External ID: HMS1262

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

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

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

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

External ID: HMS791

Protocol: Prior to screening, FITC LANA1-23 was stored lyophilized at -80 degC and freshly purified chicken nucleosomes were stored at 4 degC in 20 mM Tris pH 7.5, 600 mM NaCl, 0.2 mM EDTA, 0.5 mM B-Mercaptoethanol.

On the day of screening, FITC LANA1-23 was resuspended at 50 uM in TEN-BT buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA (pH 8.0), 2.5 mM NaCl, 5 mM Beta-mercaptoethanol, 0.01% Triton-X-100) and diluted to a final concentration of 50 nM in TEN-BT buffer plus 240 nM of purified nucleosomes (480 nM LANA peptide binding sites). 30 uL per well were dispensed in column 1-22 in Corning #3575 black 384 well plates.

Wells in column 23 contained 30 uL of the same mixture (for pilot screen) or with the addition of 10 uM monensin (for HTS) as a negative control. In column 24, 1250 nM unlabeled WT LANA1-23 peptide (for pilot screen) or 10 uM mitoxantrone (for HTS) was added to the wells as a positive control. Compounds were transferred into wells via stainless steel pin array (100 nL) and the reaction was incubated at room temperature for 10 to 45 minutes (stable for up to 2 hours). Library plates were screened in duplicate, with both assay plates in a given set prepared on the same day.

Following a room temperature incubation of 10 to 45 minutes, the assay is read on a EnVision plate reader using a 480 nM excitation filter, 535 nM S and P emission filters and D505fp/D535 dichoric mirror. mP value for FP measurement = 1000*(S-G*P)/(S+G*P) where S= , P=, G= G-factor. The G Factor = 1.

Comment: LANA 1-23 peptide containing the first 23 amino acids of the LANA protein from Kaposi's sarcoma herpesvirus (KSHV) was synthesized with an N-terminal FITC via a beta alanine linker and HPLC purified (peptide sequence: [FITC]-Beta alanine-MAPPGMRLRSGRSTGAPLTRGSC-[NH2]).

Data analysis: Z-scores were calculated for each replicate well using the mean and standard deviation of plate experimental well FP values. Compounds were considered active if the Z-score for both replicates < -2. Wells with high total fluorescence intensity (high S and P channel values) were excluded from further consideration. Activity scores were calculated based on replicate average FP Z-scores. For wells with average Z-score >= 0, the activity score was set to 0. For wells with replicate average Z-score < 0, replicate Z-score was divided by 4 and multiplied by -100. The replicate average was then used to determine the well activity score. Values > 100 (replicate average Z-score < -4) were set to 100.

External ID: HMS1303

Protocol: Prior to screening, lyophilized FITC-GIV peptide (a.a. 1671-1701) is dissolved in 100% DMSO to a concentration of 0.5 mM, then further diluted with water to a final concentration of 50 microM. One stock was prepared in batch for the entire screen and aliquoted. All protein stocks were stored at -80C. Thawing of aliquots on the day of the assay was performed rapidly at room temperature.

Prior to transfer of screening compounds, experimental wells of 384-well assay plates were pre-filled with 37.8 nM of FITC-GIV peptide (for 25 nM final concentration) in a 10 microL volume of assay buffer (50 mM Tris [pH 7.4], 100 mM NaCl, 10 mM MgCl2, 5 mM EDTA, 0.4% NP-40, 30 microM GDP, 1 mM DTT). Control wells were pre-filled with 37.8 nM of FITC-GIV peptide in 10 microL of assay buffer containing either 1% DMSO (vehicle negative control) or 45.3 microM AlCl3+15.1 mM NaF (AlF4- positive control). Each plate contained 16 negative control and 16 positive control wells. Plates were centrifuged at 1000xg for 2 minutes to eliminate bubbling on the surface. Following, 100 nL of experimental compound was pin-transferred into individual wells for each of two replicate assay plates (A&B). After pin-transfer, 5 microL of 3 microM His-tagged rat Galphai3 purified from E. coli (for 1 microM final concentration) was added to each well, and assay plates were shaken at low speed for 5 seconds. Final assay volume was 15 microL. Plates were centrifuged at 1000xg for 2 minutes to eliminate bubbling on the surface and shaken for 10 seconds prior to an incubation of 90 minutes at room temperature. Plates were protected from prolonged light exposure. All manipulations were performed at room temperature.

Assay plates were read with a PerkinElmer Wallac EnVision Multilabel 2103 plate reader using the fluorescent polarization measurement technology with a D505fp/D535 dual mirror and a 480 nm excitation filter. 535 nm emission filters were used for P and S channel reads at 11 mm measurement height with 30 flashes and detector gains of 319 and 563. G-factor 0.96.

Negative control: DMSO
Positive control: Aluminum tetrafluoride (AlF4-, composite of NaF and AlCl3)

Comment: Calculated result values and scoring of active compounds:

Z-score: Indicates the number of standard deviations an experimental condition is from the mean. It is calculated based on plate average (u) and standard deviation (s) of experimental well fluorescence polarization (FP) measurements for each replicate. Z-score (z) is defined as the quotient of the mean of the experimental well population subtracted from an individual well's FP (x), divided by the standard deviation within the experimental wells; z = (x-u)/s.

Normalized percent control: calculated by subtracting plate average positive control FP from experimental well FP, dividing by the difference between plate average negative and positive control FP, and multiplying by 100.

STD: the number of standard deviations from the plate average negative control FP for each replicate experimental well

Compound wells with FP values exceeding +5 standard deviations from the negative control sample average were flagged as potential aggregators or fluorescence quenchers due to their high polarization signal (HighPol) and were excluded from further consideration. Activity scores were calculated by averaging the replicate normalized percent of control values and subtracting from 100 (score based on single replicate if data for other replicate was excluded for any reason). Result values < 0 were set to 0 (no activity) and > 100 were set to 100 (100% activity). Compounds with activity score >= 15 were considered active.

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: GPR151_PHUNTER_AG_LUMI_1536_1X%ACT

Protocol: Assay Overview:
TThe goal of this NIH sponsored research project was to identify GPR151 agonists via high-throughput screening (HTS) efforts. In brief, the McDonald Lab transferred the GPR151 AG 384wpf assay to Scripps for implementation and miniaturization to 1,536-well format followed by screening against the Scripps Drug Discovery Library (SDDL). A counterscreen assay, employing GPR119 cells, was also implemented by Scripps to identify compounds that non-specifically affect the PathHunter detection method. This project was aided by cheminformatic efforts to help identify compounds that demonstrated authentic agonist pharmacology and non-promiscuous activity profiles across other primary screens run against the SDDL. At completion of the project, several compounds demonstrated activity in the GPR151 AG PathHunter assay. All compounds selected for titration were also subjected to LC-MS analysis to confirm mass and sample purity

Protocol Summary:
Prior to the start of the assay, cells were resuspended in growth media at 2000cells/well in 1536 well plates. This was followed by an incubation of 18 hours at 37C+5%CO2. Then 1uL of Opti-Mem added to all wells except high controls to which 3X EA reagentwas added to each of those wells (0.2X final in lysis buffer). Compounds were pinned at 11.20uM final concentration in 1.0% DMSO followed by a 90 minute incubation at 37C+5%CO2. At this stage 3uL of Promega Beta-Glo detection Buffer was added to all wells followed by a 1 hour incubation at room temperature. Finally the assay end point read was taken using the ViewLux imaging reader from PerkinElemer Lifesciences with a 5 second exposure. Raw assay data was imported into Scripps# corporate database and ascetrained for Z' greater than 0.5 in order to be processed futher.

The percent activation for each compound was calculated as follows:

Percent Response of compound= 100 * ((Test Well-Median Data Wells)/(Median High Control # Median Data Wells))
Where:
Test_Well is defined as wells containing GPR151 cells in the presence of test compound
High_Control represents wells containing GPR151 cells stimulated with 0.2X EA reagent
Low_Control is defined as wells containing GPR151 cells and DMSO
PubChem Activity Outcome and Score:
Standard Cutoff
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:
GPR151 cells (supplied by Patricia McDonald)
Pen Strep (Invtirogen 15140)
FBS (Invtirogen 16140)
Hygromycin (Invtirogen 10687010)
Lysis buffer (CisBio 62CL2FDF)
EA Reagent (DiscoverX 30-411)
G418 (Gemini Bio 400-113)
Opti-MEM (Invtirogen 31985)
Trypsin (Invtirogen 15400054)
Beta Glo (Promega E4780)
1536-well plates (Greiner Bio-One, part 789173)

Comment: Due to the size of the Scripps Molecular Screening Center compound library, this assay have been run as multiple 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.

A mathematical algorithm was used to determine what we call the standard hit cut-off to identify active compounds. Two values were calculated: (1) the average percent activation of all compounds tested in 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 was declared active. Using this "Standard Cutoff" (= 4.91%) the primary assay yielded 6,756 active compounds ("hits")."

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: MITF_INH_Alpha_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this biochemical assay is to identify MITF dimerization inhibitors that disrupt or prevent its homodimerization. This assay is a bead-based proximity assay, which employs acceptor beads and donor beads to generate a chemiluminescence signal. In this assay, Biotin-labelled MITF and His- tagged MITF homodimerization 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 reaction within the same bead resulting in emitted light at 520-620 nm. As designed, small molecule inhibitors that interfere with this interaction will increase the distance of the acceptor beads, thus leading to decreased signal. Compounds were tested in singlicate at a nominal test concentration of 2.6 micromolar.

Protocol Summary:
Prior to the start of the assay, 1.25#L of assay buffer (50mM HEPEs pH7.5, 250mM Sodium chloride, 0.1% Tween, 0.1% BSA) containing 10nM His-MITF_r259stop were dispensed into columns 1 thru column 2, and 1.25#L of assay buffer containing 10nM of His MBP MITF were dispensed into columns 3 thru column 48 of 1536 microtiter plates. Next, 10nL of test compounds in DMSO, 7,8-Dihydroxycoumarin (200#M final highest concentration) in DMSO, or DMSO alone (0.15% final concentration) were added to the appropriate wells before dispensing 1.25#L of 10#g/mL Acceptor beads into column 1 to 46 and 1.25#L of assay buffer into column 47 and 48. Plates were incubated in dark for 2hrs at room temperature. Then, dispenses of 1.25#L of 10nM Biotin-MITF into all columns, and 10#g/mL Donor beads into column 1 to 46 and 1.25#L of assay buffer into column 47 to 48 followed by 3hrs incubation in dark at RT, was done. Alphascreen signal was determined using an EnVision microplate reader (PerkinElmer, Waltham, MA) at 680nm excitation and 570nm emission.

The percent inhibition for each compound was calculated as follows:

100 *( ( Median_Low Control-Test Compound) / ( Median_Low Control - Median_High Control ) )

Where:

Test_Compound is defined as wells containing His-MITF + Biotin-MITF in the presence of test compound
High_Control is defined as wells containing His-MITF_r259stop + Biotin-MITF and DMSO.
Low_Control is defined as wells containing His-MITF + Biotin-MITF and DMSO

PubChem Activity Outcome and Score:

Standard Cutoff

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:

His-tagged MITF protein (supplied by Min Guo)
His-tagged MITF_r259stop protein (supplied by Min Guo)
Biotinylated-MITF protein (supplied by Min Guo)
7,8-Dihydroxycoumarin (Sigma, part D5564)
HEPEs (Sigma, part H3375, H3784)
Sodium chloride (Fisher, part BP358-212)
Tween-20 (Fisher, part 50146671)
DMSO (Fisher, part D159)
BSA (Fisher, part BP1600-100)
AlphaScreen Beads Kit (Perkin Elmer, part 6760619L)
1536-well plates (Greiner Bio-One, part 789175)

Comment: Due to the size of the Scripps Molecular Screening Center compound library, this assay have been run as multiple 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: CHEMBL5235756

Protocol: N/A

Comment: Journal: Eur J Med Chem
Year: 2021
Volume: 215
First Page: 113263
Last Page: 113263
DOI: 10.1016/j.ejmech.2021.113263

Target ChEMBL ID: CHEMBL1827
ChEMBL Target Name: Phosphodiesterase 5A
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: 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:

External ID: HMS750

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

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

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

External ID: 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: