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

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

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

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

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

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

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

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

External ID: HMS979

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

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

Comment: Data analysis description:

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

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

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

External ID: 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: 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: PROCASPASE3_ACT_EPIABS_1536_1X%ACT PRUN

Protocol: Assay Overview:

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

Protocol Summary:

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

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

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

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

Where:

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

The percent activation for each compound was calculated as follows:

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

Where:

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

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

PubChem Activity Outcome and Score:

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

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

List of Reagents:

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

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

External ID: 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: 2084-01_Activator_SinglePoint_HTS_Activity

Protocol: MITF Primary Screening Protocol
(TRPM-1 Promoter//Luciferase reporter assay)

Day 1, plate cells 2000 per well in 30 uL media (phenol red free DMEM/10% iFBS/Pen/Strep/L-Glutamine)

Day 2, pin 100 nL into 30 uL assay volume in white, opaque Corning 8867 barcoded 384 well plates. (will also require sentinel pinning with the positive control, parthenolide)
Incubate 24 hours at 37 degrees C in Liconic incubator

Day 3, add 20 uL 100% Promega Steady glo with Thermo Combi fluid transfer apparatus.
Shake 15 seconds on "big bear" plate shaker.
Incubate at RT for 5 minutes.
Read on Perkin-Elmer Envision with US LUM settings for 0.5 sec per well

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

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

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the mean of the normalized and corrected sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 80.

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

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

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: 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: 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: CHRM1_ANT_FLUO8_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that act as antagonists and decrease activity of the human M1 muscarinic receptor (CHRM1; M1) that have been pre-treated with a known agonist, with the end result being a decrease in intracellular calcium. In this assay, CHO-K1 cells stably expressing human M1 are loaded with the Fluo-8 calcium indicator dye. Compounds are added followed by treatment with the activator acetylcholine at a concentration that results in 80% activation (Ec80). As designed, compounds that act as CHRM1 antagonists will decrease calcium mobilization, resulting in decreased relative fluorescence of the indicator dye below that of the Ec80 of acetylcholine. 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 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 EC80 concentration of acetylcholine. Then a real time fluorescence measurement was immediately performed for the remaining 140 seconds of the assay.

Hits for this assay were determined 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 inhibition was calculated from the median ratio as follows:

%_Inhibition = ( 1 - ( Ratio Test_Compound - Median_Ratio_High_Control ) / ( Median_Ratio_Low_Control - Median_Ratio_High_Control ) ) ) * 100

Where:

Test_Compound is defined as wells containing test compound.
Low_Control is defined as wells containing Ec80 of acetylcholine and DMSO.
High_Control is defined as wells containing DMSO.

PubChem Activity Outcome and Score:

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

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

The PubChem Activity Score range for active compounds is 100-7, and for inactive compounds 80-0.

In this assay not all plates were run in the same batch. This resulted in batch-to-batch variation among the different batches of plates, thereby necessitating the use of a plate-based activity cutoff. For this reason the inactive and active scores overlap.

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: 250mM (pH 8.0); (Sigma P8761)
Potentiator: 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: HMS1364-MLP_WT

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

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

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

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

External ID: FBW7_ACT_ALPHA_1536_1X%ACT PRUN

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

The percent activation for each compound was calculated as follows:

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

A mathematical algorithm was used to determine active compounds. Two values were calculated: (1) the average percent activation of all compounds tested for the screen, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater percent activation than the cutoff parameter (1.85% in the case here) was declared active.
The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.
The activity score range for active compounds is 100-1, for inactive 1-0.
List of Reagents:
Ni Beads- PerkinEmer Lifesciences Cat#6760619R
RLFbw7-Assay Provider
WTFbw7-Assay Provider
Cyclin E peptide-Assay Provider
5M NaCl- Sigma Cat# S6546-1L
Tween20- Fisher Cat# BP337
Tris 1M pH7.4 Research Organics Cat# 9686T
BSA-Sigma Cat#A7030
?-Mercaptoethanol-SigmaM6250
1536-well plates (Corning, part 7254)

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

External ID: FLUC100

Protocol: NCGC Assay Protocol Summary:

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

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

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

Comment: Compound Ranking:

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

External ID: HMS1364-MLP_ugtP

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

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

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

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

External ID: SMAD3101

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. The plates were measured on an Acumen eX3 Explorer plate reader for GFP fluorescence (ex488/em500-530). GFP values were calculated by determining the mean GFP fluorescence of individual cells, and compiling these values for each well to determine a total well GFP signal. The %Activity was determined from the corrected fluorescence values. A titration of the known TGF-B inhibitor SB431542 was included to monitor plate performance, while unstimulated HEPG2 (-TGF-B) control wells were used to normalize %Activity of identified inhibitors; unstimulated wells corresponded to 100%Activity (full inhibition), while stimulated cell controls (+DMSO) were used to normalize 0%Activity (no inhibition).

Concentration-response curves were fitted to the signals arising from the resulting fluorescence. 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. Active inhibitors showed concentration-dependent decreases in GFP fluorescence, concordant with a decrease in TGF-B/SMAD3-driven GFP expression. Inactive compounds showed no effect on fluorescence signal.

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: HMS704

Protocol: Prior to screening, a wildtype strain of S. aureus RN4220 was freshly transformed with the plasmid pMS182 encoding constitutively expressed GFP and a DeltatarO strain of RN4220 was transformed with the plasmid pMS183 encoding constitutively expressed mCherry. Following transformation, both plasmids were maintained with chloramphenicol (Cm) at a concentration of 10 micrograms/mL. The day before screening, both strains were grown overnight (~24 h) in sterile-filtered tryptic soy broth (TSB) with 10 micrograms/mL chloramphenicol at 30 degrees C with shaking.

On the day of screening, columns 1-23 of assay plates (Corning 3710) were prefilled with 40 uL of TSB. Next, 300 nL of each compound were pin-transferred to each plate (final assay concentration of test compound was 18.75 micrograms/mL). For every compound library plate, there were four daughter plates (A & B containing mCherry-labeled DeltatarO and C & D containing GFP-labeled wild type). To equilibrate the number of cells, the overnight cultures of RN4220 and RN4220 DeltatarO were diluted to an OD600 = 2 and then diluted 1000x into TSB (Cm 10). 40 uL of the diluted cultures were added to columns 1-22 of the corresponding assay plates. Wells in column 23 contained only medium and served as the negative controls. Column 24 was filled with the positive control (erythromycin at 20 micrograms/mL). Final assay well volume was 80 uL. Plates were stacked 5 high, covered with lids (Corning 3009), and incubated at 30 degrees C overnight (~18 h).

The following day, assay plates were read using a PerkinElmer EnVision (600 nm filter).

Comment: OD600 values were normalized to the positive and negative controls to determine a normalized percent survival. Based on the normalized OD600 values, a substance was considered active with >50% survival for the DeltatarO strain (activity outcome = 1 for assay 2) and <10% survival for the wild type strain (activity outcome = 2 for assay 1). Activity scores were calculated using average % survival for each strain. Average % survival <= 0 was scored as 100 for activity; average % survival >= 100 was scored as 0 for activity. Average % survival between 0 and 100 was subtracted from 100 to generate activity scores for intermediate values (i.e. average % survival = 40 corresponds to an activity score of 60).

Note that since this is treated as a panel assay, the query for active compounds includes many that were considered active only for the DeltatarO mutant strain or for both strains. The final activity outcome (reduced survival for the wild type strain only) is a small subset of this list, based on activity against the wild type strain but not against the DeltatarO mutant strain.

External ID: SBCCG-A754-STEP-Primary-Assay

Protocol: STEP Assay HTS Protocol

A. Brief Description of the Assay:
This assay idenitfies inhibitors of STEP (STriatal-Enriched Phosphatase) enzyme. It is measured via fluorescence in 1536-well plate format.

B. Materials:
Item, Source, Cat #
STEP Enzyme Stock Solution 6.2mg/mL (178uM), Dr. Lutz Tautz, N/A
Bis-Tris, Fisher Sci, BP301-100
Tween 20, Sigma, P1379
DTT, Sigma, D9779
OMFP, Sigma, M2629-100MG
Mol. Grade Water, Mediatech, Inc., 46-000-CM
1536 well black solid flat bottom Non-Binding plate, Corning, 3724

C. Final Assay Conditions:
Reagent, Final Concentration
BIS-TRIS pH 6.0, 50 mM
Tween 20, 0.005 %
DTT, 2.5 mM
STEP, 0.5 nM
OMFP, 25 uM
Final reaction volume, 4 uL/well in 1536 well plate
Test compound concentration, 20 uM
Final DMSO concentration, 1.0%
D. Procedures:
Step#, Description
1. Prepare Reagents as described in sections F. Recipe.
2. Using LabCyte Echo, transfer 40 nL from a plate containing 2 mM test compounds into assay plate Col. 5 - 44 (final concentration of test compounds is 20 uM, 1.0% DMSO). 40nL of DMSO should be transferred to col. 1-4 for control wells.
3. Spin plates at 1000 rpm for 1 minute in centrifuge.
4. Set up Kalypsys dispenser as described in section G. Instrument settings.
5. Using the Kalypsys dispenser, add 2 uL/well of control buffer (no enzyme control) to columns 1 and 2 for the positive control wells.
6. Using the Kalypsys dispenser, add 2 uL/well of enzyme solution to col. 3-48 for the negative control and test compound wells.
7. Using the Kalypsys dispenser, add 2 uL/well of substrate solution to columns. 1-48 (all wells).
8. Spin plates at 1000 rpm for 1 minute in centrifuge.
9. Incubate plates in the dark at room temperature for 20 minutes.
10. Detect signals on Perkin Elmer Viewlux with settings as described in section G. Instrument settings.

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

F. Recipe:
Enzyme solution (STEP)
Reagent, Working Conc.
BIS-TRIS pH 6.0, 50 mM
Tween 20, 0.005 %
DTT, 5 mM
STEP, 0.5 nM

Substrate solution (OMFP)
Reagent, Working Conc.
BIS-TRIS pH 6.0, 50 mM
Tween 20, 0.005 %
OMFP, 50uM

G. Instrument settings:
Kalypsys dispenser
Step#, Description
1. Before assay starts, rinse tubing thoroughly with 5 mL of MilliQ H2O per dispensing tip.
2. Air rinse tubing.
3. Rinse and prime tubing with 1 mL of actual reagents per dispensing tip.
4. When the assay is done, clean tubing thoroughly with 5 mL of MilliQ H2O per dispensing tip.
5. Air rinse tubing.
6. Rinse tubing thoroughly with 5 mL of 25% EtOH per dispensing tip.

Perkin Elmer Viewlux
Light Energy: 10000
Measurement: Time 1 sec.
Excitation Filter: 480/20 (FITC)
Emission Filter: 540/25 (FITC)
Mirror: FITC dichroic
Sensitivity: 4.13 e - /ADU

H. Note:
1. All reagents should be made up according to its spec-sheet or otherwise in Mol. Grade Water.
2. Make up buffer minus Tween-20 in large scale and add fresh Tween-20 weekly.
3. Make up enzyme buffer minus DTT in large scale and add fresh DTT just before the assay starts.
4. Storage conditions after reagents are made up:
Reagent, Temp.
Buffer minus DTT, 4 degree
STEP, -80 degrees
Na3VO4, -80 degrees
DTT, -80 degrees
OMFP, -80 degrees (light sensitive)

The experimental values were normalized by difference between values from neutral and stimulator control wells in each plates. Then normalized data was corrected to remove systematic plate patterns due to artifacts such as dispensing and edge effect due to overnight incubation. The algorism "Assay Pattern (Multiplicative)" was applied in Genedata Screener(R) software to correct screen data. Further information about data correction is available at http://www.genedata.com/products/screener.html.
Compounds that demonstrated % activity of >= 40 % at 20 uM

Comment: Compounds that demonstrated a normalized or corrected inhibition of >= 40% at 20uM 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.

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