External ID: USP8 FAST DUB HTS Primary

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

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

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

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

External ID: USP17 FAST DUB HTS Primary

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

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

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

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

External ID: USP7 FAST DUB HTS Primary

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

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

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

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

External ID: VANDERBILT_HTS_GIRK2_HPP

Protocol: To screen for modulators of the G protein-gated inwardly-rectifying potassium channel subunit 2 (GIRK2) homomeric channels, HEK293 cells were engineered to overexpress GIRK2 and the neuropeptide Y receptor type 4 (NPY4). The purpose of NPY4 overexpression was to promote modest activation (~30% activity) of GIRK2 channels upon NPY4 activation with human pancreatic polypeptide (hPP). These cells were screened using a high-throughput thallium-flux assay in a 384-well format. High-throughput thallium flux assays are based on three principles: (1) extracellular thallium can permeate through potassium channels into cells, (2) the fluorescent dye Thallos enters and stays in the cytoplasm of HEK293 cells, and (3) the fluorescence of Thallos increases dramatically when it interacts with thallium ions. Compounds that modulate the activity of an overexpressed channel, in this case GIRK2, are identified by the change in fluorescence that results from changes in thallium influx into cells through the overexpressed channel.

The screening protocol was conducted as follows. One hour prior to screening, cells were loaded with 1.5 micromolar (muM) Thallos in assay buffer (1x Hank's Balanced Salt Solution and 20 millimolar (mM) HEPES). The liquid handling and imaging was conducted using a kinetic-imaging plate reader, Panoptic (WaveFront Biosciences). Before screening, Thallos-containing buffer was removed from cells and replaced with 20 microliters (muL) per well of assay buffer. Compounds were dissolved in assay buffer at 20 muM. After 8 seconds of imaging, 20 muL of compound solution was added per well and allowed to incubate for 150 seconds. Next, 10 muL of a solution containing 2.0 mM thallium and 3.5 nanomolar (nM) hPP in Base Buffer (125 mM NaHCO3, 1 mM MgSO4, 1.8 mM CaSO4, 5mM Glucose, and 20 mM HEPES) was added to the wells. 30 seconds later, 12 muL of base buffer containing 2.0 mM thallium and 1 muM hPP, a maximally-activating concentration, was added to each well. Data was collected for 60 seconds after this final addition. The positive control for this screen was 16.6 muM eprinomectin, a natural product previously identified as a GIRK2 activator as part of a small-scale pilot screen. Microsoft Excel was utilized for data analysis. Data were normalized, F/Fo, on a well-to-well basis to account for differences in cell number. Compound activity was analyzed by comparing the sums of control-subtracted amplitudes of fluorescence intensity at 20 seconds after each thallium addition. The top 0.5% compounds corresponding to wells that demonstrated the largest increases in fluorescence were selected as "hits" for counter screening, as described below.

All 34,131 compounds screened using the method described above are listed in column OUTCOME_SCREEN_HITS. The 167 compounds selected as hits based on the above criteria are labelled ACTIVE in this column. Fluorescent compounds were included in the inactive compounds and marked as INACTIVE.

The 167 active compounds from column OUTCOME_SCREEN_HITS were tested for non-specific activity in untransfected HEK293 cells using assay conditions otherwise identical to the primary screen. Column OUTCOME_HEK_COUNTERSCREEN lists the 39 compounds that displayed an increase in fluorescence upon thallium addition, labelled with ACTIVE.

The 128 compounds which were identified as hits in the primary screen but inactive when tested in untransfected HEK293 cells were tested for activity in GIRK2-overexpressing HEK293 cells that did not express NPY4 receptor. Column OUTCOME_GIRK2_COUNTERSCREEN lists the 8 compound that displayed an increase in fluorescence upon thallium addition, labelled with ACTIVE.

The 8 active compounds from column OUTCOME_GIRK2_COUNTERSCREEN were tested at various concentrations to determine if compound activity was concentration dependent. Column OUTCOME_GIRK2_DOSE_RESPONSE provides the efficacy of these compounds; activity of all compounds at the provided concentrations was normalized to the activity of VU0529331 at 37.5 muM, which was the highest efficacy observed during this screening. For each compound concentration listed, the efficacy of that compound provided refers to the normalized, control-subtracted fluorescence intensity at 15 seconds after the addition of thallium. Compounds that demonstrated >5% efficacy and concentration-dependent activity were labelled as ACTIVE.

Finally, column Outcome combines all of the information from the above counterscreens together, indicating the 6 hits from this GIRK2 screen.

Comment:

External ID: USP30 FAST DUB HTS Confirmation

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

Protocol for Inhibitors of USP30: Confirmation Screen
1. Compounds in 4-point log dose response were transferred from source plates to columns 1-46 of a Greiner 1536 well Black, flat bottom, polystyrene microplate (Griener 782076) starting at an initial concentration of 50 uM (columns 45-46: DMSO control)
2. Reagent, 2.5 uL of USP30 in assay buffer (final concentration of 1 nM) to columns 1-46
3. Reagent, 2.5 uL of assay buffer to columns 47-48 on all plates (No enzyme control)
4. Reagent, 2.5 uL of Ub-Rho in assay buffer (final concentration of 12 nM) to all wells
5. Time, Incubate at room temperature for 2 hours
6. Reagent, 2.5 uL of stop buffer to all wells
7. Centrifuge plates (5s, 350 rpm)
8. Detection, Fluorescence, PheraStar, FITC Module

Comment: Each compound was tested in triplicate in the dose-response confirmation assay. Normalization of raw data to positive and negative control wells, systematic pattern models, and curve fitting were applied to the data using Helios, a high-throughput screening data analysis program developed at Novartis.(Gubler et al., 2018). For measuring compound potency, a four-parameter sigmoid Hill curve model was fitted to the data (Gubler et al., 2018) and the absolute IC50 was used for potency measurements, that is the concentration of compound where the data showed 50% inhibition. If no data points achieved 50% inhibition, then the absolute IC50 was reported as >50 uM (the highest tested concentration). Maximal efficacy of each compound was reported as a percent inhibition based on normalization to plate controls.

Primary screen hits were confirmed as active if they demonstrated greater than or equal to 30% inhibition of USP30 (USP30_EFFICACY >/= 30). Active compounds were given a score based on potency.

External ID: USP17 FAST DUB HTS Confirmation

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

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

Comment: Each compound was tested in triplicate in the dose-response confirmation assay. Normalization of raw data to positive and negative control wells, systematic pattern models, and curve fitting were applied to the data using Helios, a high-throughput screening data analysis program developed at Novartis.(Gubler et al., 2018). For measuring compound potency, a four-parameter sigmoid Hill curve model was fitted to the data (Gubler et al., 2018) and the absolute IC50 was used for potency measurements, that is the concentration of compound where the data showed 50% inhibition. If no data points achieved 50% inhibition, then the absolute IC50 was reported as >50 uM (the highest tested concentration). Maximal efficacy of each compound was reported as a percent inhibition based on normalization to plate controls.

Primary screen hits were confirmed as active if they demonstrated greater than or equal to 30% inhibition of USP17 (USP17_EFFICACY >/= 30). Active compounds were given a score based on potency.

External ID: USP28 FAST DUB HTS Confirmation

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

Protocol for Inhibitors of USP28: Confirmation Screen
1. Compounds in 4-point, log dose response were transferred from source plates to columns 1-46 of a Greiner 1536 well Black, flat bottom, polystyrene microplate (Griener 782076) starting at an initial concentration of 50 uM (columns 45-46: DMSO control)
2. Reagent, 2.5 uL of USP28 in assay buffer (final concentration of 0.5 nM) to columns 1-46
3. Reagent, 2.5 uL of assay buffer to columns 47-48 on all plates (No enzyme control)
4. Reagent, 2.5 uL of Ub-Rho in assay buffer (final concentration of 12 nM) to all wells
5. Centrifuge plates (5s, 350 rpm)
6. Time, Incubate at room temperature for 30 minutes
7. Detection, Fluorescence, PheraStar, FITC Module

Comment: Each compound was tested in triplicate in the dose-response confirmation assay. Normalization of raw data to positive and negative control wells, systematic pattern models, and curve fitting were applied to the data using Helios, a high-throughput screening data analysis program developed at Novartis.(Gubler et al., 2018). For measuring compound potency, a four-parameter sigmoid Hill curve model was fitted to the data (Gubler et al., 2018) and the absolute IC50 was used for potency measurements, that is the concentration of compound where the data showed 50% inhibition. If no data points achieved 50% inhibition, then the absolute IC50 was reported as >50 uM (the highest tested concentration). Maximal efficacy of each compound was reported as a percent inhibition based on normalization to plate controls.

Primary screen hits were confirmed as active if they demonstrated greater than or equal to 30% inhibition of USP28 (USP28_EFFICACY >/= 30). Active compounds were given a score based on potency.

External ID: OTUD3 FAST DUB HTS Confirmation

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

Protocol for Inhibitors of OTUD3: Confirmation Screen
1. Compounds in 4-point, log dose response were transferred from source plates to columns 1-46 of a Greiner 1536 well Black, flat bottom, polystyrene microplate (Griener 782076) starting at an initial concentration of 50 uM (columns 45-46: DMSO control)
2. Reagent, 2.5 uL of OTUD3 in assay buffer (final concentration of 12 nM) to columns 1-46
3. Reagent, 2.5 uL of assay buffer to columns 47-48 on all plates (No enzyme control)
4. Reagent, 2.5 uL of Ub-Rho in assay buffer (final concentration of 40 nM) to all wells
5. Time, Incubate at room temperature for 2 hours
6. Reagent, 2.5 uL of stop buffer to all wells
7. Centrifuge plates (5s, 350 rpm)
8. Detection, Fluorescence, PheraStar, FITC Module

Comment: Each compound was tested in triplicate in the dose-response confirmation assay. Normalization of raw data to positive and negative control wells, systematic pattern models, and curve fitting were applied to the data using Helios, a high-throughput screening data analysis program developed at Novartis.(Gubler et al., 2018). For measuring compound potency, a four-parameter sigmoid Hill curve model was fitted to the data (Gubler et al., 2018) and the absolute IC50 was used for potency measurements, that is the concentration of compound where the data showed 50% inhibition. If no data points achieved 50% inhibition, then the absolute IC50 was reported as >50 uM (the highest tested concentration). Maximal efficacy of each compound was reported as a percent inhibition based on normalization to plate controls.

Primary screen hits were confirmed as active if they demonstrated greater than or equal to 30% inhibition of OTUD3 (OTUD3_EFFICACY >/= 30). Active compounds were given a score based on potency.

External ID: USP28 FAST DUB HTS Primary

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

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

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

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

External ID: USP10 FAST DUB HTS Primary

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

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

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

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

External ID: UCHL1 FAST DUB HTS Primary

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

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

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

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

External ID: OTUD3 FAST DUB HTS Primary

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

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

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

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

External ID: USP30 FAST DUB HTS Primary

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

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

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

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

External ID: WaveGuideAssay:441

Protocol: For initial screening, TetON human embryonic kidney cells (HEK293) expressing GluA2 and stargazin, gamma-2, were plated in 384-well BD purecoat amine-coated plates at 16k cells/well. Cells were induced with 8 ug/mL doxycycline in the presence of 30 uM NBQX and incubated O/N at 37deg in 5% CO2. They were washed with Hank's Balanced Salt Solution (HBSS) containing 20 mM HEPES, pH 7.4 and FLIPR VSD dye solution was added and incubated on the cells at room temperature for 45 minutes. The cell plate was loaded into the Hamamatsu Functional Drug Screening System 6000 (FDSS), 10 seconds of background were collected before addition of compound at 30 uM final concentration. Data collection continued at 1Hz sampling and experimentally determined EC50 glutamate stimulus was added at 300 seconds. Controls of 1 mM glutamate (maximal effect) and NBQX (antagonist) were used to calculate a Z' score per plate.

Data were processed using the Vanderbilt screening analysis platform, WaveGuide. Data were normalized by dividing the raw fluorescence for all time points by an average of the baseline fluorescence. For each well, 4 values were calculated as follows:

1. CMPDslope was the slope of the normalized value within the timeframe 11-18 seconds,
2. CMPDmaxmin was the difference in maximal and minimal normalized value within the timeframe 8-100 seconds
3. GLUslope was the slope of the normalized value within the timeframe 310-315 seconds, and
4. GLUmaxmin was the difference in maximal and minimal normalized value within the timeframe 301-380 seconds.

Compounds were marked as hits where any of the above calculated results for a compound differed by at least three standard deviations from the mean for that calculation across all compounds in the plate.

Compounds denoted as 'hits' were given the score of '100' and outcome of 'active'. All other compounds were denoted as 'inactive' and given a score of '0'.

Comment: