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: OPRM1-OPRD1_AG_LUMI_1536_1X%ACT PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that activate heterodimer formation between the mu (OPRM1) and delta (OPRD1) opioid receptors, resulting in membrane recruitment of beta-arrestin. The assay monitors GPCR-beta-arrestin proximity using low affinity fragment complementation of beta-galactosidase (beta-gal). This assay employs U2OS cells which express OPRD1, OPRM1 fused to a beta-gal peptide fragment (enzyme donor), and beta-arrestin fused to the complementary beta-gal fragment (enzyme acceptor). Cells are incubated with test compound, followed by measurement of well luminescence. As designed, compounds that induce formation of OPRD1 homodimers or OPRM1-OPRD1 (Mu-Delta) heterodimers will cause beta-arrestin recruitment, resulting in reconstitution of the beta-gal holoenzyme. The reconstituted holoenzyme can then catalyze the hydrolysis of a substrate which yields a chemiluminescent signal, resulting in increased well luminescence. Deltorphin B will be used as the high (100% RLU) control for agonists, and wells containing cells treated with DMSO will be used as the low (0% RLU) control. Compounds were tested in singlicate at a final nominal concentration of 9.3 uM.

Protocol Summary:

The U2OS-OPRM1-OPRD1 (Mu-Delta) cell line was routinely cultured in T-175 sq cm flasks at 37 C and 95% relative humidity (RH). The growth media consisted of a 1:1 mixture of Ham's F-12 Nutrient Media (F-12) and Dulbecco's Modified Eagle Media (DMEM) supplemented with 10% v/v heat-inactivated certified fetal bovine serum, 25 mM HEPES, 250 ug/mL Geneticin, 250 ug/mL Hygromycin B, 0.25 ug/mL Puromycin and 1X antibiotic mix (penicillin, streptomycin, and neomycin).

The day before the assay 1000 cells in 3 uL of cell plating media were seeded into each well of 1536 well microtiter plates and allowed to incubate at 37 C, 5% CO2, and 95 % RH for 23 hours. Next, 28 nL of test compound in DMSO, Deltorphin B (0.9 uM final concentration) in DMSO, or DMSO alone were dispensed to the appropriate wells. The plates were then incubated for 3 hours at 37 C, 5% CO2, and 95 % RH. The assay was started by adding 2 uL of PathHuntertrade mark reagent (prepared according to the manufacturer's protocol); followed by 1 hour incubation at room temperature. Then, Well Luminescence was read on the ViewLux plate reader

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:

High_Control is defined as wells containing cells, Deltorphin B and DMSO.
Test_Compound is defined as wells containing cells, test compounds and DMSO.
Low_Control is defined as wells containing cells and DMSO.

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 100-2, and for inactive compounds 2-0.

List of Reagents:

PathHuntertrade mark B-arrestin recruitment assay, containing the U2OS OPRM1 OPRD1 Beta-arrestin cell line; PathHunter Detection Kit (DiscoveRx, part, 93-0558C3)
Ham's F-12 media (Invitrogen, part 11765-054)
DMEM media (Invitrogen, part 11995-073)
Detachin (Genlantis, part T100100)
Heat Inactivated Fetal Bovine Serum (Invitrogen, part 10082-147)
Puromycin (Invitrogen, part A11138-02)
Hygromycin B (Invitrogen, part 10687-010)
Geneticin (Invitrogen, part 10131-027)
100X Penicillin-Streptomycin-Neomycin mix (Invitrogen, part 15640-055)
T-175 tissue culture flasks (Nunc, part 159910)
Agonist: Deltorphin B (Anaspec, part 62683)
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 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: 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: SBCCG-A995-CSN5-CP-Assay

Protocol: A. Materials:
(Item, source, catalog no)
SCFSkp2-Nedd8OG, derived from human proteins and expressed in E. coli
Human Csn5 enzyme, expressed and purified from HEK293T cells
Molecular Biology Grade Water, Cellgro, 46-000-CM
Tween 20, MP Biomedicals LLC, 194841
Tris pH 7.5, Fisher Scientific, ICN819638
Trehalose, Fluka, 90210
Ovalbumin, Sigma, A5503
Sodium Chloride, Fisher Scientific, S271-10
DTT, Roche, 03117006001
1536 Black solid bottom plates, Corning, 3724

B. Final Assay Conditions:
10nM SCFSkp2-Nedd8OG substrate
0.24nM Csn5 Enzyme
25mM Tris pH7.5
50mM Sodium Chloride
1mM DTT
0.01% Tween 20
25mM Trehalose
0.015 mg/mL Ovalbumin
12.5uM test compound
0.25% DMSO (from compounds)
3uL reaction volume
90 minutes incubation at room temp

C. Assay Procedures:
1. Prepare Reagents as described in section E. Recipe.
2. ) Using LabCyte Echo, transfer 5 nL 10mM test agents (final conc 16.64uM)into assay plate Cols. 5-48, and 5 nL of 100% DMSO (final conc 0.166%) into assay plate Col. 1-4.
3. Spin plates at 1000 rpm for 1 minute in centrifuge.
4. Using the Thermo Multidrop Combi, add 1.5 uL/well of control buffer to columns 1 and 2.
5. Using the Thermo Multidrop Combi, add 1.5 uL/well of enzyme solution to col. 3-48.
6. Using the Thermo Multidrop Combi, add 1.5 uL/well of substrate solution to col. 1-48.
7. Spin plates at 1000 rpm for 1 minute in centrifuge.
8. Incubate plates at room temperature for 90 minutes.
9. Read plates on BMG Pherastar FS using a Fluorescence Polarization protocol.

D. Plate Map:
Positive (High) control in column 1-2, DMSO, buffer and substrate only.
Negative (Low) control in columns 3-4, DMSO, enzyme and substrate.
Test wells in columns 5-48: compound, enzyme and substrate.

E. Recipe:
1X Assay Buffer
25mM Tris pH7.5
50mM Sodium Chloride
1mM DTT
0.01% Tween 20
25mM Trehalose
0.015 mg/mL Ovalbumin

Enzyme Solution
0.48nM Csn5 Enzyme in 1X Assay Buffer (final enzyme concentration is 0.24nM).

Substrate Solution
20nM SCFSkp2-Nedd8OG Substrate in 1X Assay Buffer (final substrate concentration is 10nM).

F. Note:
All reagents should be made up according to its spec-sheet or otherwise in Mol. Grade Water.
Storage conditions after reagents are made up:
Reagent, Temp
Csn5 enzyme, -80 degrees
SCFSkp2-Nedd8OG Substrate, -80 degrees (light sensitive)
Tris pH7.5, 4 degrees
Sodium Chloride, 4 degrees
DTT, -80 degrees
Tween 20, room temp
Trehalose, -80 degrees
Ovalbumin, -80 degrees

Comment: Compounds with corrected %Activity >=50% and 0.5 =< FRatio <= 1.5 are considered to be active in this assay.

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

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: SBCCG-A996-CSN5-MMP2-CP-Assay

Protocol: Assay materials:
1) human MMP-2 enzyme (Cat # 72005) was purchased from Anaspec
2) 520 MMP FRET Substrate VI (Cat # 60573-01) was purchased from Anaspec
3) APMA (Cat A9563) was purchased from Sigma-Aldrich
4) Assay Buffer: 50 mM Tris-HCl pH 7.6 , 5 mM CaCl2 ATP, 0.05% BRIG-35
5) Aurora 1536 well black high base plate were obtained from Nexus Biosystems (Cat # 00019120BX)

Procedure for SAR dose-response assay:
1) Thaw MMP-2 enzyme, Substrate VI, and APMA on ice.
2) Activate MMP2: Make 1 M APMA solution in DMSO. Dilute this 1:100 in dH2O (NOT assay buffer) (10mM stock solution). From 10mM APMA stock, combine with MMP2 enzyme to a final concentration of 1 mM APMA: 8333.33pg/uL MMP2 in assay buffer. Incubate at 37 degrees C for 60 minutes. Then dilute mixture 1:416.66 in assay buffer (20pg/uL MMP2) and store on ice until ready to dispense into assay plates.
3) Dilute Substrate VI 20 1200nM in assay buffer. Store on ice until ready to dispense into plates.
4) Using LabCyte Echo, transfer 5 nL 10mM test agents (final conc 16.64uM) into assay plate Cols. 5-48, and 5 nL of 100% DMSO (final conc 0.166%) into assay plate Col. 1-4.
5) Using Thermo Scientific MultiDrop Combi dispense 1.5 uL of Assay Buffer in columns 1 and 2, and 1.5 uL of MMP-2 solution to columns 3-48. (final concentration: 10 pg/muL MMP-2)
6) Using Thermo Scientific MultiDrop Combi dispense 1.5uL of substrate VI into all wells (columns 1-48) (final concentration: 600 nM Substrate VI)
7) Spin plates at 1500 rpm for 1 minute on Eppendorf centrifuge 5810.
8) Incubate for 60 minutes at room temp.
9) Read plates on BMG Pherastar at Ex/Em 485/520 nm in fluorescence intensity mode

Comment: Compounds with corrected NZ score >=5 or NZ score<=-5 or % Activity>=15 are to be considered to be active in this assay.

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

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: CHRM1_AG_FLUO8_1536_1X%ACT PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that act as agonists of the human M1 muscarinic receptor (CHRM1; M1). In this assay, CHO-K1 cells stably expressing human M1 are loaded, intracellularly with the calcium indicator dye, Fluo-8, followed by treatment with agonist control or test compounds. As designed, compounds that act as CHRM1 agonists will increase intracellular calcium mobilization, resulting in increased relative fluorescence of the indicator dye and well fluorescence. Compounds are tested in singlicate at a final nominal concentration of 3 uM.

Protocol Summary:

The CHO-hM1 cell line was routinely cultured in T-175 sq cm flasks at 37 C and 95% relative humidity (RH). The growth media consisted of Ham's F-12 Nutrient Media (F-12) supplemented with 10% v/v heat-inactivated qualified fetal bovine serum, 20 mM HEPES, 50 ug/mL Geneticin, and 1X antibiotic mix (penicillin and streptomycin).

The day before the assay 3000 cells in 3 uL of growth media were seeded into each well of 1536 well microtiter plates and allowed to incubate at 37 C, 5% CO2, and 95 % RH for 17-24 hours. Next, 2 uL of the fluorogenic Fluo-8 intracellular calcium indicator mixture (prepared according to the manufacturer's protocol) was added to each well. Plates were then incubated for 1 hour at 37 C, 5% CO2, and 95 % RH, followed by 30 minute incubation at room temperature. Then, 15 nL of test compound in DMSO were dispensed to appropriate wells. The assay was started by performing a basal read of plate fluorescence (470-495 nm excitation and 515-575 nm emission) for 5 seconds on the FLIPR Tetra (Molecular Devices). Then a real time fluorescence measurement was immediately performed for the remaining 140 seconds of the assay. A ratio for each well was calculated to normalize assay data, according to the following mathematical expression:

Ratio = I_Max / I_Min

Where:

I_Max represents the maximum measured fluorescence emission intensity over the 140 second read.
I_Min represents the minimum (basal) measured fluorescence emission intensity before compound was added.

The percent activation was calculated from the median ratio 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 Acetylcholine (EC100) and DMSO.

PubChem Activity Outcome and Score:

A mathematical algorithm was used to determine nominally activating compounds in the primary screen. Two values were calculated for each assay plate: (1) the average percent activation of test compound wells and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter for the entire run, i.e. any compound that exhibited greater % activation than the entire screen's 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 100-1, and for inactive compounds 1-0.

List of Reagents:

Cell line: Chinese Hamster Ovary (CHO) cells containing hM1 receptor; (Conn Lab)
Calcium sensitive dye: Fluo-8 No Wash Calcium Assay Kit; (AAT Bioquest, part 36316)
Growth media: Ham's F-12; 10% FBS, 20mM HEPES, 50 ug/mL G418
Assay media: Ham's F-12, 10% FBS, 20 mM HEPES
Assay plates: Aurora black/clear 1536well FLIPR plate; (Aurora, part 00019326)
Probenecid: 250 mM (pH 8.0); (Sigma P8761)
Agonist: Acetylcholine (50 mM stock in water); Sigma A9187

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: HCS to Identify Inhibitors of Dynein Mediated Cargo Transport on Microtubules: Confirmation Assay, R21NS057026

Protocol: Glucocorticoid Nuclear Hormone Receptor-EGFP (GNHR-EGFP) Translocation HCS Assay Protocol.

Cell Model.
The Mouse mammary adenocarcinoma cell line (3617.4) stably expressing a rat glucocorticoid nuclear hormone receptor green fluorescent fusion protein (GNHR-EGFP) under the control of a tetracycline (tet)-regulated promoter was utilized for the HCS assay (Elbi et al, 2004, Sci STKE. 238:pl10.).
Automated Imaging Platform.
The ArrayScan VTi houses a Zeiss 200M inverted microscope outfitted with 5X/0.25 NA, 10X/0.3NA, 20X/0.4 NA and 40X 0.5 NA Zeiss objectives. Illumination is provided by a full spectrum (300-2000 nM) Hg-halide arc lamp source (EXFO, Quebec, Canada) and fluorescence is detected by a high sensitivity cooled Orca CCD Camera (Photometrics Quantix). The ArrayScan VTi uses an image-based auto-focus system, and has the capability of imaging multi-wavelength fluorescence, up to six excitation and emission channels excited and acquired sequentially. Channel selection is accomplished using a fast excitation filter wheel combined with a multi-band emission filter.

Images were acquired using a 10 x 0.3NA objective in two fluorescent channels using the XF100 filter set for Hoechst in Channel 1 and GFP (FITC) in Channel 2. The ArrayScan VTI was set up to acquire 2 fields of view or 100 valid objects (nuclei) whichever came first.

Image Analysis Algorithm.
The Molecular Translocation algorithm was used to quantify the relative distribution of the GNHR-EGFP between two cellular compartments, the cytoplasm and the nucleus. The nucleic acid dye Hoechst 33342 was used to stain and identify the nucleus, and this fluorescent signal was used to focus the instrument and to define a nuclear mask. The mask is eroded to reduce cytoplasmic contamination within the nuclear area, and the reduced mask is used to quantify the amount of target channel GNHR-EGFP fluorescence within the nucleus. The nuclear mask was then dilated to cover as much of the cytoplasmic region as possible without going outside the cell boundary. Removal of the original nuclear region from this dilated mask creates a ring mask that covers the cytoplasmic region outside the nuclear envelope. The image analysis algorithm outputs quantitative data such as the total or average fluorescent intensities of the GNHR-EGFP signal in the nucleus (Circ) or cytoplasm (Ring) on a per cell basis, that may also be reported as an overall well averaged value.

Detailed Protocol of the GNHR-translocation Assay.

A. Mouse mammary adenocarcinoma cell line 3617.4 Culture Media:
DMEM + 10% FBS + 100 uM Nonessential amino acids 100 uM + 1 mM Sodium Pyruvate + 2 mM L-Glutamine + Penicillin-streptomycin + G418/Geneticin 0.96 mg/mL + Tetracycline 5 mg/mL.

B. Mouse mammary adenocarcinoma cell line 3617.4 Induction Media
DMEM + 10% charcoal stripped FBS + 100 uM Nonessential amino acids 100 uM + 1 mM Sodium Pyruvate + 2 mM L-Glutamine + Penicillin-streptomycin + G418/Geneticin 0.96 mg/mL.

GNHR-EGFP Translocation Protocol
1. Passage and maintain 3617.4 GNHR-EGFP-GR cells in culture medium with Tetracycline and Geneticin.
2. Aspirate culture media and incubate in Induction medium (no Tet) for 15 min at 37 C, 5% CO2 and 95% humidity
3. Wash cells three times with HBSS and harvest cells by trypsinization.
4. Centrifuge cells at 500 x g for 5 min and re-suspend in 10 mL induction medium
5. Count viable cells via trypan exclusion using a haemocytometer
6. Re-suspend cells to 41,666 cells/mL in GNHR-EGFPInduction medium
7. Add 60 uL of cell suspension per well to the 384-well black walled clear bottom plates (Greiner Bio-one Cat # 781091) for a final density of 2500 cells/well using the Zoom dispenser.
8. Incubate plates for 48 hrs at 37 C, 5% CO2 and 95% humidity
9. Transfer 20 uL of pre-diluted compounds to assay plates using the Vprep or EP3 liquid handler outfitted with a 384-well transfer head
10. Incubate plates for 1.0 hrs at 37 C, 5% CO2 and 95% humidity
11. Add 20 uL of 5.0 uM Dexamethasone (final in well 1.0 uM) to assay plates using the Vprep or EP3 liquid handler outfitted with a 384-well transfer head.
12. Incubate for 30 min at 37 C, 5% CO2 and 95% humidity.
13. Aspirate media and fix cells for 10 min with 50 uL 3.7% Formaldehyde, 2 ug/mL (final) Hoechest 33342 in PBS without Ca2+ and Mg2+, pre-warmed to 37 C.
14. Aspirate fixation solution and wash cells 1x with 50 uL PBS.
15. Aspirate PBS, add 50 uL PBS and seal plate on Abgene plate sealer.
16. Acquire images on the ArrayScan VTI and perform image analysis using the established Molecular Translocation algorithm.

Comment: HCS Parameters Reported:
1. MCRAID-CH2, Mean Circ (Nucleus) - Ring (Cytoplasm) Average GNHR-EGFP Intensity Difference in Channel 2
2. MRAI-CH2, Mean Ring (Cytoplasm) Average GNHR-EGFP Intensity in Channel 2
3. MCAI-CH2, Mean Circ (Nucleus) Average GNHR-EGFP Intensity in Channel 2
4. SCCPVF, Selected cell counts per valid field of view
Confirmation of inhibition of Dex-induced GNHR-EGFP translocation in 2 independent tests at 20 uM.
Duplicate GNHR Confirmation Activity Scoring System
PUBCHEM_ACTIVITY_OUTCOME
1 - Substance is considered inactive when
%inhibition of MCRAID-CH2 < 50% in both runs AND
Z-score_MRAI-CH2 > 4 in one of two runs OR
Z-score_MCAI-CH2 >4 in one of two runs OR
Z-score_SCCPVF < -4 in one of two runs


2 - Substance is considered active when
%inhibition of MCRAID-CH2 >= 50% in both runs AND
Z-score_MRAI-CH2 <= 4 in both runs AND
Z-score_MCAI-CH2 <= 4 in both runs AND
Z-score_SCCPVF >=-4 in both runs
3 - Substance activity outcome is inconclusive when
%inhibition of MCRAID-CH2 >= 50% in one of two runs AND

Z-score_MRAI-CH2 <= 4 in both runs AND
Z-score_MCAI-CH2 <= 4 in both runs AND
Z-score_SCCPVF >=-4 in both runs
PUBCHEM_ACTIVITY_SCORE
If %inhibition of MCRAID-CH2 < 50% in both runs AND
Z-score_MRAI-CH2 > 4 in one of two runs OR
Z-score_MCAI-CH2 >4 in one of two runs OR
Z-score_SCCPVF < -4 in one of two runs, then the score is 0

If %inhibition of MCRAID-CH2 >= 50% in both runs AND
Z-score_MRAI-CH2 <= 4 in both runs AND
Z-score_MCAI-CH2 <= 4 in both runs AND
Z-score_SCCPVF >= -4 in both runs, the score is 45
If %inhibition of MCRAID-CH2 >= 50% in one of two runs AND

Z-score_MRAI-CH2 <= 4 in both runs AND
Z-score_MCAI-CH2 <= 4 in both runs AND
Z-score_SCCPVF >=-4 in both runs, the score is 40

Follow-up Concentration Response IC50 < 20 uM in the GNHR-EGFP translocation assay.
Structural Verification
Indications of Specificity & Selectivity ~ PubChem X-target Query
Evidence of SAR.

Secondary Testing Paradigm.

Confirmed concentration dependent actives in the GNHR-EGFP translocation assay would be counter screened in the p53 translocation after mild DNA damage assay to confirm that they inhibited dynein-mediated transport events.

Dr. Day plans to implement the necessary biochemical screens to confirm that a small molecule's molecular target is indeed cytoplasmic dynein. These include microtiter plate-based colorimetric, turbidimetric and fluorescence polarization analyses of the direct action of library chemicals on recombinant dynein heavy chain, glucocorticoid receptor ligand binding domain, HSP70 and HSP90, and on isolated myosin and tubulin.

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: 2147-01_Inhibitor_SinglePoint_HTS_Activity

Protocol: Protocol:
The FadD28 is purified through Ni-NTA affinity column due to a 6x Histidine tag. Purified protein is stored at -80 degrees C (D.J. Wilson, and C.C. Aldrich. Anal. Biochem. 404 (2010) 56-63)
I. Solution preparation (For a run of 121 plates):
1) Prepare 700mL of 200nM compound 11 (TAMRA labeled substrate) in FP buffer. Take 1.4mL of 100uM compound 11 in 100% DMSO in 700mL FP buffer
2) Prepare 50mL compound24 (non-labeled substrate) in FP buffer. Take 200uL of 10mM compound 24 in 100% DMSO in 50mL FP buffer
3) Prepare 350mL 4uM FadD28 in FP buffer. Take 1.746mL of 802uM FadD28 in 350mL FP buffer
II. Setup reagents on automation instrument
1) Add the solutions of compound24, FadD28 and FP buffer to the bottles of BioRaptr (Beckman) based on the dispense table
2) Add compound 11 solution to the bottle of combi NL (Thermo Scientific)
3) Add 1536-well assay ready plates (ARPs) to incubator
III. Run automation protocol
1) Dispense 3uL/well of the solutions from BioRaptr based on the dispense table to 1536-well assay ready plates (ARPs) (Aurora, Cat#: 00019180BX)(Positive control wells receive 1.5uL/well of compound24 and 1.5uL/well of FadD28 solution; all the other wells receive 1.5uL/well of FP buffer and 1.5uL/well of FadD28 solution)
2) Incubate the plates for 10mins at 25 degrees C
3) Dispense 3uL/well of 200nM compound11 solution to the plates by Combi NL
4) Incubate the plates for 30mins at 25 degrees C
5) Read the plates on ViewLux (PerkinElmer) with excitation wavelength of 525nm, emission wavelength of 598 nm and dichroic mirror of 550nm
Final concentration: 100nM compound11, 1uM FadD28, 10uM compound24 (positive control), compound concentration for primary screen: 12.5uM
FP buffer: 30mM Tris-HCl, pH7.5, 1mM MgCl2, 1mM DTT, 0.0025% Igepal CA630

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

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.

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


PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the maximum 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 8.

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

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

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

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

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

External ID: SBCCG-A997-CSN5-thrombin-CP-Assay

Protocol: Assay materials:
1) Ub4-pepOG protein substrate was provided by Dr. Deshaies laboratory
2) Thrombin from bovine plasma (Cat # T4648) was purchased from Sigma-Aldrich.
3) Epoxomicin (Cat # BML-PI127-0100) was purchased from BioMol/ENZO Life Sciences
4) Assay Buffer: 50 mM Tris-HCl pH 8.0 , 0.05 mM ATP, 0.05 mM Magnesium Chloride,
1 mM DTT, 0.01% NP-40
5) Epoxomicin Solution: 1.5 muM Epoxomicin in 1x Assay Buffer
6) Aurora 1536 well black high base plate were obtained from Nexus Biosystems (Cat # 00019120BX)

Procedure for validation assay:
1) Using LabCyte Echo, transfer 5 nL 10mM test agents (final conc 16.64uM) into assay plate Cols. 5-48, and 5 nL of 100% DMSO (final conc 0.166%) into assay plate Col. 1-4.
2) Pre-incubate 160 U/mL Thrombin at room temperature for 1 hour, then dilute 10-fold in pre-chilled 1x Assay Buffer.
3) Using Thermo Scientific MultiDrop Combi dispense 1.5uL of Assay Buffer in columns 1 and 2, and 1.5uL of Thrombin solution to columns 3-48. (final concentration: 8 U/mL Thrombin).
4) Using Thermo Scientific MultiDrop Combi dispense 2 mul of Ub4-pepOG substrate into all wells (columns 1-48) (final concentration: 10 nM Ub4-pepOG).
5) Spin plates at 1500 rpm for 1 minute on Eppendorf centrifuge 5810.
6) Incubate for 80 minutes at room temp.
7) Read plates on Perkin Elmer Envision with dual mirror at Ex/Em 480/535 nm in fluorescence polarization mode

Comment: Compounds with corrected %Activity >=15% and 0.5 =< FRatio <= 1.5 are considered to be active in this assay.

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

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: CHRM1_PAM_FLUO8_1536_1X%ACT PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that act as positive allosteric modulators (PAMs) and increase activity of the human M1 muscarinic receptor (CHRM1; M1) in cells pre-treated with a known agonist. In this assay, CHO-K1 cells stably expressing human M1 are loaded with the Fluo-8 calcium indicator dye, followed by addition of test compounds and subsequent treatment with the activator acetylcholine at a concentration that results in 20% activation (EC20). As designed, compounds that act as CHRM1 PAMs will increase calcium mobilization, resulting in increased intracellular calcium and relative fluorescence of the indicator dye beyond that of the EC20 of acetylcholine. Compounds are tested in singlicate at a final nominal concentration of 3 micromolar.

Protocol Summary:

The CHO-hM1 cell line was routinely cultured in T-175 sq cm flasks at 37 degrees C and 95% relative humidity (RH). The growth media consisted of Ham's F-12 Nutrient Media (F-12) supplemented with 10% v/v heat-inactivated qualified fetal bovine serum, 20 mM HEPES, 50 micrograms/mL Geneticin, and 1X antibiotic mix (penicillin and streptomycin).

The day before the assay 3000 cells in 3 microliters of growth media were seeded into each well of 1536 well microtiter plates and allowed to incubate at 37 degrees C, 5% CO2, and 95 % RH for 17-24 hours. Next, 2 microliters of the fluorogenic Fluo-8 intracellular calcium indicator mixture (prepared according to the manufacturer's protocol) was added to each well. Plates were then incubated for 1 hour at 37 degrees C, 5% CO2, and 95 % RH, followed by 30 minute incubation at room temperature. Then, 15 nL of test compound in DMSO were transferred to appropriate wells. The assay was started by performing a basal read of plate fluorescence (470-495 nm excitation and 515-575 nm emission) for 5 seconds on the FLIPR Tetra (Molecular Devices) prior to all wells being treated with an EC20 concentration of acetylcholine. Then a real time fluorescence measurement was immediately performed for the remaining 140 seconds of the assay. A ratio for each well was calculated to normalize assay data, according to the following mathematical expression:

Ratio = I_Max / I_Min

Where:

I_Max represents the maximum measured fluorescence emission intensity over the 140 second read and;
I_Min represents the minimum (basal) measured fluorescence emission intensity before compound was added.

The percent activation was calculated from the median ratio 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 Acetylcholine at EC20 and DMSO.
High_Control is defined as wells containing Acetylcholine (EC100) and DMSO.

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

PubChem Activity Outcome and Score:

The inactive compounds of this assay have an activity score range of 0 to 78 and the active compounds have an activity score range of 50 to 100.

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

List of Reagents:

Cell line: Chinese Hamster Ovary (CHO) cells containing hM1 receptor; (Conn Lab)
Calcium sensitive dye: Fluo-8 No Wash Calcium Assay Kit; (AAT Bioquest, part 36316)
Growth media: Ham's F-12; 10% FBS, 20mM HEPES, 50?g/mL G418
Assay media: Ham's F-12, 10% FBS, 20 mM HEPES
Assay plates: Aurora black/clear 1536well FLIPR plate; (Aurora, part 00019326)
Probenecid: 250mM (pH 8.0); (Sigma P8761)
Potentiator: Acetylcholine (50mM stock in water); Sigma A9187

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: 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: SBCCG-A1015-NR3A-Primary-Assay

Protocol: A.#Brief Description of the Assay
This assay attempts to identify small molecule compound binding to NR3A LBD using differential scanning fluorimetry assay. The assay is performed format using Applied Biosystems 384-well plates (cat #4309849) on ViiA7 qPCR instrument (Thermo-Fisher Scientific).

B.#Assay reagent components:
1.#Assay buffer: 20 mM HEPES, pH 7.5, 200 mM NaCl
2.#NR3A LBD working solution in the assay buffer
3.#NR3A LBD with glycine working solution in the assay buffer
4.#Sypro Orange working solution in the assay buffer
5.#Compounds in 100% DMSO

C.#Step-by-step protocol:
1.#Reagent dispenses
a.#Add compound aliquots to the wells in columns 3-24
b.#Add DMSO aliquots to the wells in columns 1-2
c.#Dispense 5 uL of NR3A LBD working solution into columns 2-24
d.#Dispense 5 uL of NR3A LBD with glycine working solution to column 1
e.#Dispense 5 uL of Sypro Orange with glycine solution to columns 1-24
2.#Perform assay by ramping temperature and measuring concomitant changes in fluorescence
3.#Determine Tm corresponding to the maximum of the first derivative of fluorescence
D.#Final concentration of reagents in the assay wells
1.#1.25 uM NR3A LBD (all wells)
2.#x5 Sypro Orange (all wells)
3.#25 uM tested compounds (wells in columns 3-24)
4.#0.25% DMSO (all wells)
E.#Plate Map:
1.#Positive (high Tm) control in column 1
2.#Negative (low Tm) control in column 2.
3.#Test wells in columns 3-24.

Comment: %Activity = (Melting temperature of compound well - Average melting temperature of negative control well)/(Melting temperature of positive control wells - Melting temperature of negative control wells)*100%.

Compounds that demonstrated %Activity >= 10% at 25 uM concentration are defined as actives in this assay.

Activity Scoring
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 active, then the assigned score is 20

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: PolI100

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

Comment: Compound Ranking:

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

External ID: GDH-TPI_INH_ABS_1536_1X%INH CSRUN

Protocol: Assay Overview:

The purpose of this biochemical counterscreen is to determine whether compounds act as absorbance assay artifacts or are non-selective. This assay also serves as a counterscreen for a set of ongoing high throughput primary experiments entitled, "Absorbance-based biochemical primary high throughput screening assay to identify inhibitors of the aldolase of M. tuberculosis."

This counterscreen is similar in format to the aforementioned assay with the only two following differences: (i) the fructose-1,6-bisphosphate substrate is replaced with glyceraldehyde 3 phosphate, a product of its conversion by FBA and (ii) no (FBA) is used. The counterscreen hence recapitulates the two steps involved in the monitoring of FBA activity through the conversion of FB into the triose product glyceraldehyde 3 phosphate (G3P), which would be converted to dihydroxyacetone phosphate (DHAP) by the helper enzyme triose phosphate isomerase (TPI). A second helper enzyme, glycerophosphate dehydrogenase (GDH), converts the dihydroxyacetone phosphate to glycerol-3-phosphate with the concomitant oxidation of NADH to NAD, which is monitored by measuring the absorbance at 340 nm. In this new assay format, the A340 is independent of FBA activity, hence compounds that reduce absorbance at 340 nm are either absorbance artifacts or helper enzyme inhibitors that will not be pursued. Compounds are tested in singlicate at a final nominal concentration of 4.78 uM.

Protocol Summary:

Prior to the start of the assay, 5 uL /well of Buffer A (50 mM HEPES, 0.01% Triton X-100, 10% Glycerol, pH8.0) supplemented with 400 nM ZnCl2,240 uM NADH and the helper enzymes GDH-TPI (4 U/mL) was dispensed into all wells of a 1536-well plate except the "No enzyme" wells that contained the same supplemented buffer but no GDH-TPI enzymes. Next, 48 nL of test compounds were then delivered in each well using a PinTool. The assay was then initiated by dispensing 5 uL of Buffer A supplemented with 240 uM of the substrate glyceraldehyde-3-phosphate (G3P). Plates were incubated at room temperature for 20 minutes before A340 was measured using the EnVision plate reader (Perkin Elmer).

The percent inhibition for each compound was calculated as follows:

%Inhibition = 100 * ( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control ) )

Where:

Test_Compound is defined as wells treated with test compounds.
Low_Control is defined as wells treated with DMSO.
High_Control is defined as wells with no GDH-TPI enzyme.

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 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 the cutoff parameter was declared active.

PubChem Activity Outcome and Score:

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

The PubChem Activity Score range for active compounds is 100-13, and for inactive compounds 13-0.

List of Reagents:

ZnCl2 (Fisher Scientific, part Z33-500)
NADH (EMD Biosciences, part 481913)
GDH-TPI (Sigma, part G1881)
HEPES (EMD Biosciences, part EM-5310)
Triton X-100 (Sigma, part T8787)
Glycerol (Fisher, part AC327255000)
Glyceraldehyde-3-phosphate (Sigma, part D7137)
1536-well plates (Aurora, part 1091-11020-S)

Comment: Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that quench or emit absorbance 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: PolE100

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

Comment: Compound Ranking:

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

External ID: Concentration-Response Confirmation Assays for HCS to Identify Inhibitors of Dynein Mediated Cargo Transport on Microtubules: R21NS057026

Protocol: Glucocorticoid Nuclear Hormone Receptor-EGFP (GNHR-EGFP) Translocation HCS Assay Protocol.

Cell Model.
The Mouse mammary adenocarcinoma cell line (3617.4) stably expressing a rat glucocorticoid nuclear hormone receptor green fluorescent fusion protein (GNHR-EGFP) under the control of a tetracycline (tet)-regulated promoter was utilized for the HCS assay (Elbi et al, 2004, Sci STKE. 238:pl10.).
Automated Imaging Platform.
The ArrayScan VTi houses a Zeiss 200M inverted microscope outfitted with 5X/0.25 NA, 10X/0.3NA, 20X/0.4 NA and 40X 0.5 NA Zeiss objectives. Illumination is provided by a full spectrum (300-2000 nM) Hg-halide arc lamp source (EXFO, Quebec, Canada) and fluorescence is detected by a high sensitivity cooled Orca CCD Camera (Photometrics Quantix). The ArrayScan VTi uses an image-based auto-focus system, and has the capability of imaging multi-wavelength fluorescence, up to six excitation and emission channels excited and acquired sequentially. Channel selection is accomplished using a fast excitation filter wheel combined with a multi-band emission filter.

Images were acquired using a 10 x 0.3NA objective in two fluorescent channels using the XF100 filter set for Hoechst in Channel 1 and GFP (FITC) in Channel 2. The ArrayScan VTI was set up to acquire 2 fields of view or 100 valid objects (nuclei) which ever came first.

Image Analysis Algorithm.
The Molecular Translocation algorithm was used to quantify the relative distribution of the GNHR-EGFP between two cellular compartments, the cytoplasm and the nucleus. The nucleic acid dye Hoechst 33342 was used to stain and identify the nucleus, and this fluorescent signal was used to focus the instrument and to define a nuclear mask. The mask is eroded to reduce cytoplasmic contamination within the nuclear area, and the reduced mask is used to quantify the amount of target channel GNHR-EGFP fluorescence within the nucleus. The nuclear mask was then dilated to cover as much of the cytoplasmic region as possible without going outside the cell boundary. Removal of the original nuclear region from this dilated mask creates a ring mask that covers the cytoplasmic region outside the nuclear envelope. The image analysis algorithm outputs quantitative data such as the total or average fluorescent intensities of the GNHR-EGFP signal in the nucleus (Circ) or cytoplasm (Ring) on a per cell basis, that may also be reported as an overall well averaged value.

Detailed Protocol of the GNHR-translocation Assay.

A. Mouse mammary adenocarcinoma cell line 3617.4 Culture Media:
DMEM + 10% FBS + 100 uM Nonessential amino acids 100 uM + 1 mM Sodium Pyruvate + 2 mM L-Glutamine + Penicillin-streptomycin + G418/Geneticin 0.96 mg/mL + Tetracycline 5 mg/mL.

B. Mouse mammary adenocarcinoma cell line 3617.4 Induction Media
DMEM + 10% charcoal stripped FBS + 100 uM Nonessential amino acids 100 uM + 1 mM Sodium Pyruvate + 2 mM L-Glutamine + Penicillin-streptomycin + G418/Geneticin 0.96 mg/mL.

GNHR-EGFP Translocation Protocol
1. Passage and maintain 3617.4 GNHR-EGFP-GR cells in culture medium with Tetracycline and Geneticin.
2. Aspirate culture media and incubate in Induction medium (no Tet) for 15 min at 37 C, 5% CO2 and 95% humidity
3. Wash cells three times with HBSS and harvest cells by trypsinization.
4. Centrifuge cells at 500 x g for 5 min and re-suspend in 10 mL induction medium
5. Count viable cells via trypan exclusion using a haemocytometer
6. Re-suspend cells to 41,666 cells/mL in GNHR-EGFPInduction medium
7. Add 60 uL of cell suspension per well to the 384-well black walled clear bottom plates (Greiner Bio-one Cat # 781091) for a final density of 2500 cells/well using the Zoom dispenser.
8. Incubate plates for 48 hrs at 37 C, 5% CO2 and 95% humidity
9. Transfer 20 uL of pre-diluted compounds to assay plates using the Vprep or EP3 liquid handler outfitted with a 384-well transfer head
10. Incubate plates for 1.0 hrs at 37 C, 5% CO2 and 95% humidity
11. Add 20 uL of 5.0 uM Dexamethasone (final in well 1.0 uM) to assay plates using the Vprep or EP3 liquid handler outfitted with a 384-well transfer head.
12. Incubate for 30 min at 37 C, 5% CO2 and 95% humidity.
13. Aspirate media and fix cells for 10 min with 50 uL 3.7% Formaldehyde, 2 ug/mL (final) Hoechest 33342 in PBS without Ca2+ and Mg2+, pre-warmed to 37 C.
14. Aspirate fixation solution and wash cells 1x with 50 uL PBS.
15. Aspirate PBS, add 50 uL PBS and seal plate on Abgene plate sealer.
16. Acquire images on the ArrayScan VTI and perform image analysis using the established Molecular Translocation algorithm.

Comment: GNHR duplicate IC50 confirmation PubChem scoring system:
PUBCHEM_ACTIVITY_OUTCOME
1 - Substance is considered inactive when IC50s of two runs > 50uM and the mean IC50#s is > 50 uM
2 - Substance is considered active when IC50s of two runs < 50uM and the mean IC50#s is < 50uM
3 - Substance is considered inconclusive when IC50 of one run > 50uM
PUBCHEM_ACTIVITY_SCORE
20 - Compounds that were inactive in duplicate 10-pt dose response assays with a mean IC50 > 50 uM.
40 - Compounds that were active in one of the duplicate 10-pt dose response assays with an IC50 >25 uM but exhibited an IC50 < 50 uM in the other runs.
50 - Compounds that were active in duplicate 10-pt dose response assays with a mean IC50 in the 25 to 50 uM range
60 - Compounds that were active in duplicate 10-pt dose response assays with a mean IC50 in the 15 to 25 uM range
70 - Compounds that were active in duplicate 10-pt dose response says with a mean IC50 in the 10 to 15 uM range
80 - Compounds that were active in duplicate 10-pt dose response assays with a mean IC50 in the 5 to 10 uM range
90 - Compounds that were active in duplicate 10-pt dose response assays with a mean IC50 in the 0 to 5 uM range

Secondary Testing Paradigm.

Confirmed concentration dependent actives in the GNHR-EGFP translocation assay would be counter screened in the p53 translocation after mild DNA damage assay to confirm that they inhibited dynein-mediated transport events.

Dr Day plans to implement the necessary biochemical screens to confirm that a small molecule's molecular target is indeed cytoplasmic dynein. These include microtiter plate-based colorimetric, turbidimetric and fluorescence polarization analyses of the direct action of library chemicals on recombinant dynein heavy chain, glucocorticoid receptor ligand binding domain, HSP70 and HSP90, and on isolated myosin and tubulin.

External ID: PolK100

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

Comment: Compound Ranking:

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

External ID: SMAD3201

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

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

Comment: Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description".

2. For all inactive (non-toxic) compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active (toxic) compounds, a score range was given for each curve class type given above. Active (toxic) compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.

External ID: 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: 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: CHOCYTOX_INH_LUMI_1536_3X%INH CSRUN

Protocol: Assay Overview:

The purpose of this assay is to determine whether compounds identified as active in a set of previous experiments entitled, "Fluorescence-based cell-based primary high throughput screening assay to identify inhibitors of TLR9-MyD88 binding" (AID 504734) are cytotoxic to CHO cells, or are non-selective due to their inhibition of cell viability/growth. In this assay the cell line used in the primary HTS assay, TLR9 MyD88 CHO cells, are incubated with test compounds, followed by determination of cell viability. The assay utilizes the CellTiter-Glo luminescent reagent to measure intracellular ATP in viable cells. Luciferase present in the reagent catalyzes the oxidation of beetle luciferin to oxyluciferin and light in the presence of cellular ATP. Well luminescence is directly proportional to ATP levels and cell viability. As designed, compounds that reduce cell viability will reduce ATP levels, luciferin oxidation and light production, resulting in decreased well luminescence. Doxurubicin was used as the positive control. Compounds are tested in triplicate at a final nominal concentration of 4.98 uM.

Protocol Summary:

The stably transfected TLR9 MyD88 CHO cells were cultured in T-175 sq cm flasks at 37 C and 95% relative humidity (RH). The growth media consisted of Dulbecco's Modified Eagle's Media (DMEM) containing 10% v/v fetal bovine serum, 0.1 mM NEAA, 1 mM Sodium Pyruvate, 10 mM HEPES, 5 mM L-Glutamine, 200 ug/mL geneticin (G418) and 1X Penicillin/Streptomycin.

Prior to the start of the assay, cells were suspended to a concentration of 100,000 cells/ml in DMEM (Phenol Red Free), containing 10% v/v fetal bovine serum, 0.1 mM NEAA, 1 mM Sodium Pyruvate, 10 mM Hepes, 5 mM L-Glutamine and 1X Penicillin/Streptomycin. To start the assay, 5 uL of assay media was dispensed into the first two columns of a 1536 well plate and 5 ul of cell suspension to the remaining wells (500 cells per well). The assay was started immediately by dispensing 25 nL of test compound in DMSO, Doxurubicin (50 uM final concentration) or DMSO alone (0.5% final concentration) to the appropriate wells. The plates were then incubated for 48 hours at 37 C, 5% CO2 and 95% RH).

Following the two day incubation, plates were equilibrated to room temperature for 10 minutes and 5 ul of CellTiter-Glo reagent was added to each well. Plates were centrifuged and incubated at room temperature for 10 minutes. Well luminescence was measured on the ViewLux plate reader.

The percent inhibition for each compound was calculated as follows:

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

Where:

Test_Compound is defined as wells containing test compound.
Low_Control is defined as wells containing 0.5% DMSO. (0% inhibition)
High_Control is defined as wells containing 50 uM doxorubicin in 0.5% DMSO (100% inhibition).

The average percent inhibition and standard deviation of each compound tested were calculated. In addition, two values were calculated as well: (1) the average percent inhibition of the wells containing DMSO only, and (2) three times their standard deviation. The sum of these two values was used as a hit cutoff parameter. Any compound that exhibited a percent inhibition greater than the calculated hit cutoff 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 100-10, and for inactive compounds 9-0.

List of Reagents:

Doxorubicin (Sigma, part D1515)
DMEM, High Glucose (Invitrogen, part 11965-092)
DMEM, High Glucose, Phenol Red Free (Invitrogen, part 21063-029)
Fetal Bovine Serum (Invitrogen, part 26140-079)
NEAA (Invitrogen, part 11140-050)
Hepes (Invitrogen, part 15630-080)
Pen/Strep (Invitrogen, part 15640-055)
L-Glutamine (Invitrogen, part 25030-081)
Sodium Pyruvate (Invitrogen, part 11360-070)
Geneticin (Invitrogen, part 10131027)
T-175 Flasks (Nunc, part 159910)
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 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. The MLSMR was not able to provide all compounds selected for testing in this assay.

External ID: SBCCG-A764-CF-PAF-Primary-Assay

Protocol: Assay Materials:
KKLEB-NFkB-GFP cells (Assay Provider)
PAF(Assay Provider)
Fetal Bovine Serum (Hyclone SH30396.03)
Penicillin Streptomycin solution
L-glutamine (100X)
TrypLE (Invitrogen 12563)
DPBS without calcium and magnesium (1X)
Corning culture flasks
Black CellBind 1536-well plates (Corning 3833)
ATPlite (Perkin Elmer 6016739)

I. Cell Suspension
1- Dispense 3 uL/well of cells at 5X10;5 cells/mL to the whole plate (plate cells in 2% FBS assay media).
2- Spin down plates on Eppendorf centrifuge 5810 at 500 rpm for 1 minute.

II. Compound Addition:
3- Transfer test compounds to columns 5-48 and DMSO to columns 1-4 using the Labcyte ECHO 555.
4- Transfer volume of test compound and DMSO is 15nL, making 5uM compound concentration at 0.25% DMSO final.
5-Spin down plates on Vspin at 1000 rpm for 1 minute.
6-Put Kalypsys metal lids on plates, incubate plates at 37 degrees C with 5% CO2 for 2 hours.

III. Reagent Addition
7- Dispense 3 uL/well of serum free assay media to columns 1 and 2.
8- Dispense 3 uL/well of PAF (dilute in serum free assay media) to columns 3-48.
9- Spin down plates without lids on Vspin at 2000 rpm for 2 min
10- Put Kalypsys metal lids on plates, and incubate plates at 37 degrees C with 5% CO2 overnight.

IV. Reading plates:

11-Spin plates upside down with a container at 1000 rpm for 15 sec. Dab them with a tissue to dry them and Read immediately on envision for GFP fluorescence.
12-Dispense 6 uL/well of ATPlite (diluted in DPBS 1:1).
13-Spin down plates on Eppendorf centrifuge 5810 at 2000 rpm for 2 minutes without lids.
14-Incubate plates for 10 min at RT and run Luminescence read on Viewlux.

Comment: Compounds that demonstrated a corrected %Activity of >= 50% at 5 uM concentration are defined as actives in this assay.

The experimental values were normalized by the difference between values from neutral and stimulator control wells in each plate. Then normalized data was corrected to remove systematic plate patterns due to artifacts such as dispensing tip issues etc. Further information about data correction is available at http://www.genedata.com/products/screener.html.

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: 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: CHOCYTOX_INH_LUMI_1536_3XIC50 DCSRUN

Protocol: Assay Overview:

The purpose of this assay is to determine whether compounds identified as active in a set of previous experiments entitled, "Fluorescence-based cell-based primary high throughput screening assay to identify inhibitors of TLR9-MyD88 binding" (AID 504734); that confirmed activity in a set of experiments entitled, "Fluorescence-based cell-based high throughput confirmation assay for inhibitors of TLR9-MyD88 binding" (AID 540250); and that were inactive in a set of experiments entitled, "Counterscreen for inhibitors of TLR9-MyD88 binding: fluorescence-based cell-based high throughput assay to identify non-selective inhibitors of the beta-lactamase enzyme (BLA)" (AID 540251) were non-selective or assay artifacts due to inhibition of CHO cell viability or growth.

In this assay, the cell line used in the primary HTS assay, TLR9 MyD88 CHO cells, are incubated with test compounds, followed by determination of cell viability. The assay utilizes the CellTiter-Glo luminescent reagent to measure intracellular ATP in viable cells. Luciferase present in the reagent catalyzes the oxidation of beetle luciferin to oxyluciferin and light in the presence of cellular ATP. Well luminescence is directly proportional to ATP levels and cell viability. As designed, compounds that reduce cell viability will reduce ATP levels, luciferin oxidation and light production, resulting in decreased well luminescence. Compounds are tested in triplicate using a 10-point, 1:3 dilution series, starting at a nominal test concentration of 49.8 uM.

Protocol Summary:

The stably transfected TLR9 MyD88 CHO cells were cultured in T-175 sq cm flasks at 37 C and 95% relative humidity (RH). The growth media consisted of Dulbecco's Modified Eagle's Media (DMEM) containing 10% v/v dialyzed fetal bovine serum, 0.1 mM NEAA, 1 mM Sodium Pyruvate, 10 mM HEPES, 5 mM L-Glutamine, 500 ug/mL geneticin (G418) and 1X Penicillin/Streptomycin.

Prior to the start of the assay, cells were suspended to a concentration of 100,000 cells/ml in DMEM (Phenol Red Free), containing 10% v/v fetal bovine serum, 0.1 mM NEAA, 1 mM Sodium Pyruvate, 10 mM Hepes, 5 mM L-Glutamine and 1X Penicillin/Streptomycin. To start the assay, 5 uL of assay media was dispensed into the first two columns of a 1536 well plate and 5 ul of cell suspension to the remaining wells (500 cells per well). The assay was started immediately by dispensing 25 nL of test compound in DMSO, Doxurubicin (50 uM final concentration) or DMSO alone (0.5% final concentration) to the appropriate wells. The plates were then incubated for 48 hours at 37 C, 5% CO2 and 95% RH).

Following the two day incubation, plates were equilibrated to room temperature for 10 minutes and 5 ul of CellTiter-Glo reagent was added to each well. Plates were centrifuged and incubated at room temperature for 10 minutes. Well luminescence was measured on the ViewLux plate reader. The percent inhibition for each compound was calculated as follows:

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

Where:

Test_Compound is defined as wells containing test compound.
Low_Control is defined as wells containing 0.5% DMSO. (0% inhibition)
High_Control is defined as wells containing 50 uM doxorubicin in 0.5% DMSO (100% inhibition).

For each test compound, percent inhibition was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (Symyx Technologies Inc). The reported IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% inhibition level of the Y-intercept value. In cases where the highest concentration tested (i.e. 49.8 uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 49.8 uM.

PubChem Activity Outcome and Score:

Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 uM were considered active.

Any compound with a percent activity value < 50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >= 50% at any test concentration was assigned an activity score greater than zero. Activity score was then ranked by the potency, with the most potent compounds assigned the highest activity scores.

The PubChem Activity Score range for active compounds is 100-81, and for inactive compounds 80-0.

List of Reagents:

Stably-transfected CHO cell line coexpressing TLR9-Bla(b) and MyD88-Bla(a) (supplied by Assay Provider)
Cell Titer Glo (Promega, part G7573)
Doxorubicin (Sigma Chemical, part D1515)
DMEM, High Glucose (Invitrogen, part 11965-092)
DMEM, High Glucose, Phenol Red Free (Invitrogen, part 21063-029)
Fetal Bovine Serum (Invitrogen, part 26140-079)
NEAA (Invitrogen, part 11140-050)
Hepes (Invitrogen, part 15630-080)
Pen/Strep (Invitrogen, part 15640-055)
L-Glutamine (Invitrogen, part 25030-081)
Sodium Pyruvate (Invitrogen, part 11360-070)
Geneticin (Invitrogen, part 10131027)
T-175 Flasks (Nunc, part 159910)
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 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. The MLSMR was not able to provide all compounds selected for testing in this assay.