, 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: USP28 FAST DUB HTS Primary

Protocol: Deubiquitinases were purified from E. Coli. Ub-Rho substrate was purchased from Boston BioChem (Cat #: U-555). Assay buffer consisted of 50 mM Tris pH 8, 50 mM NaCl, 0.002% Tween20, 5 mM DTT in MilliQ water. Stop buffer consisted of assay buffer + 0.2% trifluoroacetic acid.

Protocol for Inhibitors of USP28: Primary Screen
1. Compound, 25 nL of screening compounds (final concentration of 25 uM) to columns 1-46 of a Greiner 1536 well Black, flat bottom, polystyrene microplate (Griener 782076) (columns 45-46: DMSO control)
2. Reagent, 2.5 uL of assay buffer to columns 47-48 on all plates (No enzyme control)
3. Reagent, 2.5 uL of USP28 in assay buffer (final concentration of 0.5 nM) to columns 1-46
4. Reagent, 2.5 uL of Ub-Rho in assay buffer (final concentration of 12 nM) to all wells
5. Time, Incubate at room temperature for 30 minutes
6. Reagent, 2.5 uL of stop buffer to all wells
7. Centrifuge plates (5s, 350 rpm)
8. Detection, Fluorescence, PheraStar, FITC Module

Comment: Normalization of raw data to positive and negative control wells were applied to the data using Helios, a high-throughput screening data analysis program developed at Novartis.(Gubler et al., 2018)

Compounds demonstrating greater than or equal to 30% inhibition of USP28 (USP28_EFFICACY >/= 30) were called as active, compounds demonstrating activity below this threshold were called as inactive.

External ID: USP10 FAST DUB HTS Primary

Protocol: Deubiquitinases were purified from E. Coli. Ub-Rho substrate was purchased from Boston BioChem (Cat #: U-555). Assay buffer consisted of 50 mM Tris pH 8, 50 mM NaCl, 0.002% Tween20, 5 mM DTT in MilliQ water. Stop buffer consisted of assay buffer + 0.2% trifluoroacetic acid.

Protocol for Inhibitors of USP10: Primary Screen
1. Compound, 20 nL of screening compounds (final concentration of 20 uM) to columns 1-46 of a Greiner 1536 well Black, flat bottom, polystyrene microplate (Griener 782076) (columns 45-46: DMSO control)
2. Reagent, 2.5 uL of assay buffer to columns 47-48 on all plates (No enzyme control)
3. Reagent, 2.5 uL of USP10 in assay buffer (final concentration of 15 nM) to columns 1-46
4. Reagent, 2.5 uL of Ub-Rho in assay buffer (final concentration of 15 nM) to all wells
5. Time, Incubate at room temperature for 60 minutes
6. Detection, Fluorescence, PheraStar, FITC Module

Comment: Normalization of raw data to positive and negative control wells were applied to the data using Helios, a high-throughput screening data analysis program developed at Novartis.(Gubler et al., 2018)

Compounds demonstrating greater than or equal to 30% inhibition of USP10 (USP10_EFFICACY >/= 30) were called as active, compounds demonstrating activity below this threshold were called as inactive.

External ID: UCHL1 FAST DUB HTS Primary

Protocol: Deubiquitinases were purified from E. Coli. Ub-Rho substrate was purchased from Boston BioChem (Cat #: U-555). Assay buffer consisted of 50 mM Tris pH 8, 50 mM NaCl, 0.002% Tween20, 5 mM DTT in MilliQ water. Stop buffer consisted of assay buffer + 0.2% trifluoroacetic acid.

Protocol for Inhibitors of UCHL1: Primary Screen
1. Reagent, 2.5 uL of UCHL1 in assay buffer (final concentration of 1 nM) to columns 1-46 of a Greiner 1536 well Black, flat bottom, polystyrene microplate (Griener 782076)
2. Compound, 20 nL of screening compounds (final concentration of 20 uM) to columns 1-46 (columns 45-46: DMSO control)
3. Reagent, 2.5 uL of assay buffer to columns 47-48 on all plates (No enzyme control)
4. Time, Incubate at room temperature for 60 minutes
5. Reagent, 2.5 uL of Ub-Rho in assay buffer (final concentration of 7 nM) to all wells
6. Time, Incubate at room temperature for 10 minutes
7. Detection, Fluorescence, PheraStar, FITC Module

Comment: Normalization of raw data to positive and negative control wells were applied to the data using Helios, a high-throughput screening data analysis program developed at Novartis.(Gubler et al., 2018)

Compounds demonstrating greater than or equal to 30% inhibition of UCHL1 (UCHL1_EFFICACY >/= 30) were called as active, compounds demonstrating activity below this threshold were called as inactive.

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: OTUD3 FAST DUB HTS Primary

Protocol: Deubiquitinases were purified from E. Coli. Ub-Rho substrate was purchased from Boston BioChem (Cat #: U-555). Assay buffer consisted of 50 mM Tris pH 8, 50 mM NaCl, 0.002% Tween20, 5 mM DTT in MilliQ water. Stop buffer consisted of assay buffer + 0.2% trifluoroacetic acid.

Protocol for Inhibitors of OTUD3: Primary Screen
1. Compound, 25 nL of screening compounds (final concentration of 25 uM) to columns 1-46 of a Greiner 1536 well Black, flat bottom, polystyrene microplate (Griener 782076) (columns 45-46: DMSO control)
2. Reagent, 2.5 uL of assay buffer to columns 47-48 on all plates (No enzyme control)
3. Reagent, 2.5 uL of OTUD3 in assay buffer (final concentration of 12 nM) to columns 1-46
4. Reagent, 2.5 uL of Ub-Rho in assay buffer (final concentration of 40 nM) to all wells
5. Time, Incubate at room temperature for 2 hours
6. Reagent, 2.5 uL of stop buffer to all wells
7. Centrifuge plates (5s, 350 rpm)
8. Detection, Fluorescence, PheraStar, FITC Module

Comment: Normalization of raw data to positive and negative control wells were applied to the data using Helios, a high-throughput screening data analysis program developed at Novartis.(Gubler et al., 2018)

Compounds demonstrating greater than or equal to 30% inhibition of OTUD3 (OTUD3_EFFICACY >/= 30) were called as active, compounds demonstrating activity below this threshold were called as inactive.

External ID: HMS1364-MLP_WT

Protocol: The day before screening, RN4220 was grown overnight (16-18 h) in sterile-filtered tryptic soy broth (TSB) at 30 degrees C with shaking.

On the day of screening, columns 1-24 of assay plates (Corning 3710, clear) were prefilled with 30 uL of TSB using the Combi. Next, 100 nL of each compound was pin-transferred to each plate. For every compound library plate, there were two daughter plates (A & B). Overnight cultures were diluted 625x into TSB. 50 uL of the diluted cultures were added to columns 1-24 of the corresponding assay plates. Wells in column 2 contained only medium (with culture) and served as the negative controls. Positive control (0.8 uL of erythromycin, 10 ug/mL) was added to column 1 using the Hewlett Packard D300. Final assay well volume was 80 uL. Plates were spun down and stacked 5 high, covered with lids (Corning 3009), and incubated at 30 degrees C overnight (~16 h).

The following day, assay plates were removed from the incubator and cell density quantitated using a PerkinElmer EnVision (600 nm filter).

Comment: Z-scores were calculated for every well based on plate average and standard deviation of experimental well absorbance (OD600). Wells were considered active for reduced WT survival if Z-score for both replicates <= -3 and absorbance for both replicates <= 0.18. Percent inhibition was calculated by subtracting well absorbance from negative control plate average absorbance, dividing by the difference between plate negative and positive control plate average absorbance, and multiplying by 100. The replicate values were then averaged to determine activity scores. Resulting values > 100 were set to 100 and < 0 were set to 0, where 100 = 100% activity.

External ID: USP30 FAST DUB HTS Primary

Protocol: Deubiquitinases were purified from E. Coli. Ub-Rho substrate was purchased from Boston BioChem (Cat #: U-555). Assay buffer consisted of 50 mM Tris pH 8, 50 mM NaCl, 0.002% Tween20, 5 mM DTT in MilliQ water. Stop buffer consisted of assay buffer + 0.2% trifluoroacetic acid.

Protocol for Inhibitors of USP30: Primary Screen
1. Compound, 25 nL of screening compounds (final concentration of 25 uM) to columns 1-46 of a Greiner 1536 well Black, flat bottom, polystyrene microplate (Griener 782076) (columns 45-46: DMSO control)
2. Reagent, 2.5 uL of assay buffer to columns 47-48 on all plates (No enzyme control)
3. Reagent, 2.5 uL of USP30 in assay buffer (final concentration of 1 nM) to columns 1-46
4. Reagent, 2.5 uL of Ub-Rho in assay buffer (final concentration of 12 nM) to all wells
5. Time, Incubate at room temperature for 2 hours
6. Reagent, 2.5 uL of stop buffer to all wells
7. Centrifuge plates (5s, 350 rpm)
8. Detection, Fluorescence, PheraStar, FITC Module

Comment: Normalization of raw data to positive and negative control wells were applied to the data using Helios, a high-throughput screening data analysis program developed at Novartis.(Gubler et al., 2018)

Compounds demonstrating greater than or equal to 30% inhibition of USP30 (USP30_EFFICACY >/= 30) were called as active, compounds demonstrating activity below this threshold were called as inactive.

External ID: FBW7_ACT_ALPHA_1536_1X%ACT PRUN

Protocol: Assay Overview:
FBW7 assay principle. In this assay, either mutant or wild type (w.t.) FBW7 interact with phosphorylated cyclin E peptide (cycE~P), which will bring donor and acceptor beads into close proximity. Laser excitation of the donor beads converts oxygen to an excited singlet state. Reaction of the singlet oxygen with the acceptor beads further activates a chemiluminescence/fluorescence reaction within the same bead resulting in emitted light at 520-620 nm. Small molecule activators that enhance the mutant FBW7 interaction with the cycE~P decrease the distance of the acceptor beads, thus leading to increased signal being emitted signal.
Protocol Summary:
There are six steps in this 1536 well assay format which are listed in order. First, 2.5uL/well of a 2X working solution containing RLFbw7 [12.5nM final], Cyclin E peptide [12.5nM final], and Ni beads [5ug/mL final], in assay buffer (25mM Tris-HCl pH 7.4 + 100mM NaCl, 0.1% Tween-20, 5mM ?-Mercaptoethanol and 0.05% BSA) was dispensed into columns 1-44. Then 2.5uL/well of a 2X working solution containing WTFbw7 [12.5nM final], Cyclin E peptide [12.5nM final], and Ni beads [5ug/mL final], in assay buffer was dispensed into columns 45-48. Using the pintool transfer device 134nL of compound or control was added to each well. This achieved a nominal screening concentration of 26.1uM and 2.6% DMSO concentration. This was followed by the addition of SA beads to all wells at 5ug/mL final concentration in assay buffer. The assay was then incubated for 20 hours in a temperature controlled 25C environment followed by Alphascreen detection using the PerkinElmer EnVision.

The percent activation for each compound was calculated as follows:

100 *( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control ) )
Where:
Test_Compound is defined as wells containing RLFbw7 (mutant), cyclin E peptide and Nickel acceptor beads in the presence of test compound
High_Control is defined as wells containing WTFbw7 (wild type), cyclin E peptide and Nickel acceptor beads
Low_Control is defined as the median of the wells containing DMSO, RLFbw7 (mutant), cyclin E peptide and Nickel acceptor beads
PubChem Activity Outcome and Score:

A mathematical algorithm was used to determine active compounds. Two values were calculated: (1) the average percent activation of all compounds tested for the screen, 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 percent activation than the cutoff parameter (1.85% in the case here) was declared active.
The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.
The activity score range for active compounds is 100-1, for inactive 1-0.
List of Reagents:
Ni Beads- PerkinEmer Lifesciences Cat#6760619R
RLFbw7-Assay Provider
WTFbw7-Assay Provider
Cyclin E peptide-Assay Provider
5M NaCl- Sigma Cat# S6546-1L
Tween20- Fisher Cat# BP337
Tris 1M pH7.4 Research Organics Cat# 9686T
BSA-Sigma Cat#A7030
?-Mercaptoethanol-SigmaM6250
1536-well plates (Corning, part 7254)

Comment: Due to the size of the Scripps Molecular Screening Center 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, 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 Scripps Molecular Screening Center.

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.

External ID: HMS1364-MLP_ugtP

Protocol: The day before screening, RN4220 ugtP was grown overnight (16-18 h) in sterile-filtered tryptic soy broth (TSB) at 30 degrees C with shaking.

On the day of screening, columns 1-24 of assay plates (Corning 3710, clear) were prefilled with 30 uL of TSB using the Combi. Next, 100 nL of each compound was pin-transferred to each plate. For every compound library plate, there were two daughter plates (A & B). Overnight cultures were diluted 625x into TSB. 50 uL of the diluted cultures were added to columns 1-24 of the corresponding assay plates. Wells in column 2 contained only medium (with culture) and served as the negative controls. Positive control (0.8 uL of erythromycin, 10 ug/mL) was added to column 1 using the D300. Final assay well volume was 80 uL. Plates were spun down and stacked 5 high, covered with lids (Corning 3009), and incubated at 30 degrees C overnight (~16 h).

The following day, assay plates were removed from the incubator and cell density quantitated using a PerkinElmer EnVision (600 nm filter). Library plates were screened in duplicate, with both assay plates in a given set prepared on the same day.

Comment: Z-scores were calculated for every well based on plate average and standard deviation of experimental well absorbance (OD600). Wells were considered active for reduced ugtP survival if Z-score for both replicates < -3 and absorbance for both replicates <= 0.15. Percent inhibition was calculated by subtracting well absorbance from negative control plate average absorbance, dividing by the difference between plate negative and positive control plate average absorbance, and multiplying by 100. The replicate values were then averaged to determine activity scores. Resulting values > 100 were set to 100 and < 0 were set to 0, where 100 = 100% activity.

External ID: PLCB3_INH_QFRET_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this biochemical assay is to identify compounds that act as inhibitors of the activity of phospholipase C isozymes, PLC-B3. In this assay, PLC-B3 isozyme is incubated with test compounds and fluorogenic reporter WH-15. As designed, test compounds that act as PLC-B3 inhibitors will prevent the hydrolysis of WH-15 fluorogenic reporter, thus preventing the release of IP3, a quinomethide derivative, and 6-aminoquinoline, which is highly fluorescent, leading to decreasing well fluorescence. Compounds are tested in singlicate at a nominal test concentration of 12.2 micromolar.

Protocol Summary:

Prior to the start of the assay, 2 microliters of PLC-B3 at a final concentration of 0.4ng/ul (in 50 mM HEPES pH 7.2, 70 mM KCl, 3mM CaCL2, 3mM EGTA, 2mM DTT, 0.04mg/mL acid-free BSA, with Cholate 0.5%) are dispensed into 1536 microtiter plates, 1 microliter of assay buffer is dispensed into columns 4-48 and 1 microliter of 0.2M EGTA is added to columns 1-3. Compounds are added to plate (final concentration 12.2uM) and incubated for 10 minutes at 25 degrees Celsius. The assay start by the addition of 2 microliter of WH-15 fluorogenic reporter at a final concentration 10uM in Assay Buffer to all wells. Plates were centrifuged and after 90 min of incubation at 25 degrees Celsius fluorescence is measured at 355nm excitation and 535nm emmision.

The percent inhibition for each compound was calculated as follows:

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

Where:

Test_Compound is defined as wells containing PLCB3 in the presence of test compound and WH15 fluoreogenic reporter.
High_Control is defined as wells containing PLCB3, WH15 fluoreogenic reporter and EGTA.
Low_Control is defined as the median of the wells containing test compounds.

A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Four values were calculated: (1) the average percent inhibition of all high controls tested plus three times the standard deviation of the high controls, (2) the average percent inhibition of all low controls tested minus three times the standard deviation of the low controls, (3) the average percent inhibition of all compounds tested between (1) and (2), and (4) three times their standard deviation. The sum of two of these values, (3) and (4), was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition/activity 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 activity score range for active compounds is 100-13, for inactive 13-0.

List of Reagents:

PLCB3 isozyme (Supplied by Assay Provider)
WH-15 fluorogenic reporter (Supplied by KXTBio)
HEPES (Fisher, BP310)
Sodium cholate hydrate (Sigma, C6445)
CaCl2 (Sigma, 06991)
EGTA (Fisher, O2783)
DTT (Fisher, BP172)
KCl (Sigma, P9541)
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, 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: ADAM10_INH_QFRET_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that inhibit ADAM10. This assay employs a fluorophore and quencher pair. F =EDANS fluorophore, Q = DABCYL quencher. When intact, EDANS emission at 460nm is quenched by DABCYL via fluorescence resonance energy transfer. Upon cleavage of the scissile bond (A~V) by ADAM protease, the distance between fluorophore and quencher increases resulting in fluorescence increase at 460nm. Compounds are tested in singlicate at a nominal final nominal concentration of 6.95 micromolar.

Protocol Summary:

Prior to the start of the assay, 2.5 microliters 2X ADAM10 enzyme (20 nM in Assay Buffer: 50 mM HEPES, 0.01% Brij, pH 7.5) are dispensed into 1536 microtiter plates. Compounds are added to plate (final concentration TBD) and incubated for 30 minutes at 25 degrees Celsius. The assay is started by dispensing 2.5 microliter of2X DM2 substrate (20 uM in Assay Buffer) to all wells. Plates are centrifuged and after 3 hours of incubation at 25 degrees Celsius, fluorescence is measured (excitation = 359nm, emission = 460nm).

The % inhibition for each well was then calculated as follows:

%_Inhibition = ( RFU_Test_Compound - MedianRFU_Low_Control ) / ( MedianRFU_High_Control - MedianRFU_Low_Control ) * 100

Where:

Test_Compound is defined as wells containing test compound.
High_Control is defined as wells treated with 1 micromolar Marimastat
Low_Control is defined as wells containing DMSO.

Interval Cutoff:

A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Four values were calculated: (1) the average percent inhibition of all high controls tested plus three times the standard deviation of the high controls, (2) the average percent inhibition of all low controls tested minus three times the standard deviation of the low controls, (3) the average percent inhibition of all compounds tested between (1) and (2), and (4) three times their standard deviation. The sum of two of these values, (3) and (4), was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition/activity 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 activity score range for active compounds is 100-19, for inactive 19-0.

List of Reagents:

ADAM10 enzyme (R&D Systems, part # 936-AD)
EDANS-DABCYL DM2 peptide substrate (Supplied by Assay Provider)
0.5M HEPES solution, pH7.5 (Teknova, part #101319-900)
Brij-35 (Sigma-Aldrich, part # P1254)
1536 well plate (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. 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, 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-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: 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: COUPTFII_INH_LUMI_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this cell-based assay is to identify compounds that can inhibit COUP-TFII transcriptional activity. The expression vectors for COUP-TFII (pcDNA6.2-COUP-TFII) and the luciferase reporter NGFIA-Luc (pXP2-168) are transiently cotransfected into HEK-293T cells.

COUP-TFII has been shown to efficiently activate NGFI-A-Luc expression (17) and the readout can be measured by a luminometer. Small molecules that inhibit COUP-TFII transcriptional activity will decrease the promoter activity that can be detected by luciferase assay. As designed, compounds that inhibit COUP-TFII activity will decrease luciferase activity, resulting in decreased well luminescence.

Protocol Summary:

HEK293 cells were routinely cultured in T-175 flasks containing 25 mL of DMEM media supplemented with 10% v/v fetal bovine serum and 1% v/v antibiotic-antimycotic mix at 37 C, 5% CO2 and 95% relative humidity (RH). The day prior to run the assay, the HEK293 cells were harvested using 5 mL of TrypLE reagents and seeded in fresh media at a density of 10 million cells per T175 flask. The following day, cells were transfected with 1 mL of serum-free OptiMEM containing 12 ug of the plasmid encoding for COUP-TF2, 8 ug of the pX2-168 luciferase reporter plasmid, and 60 uL of X-tremeGene 9 transfection reagent. Twenty four hours post transfection, cells were harvested using 5 mL of preheated TrypLE and resuspended at a concentration of 800,000 cells per mL in phenol-red free DMEM supplemented as above. In the absence of a pharmacological positive control, COUP-TF2 inhibition was mimicked by transfecting cells in absence of the COUP-TF2 expressing vector (i.e. reporter plasmid only).

The assay was started by dispensing 5 uL of cell suspension into each well of white, sterile, solid-bottom 1536-well plates. The first three columns received control cells (no COUP-TF2 expressed) whereas the rest of the plate was dispensed with COUP-TF2-transfected cells. After addition of cells the plates were spun down. The plates were incubated for 4 hours and then treated with 43 nL/well of test compounds or DMSO (final concentration 0.65%) on COUP-TF2 cells and Control cells. Plates were incubated for eighteen hours at 37 C, 5% CO2 and 95% RH. Plates were then removed from the incubator and equilibrated to room temperature for 10 minutes. Luciferase activity was detected by addition of 5 uL of One-Glo reagent to each well. After a 10 minute incubation time, light emission was measured with the ViewLux reader (PerkinElmer).

The percent inhibition for each compound was calculated as follows:

%_Inhibition = ( ( Test_Compound - Median_Sample_Field ) / ( Median_High_Control - Median_Sample_Field ) ) * 100

Where:

High_Control is defined as wells containing cells transfected with reporter plamid only (pX2-168)
Test_Compound is defined as well containing cells cotransfected with pcDNA6.2-COUP-TFII and pX2-168 in the presence of test compounds
Sample_Field is defined as wells containing cells cotransfected with pcDNA6.2-COUP-TFII and pX2-168 in the presence of test compounds

PubChem Activity Outcome and Score:

A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Two values were calculated for each assay plate: (1) the average percent inhibition of test compound wells that are within the High and Low Controls (i.e. higher than the average plues three standard deveiations of the Low Control wells and lower than the average minus three stardard deviation of the High Control 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.

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 100-34, and for inactive compounds 34-0.

List of Reagents:

pX2-168 luciferase reporter plasmid (Assay Provider)
pcDNA6.2-COUP-TFII plasmid (Assay Provider)
HEK293 cells (ATCC, part CRL-1573)
DMEM (Invitrogen, part 21063)
FBS (Hyclone, part SH30088.03)
Antibiotic-Antimycotic 100X Liquid Solution (Gibco, part 15240)
X-tremeGENE 9 DNA Transfection Reagent (Roche, part 06365809001)
OptiMEM (Invitrogen, part 31985)
TrypLE Trypsin Replacement Enzyme (Invitrogen, part 12604)
OneGlo (Promega, part E6130)
1536-well plates (Corning, part 7298)

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: 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: CHEMBL4373363

Protocol: N/A

Comment: Journal: Bioorg Med Chem Lett
Year: 2019
Volume: 29
Issue: 3
First Page: 449
Last Page: 453
DOI: 10.1016/j.bmcl.2018.12.025

Target ChEMBL ID: CHEMBL2808
ChEMBL Target Name: LXR-alpha
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: stopgo-p2-SytraCBC-dual-FF

Protocol: Reagents:
StopGo2A HEK-NPG-Luc cells and other necessary media and controls were provided by Dr. Atkins's lab. Compound PTC124 was used as a control. Dose response was assessed using a 1:2 dilution of control compound, with the top dose of 50uM final concentration. Background signal was normalized to wells treated with DMSO only.

Assay Protocol:
In brief, four microliters (2400 cells/well) of StopGo2A HEK-NPG-Luc cells were plated and incubated overnight at 37C. Twenty-three nanoliters of compounds and control were added to the wells using a 1536 array Kalypsys pintool workstation. After an over night incubation (at 37C) with compounds / control, 2.5 uL of Dual/Glo Firefly reagent was added. Firefly luciferase signal was obtained using the PerkinElmer ViewLux plate reader after 20min of room temperature incubation. Dual/Glo Renilla Lucifrase reagents were then added followed by additional 20 min room temperature incubation. Finally, the renilla luciferase signal was obtained using the PerkinElmer ViewLux reader.

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

Compound Ranking:
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, with a ratio activity curve class of 4, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. For a ratio activity curve class = -1.1, score = 80+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -1.2 && abs(ratio.max_response) > 6*10, score = 60+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -2.1 || ( ratio.curve_class==-2.2 && abs(ratio.max_response) > 6*10), score = 40+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -1.2 || ratio.curve_class == -2.2, score = 20+abs((log_ac50+4.5)*inf_activity/20). Inconclusive compounds, with a donor curve class other than 4, have PUBCHEM_ACTIVITY_SCORE of 10. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39.

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: CHEMBL4373365

Protocol: N/A

Comment: Journal: Bioorg Med Chem Lett
Year: 2019
Volume: 29
Issue: 3
First Page: 449
Last Page: 453
DOI: 10.1016/j.bmcl.2018.12.025

Target ChEMBL ID: CHEMBL4093
ChEMBL Target Name: LXR-beta
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: stopgo-p2-SytraCBC-dual-Ren

Protocol: Reagents:
StopGo2A HEK-NPG-Luc cells and other necessary media and controls were provided by Dr. Atkins's lab. Compound PTC124 was used as a control. Dose response was assessed using a 1:2 dilution of control compound, with the top dose of 50uM final concentration. Background signal was normalized to wells treated with DMSO only.

Assay Protocol:
In brief, four microliters (2400 cells/well) of StopGo2A HEK-NPG-Luc cells were plated and incubated overnight at 37C. Twenty-three nanoliters of compounds and control were added to the wells using a 1536 array Kalypsys pintool workstation. After an overnight incubation (at 37C) with compounds / control, 2.5 uL of Dual/Glo Firefly reagent was added. Firefly luciferase signal was obtained using the PerkinElmer ViewLux plate reader after 20min of room temperature incubation. Dual/Glo Renilla Lucifrase reagents were then added followed by additional 20 min room temperature incubation. Finally, the renilla luciferase signal was obtained using the PerkinElmer ViewLux reader.

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

Compound Ranking:
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, with a ratio activity curve class of 4, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. For a ratio activity curve class = -1.1, score = 80+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -1.2 && abs(ratio.max_response) > 6*10, score = 60+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -2.1 || ( ratio.curve_class==-2.2 && abs(ratio.max_response) > 6*10), score = 40+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -1.2 || ratio.curve_class == -2.2, score = 20+abs((log_ac50+4.5)*inf_activity/20). Inconclusive compounds, with a donor curve class other than 4, have PUBCHEM_ACTIVITY_SCORE of 10. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39.

External ID: CHEMBL3057094

Protocol: N/A

Comment: Journal: Med Chem Res
Year: 2012
Volume: 21
Issue: 1
First Page: 27
Last Page: 37
DOI: 10.1007/s00044-010-9489-9

Target ChEMBL ID: CHEMBL375
ChEMBL Target Name: Mus musculus
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular