External ID: FATTTLab-Algae-Lipid

Protocol: Cells were pre-grown to mid-log phase in TAP media. For primary screen, cells were seeded at low density of 500,000 / well in 384-well plate. Compound in DMSO was added to each well at a final concentration of 10 micromolar and cells were allowed to grow for 72 hours. At the end of assay, Nile Red (30 uM f.c.) was added and plates were incubated at 37 C for 60 mins in dark. Fluorescence intensity was measured. Readouts were reported as normalized fold change in the intensity of treated versus control.

Comment: Reference:
1. Wase, N., Tu, B., Black, P. N. & DiRusso, C. C. Phenotypic screening identifies Brefeldin A/Ascotoxin as an inducer of lipid storage in the algae Chlamydomonas reinhardtii. Algal Research 11, 74-84 (2015).

2. Identification and metabolite profiling of chemical activators of lipid accumulation in green algae
#Nishikant Wase, Boqiang Tu, James W Allen, Paul N Black, Concetta C DiRusso
#Plant Physiology Jun 2017, pp.00433.2017; DOI: 10.1104/pp.17.00433
#http://www.plantphysiol.org/content/early/2017/06/26/pp.17.00433

Patent:
DiRusso, C., & Wase, N. (2016). Compounds for Increasing Lipid Synthesis and Storage. United States#NUtech Ventures (Lincoln, NE, US) http://www.freepatentsonline.com/y2016/0312253.html

For additional Information please contact:

Prof. Concetta C. DiRusso
Department of Biochemistry
University of Nebraska-Lincoln
cdirusso2@unl.edu

Nishikant Wase, PhD
Department of Biochemistry
University of Nebraska-Lincoln
nishikant.wase@gmail.com

External ID: IP6K1-p1

Protocol: PROTOCOL TABLE
SEQUENCE No. (e.g., 1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE and DESCRIPTION.

1. Reagent, 3 uL of 1.3 mM ATP and 130 uM IP6 mixture in assay buffer was dispensed to a white, 1536-well assay plate.
2. Compound, 23 nL of compounds of the top two doses (57.5 uM and 19.1 uM final concentration) of the libraries were dispensed into the mixture using the Kalypsis pintool.
3. Reagent, 1 uL of 2.4 uM IP6K1 was dispensed to the assay plates.
4. Incubation, 2 hr incubation at room temperature.
5. Reagent, 2 uL of ADP-Glo reagent was added to the wells.
6. Incubation, 1 hr incubation at room temperature.
7. Reagent, 4 uL or ADP-Glo substate was added to the wells.
8. Incubation, 45 min incubation at room temperature.
9. Detection, luminescence signal was detected using the ViewLux microplate imager (PerkinElmer).

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

Compound Ranking:
1. Majority of the compounds were tested at 58uM and 11uM. Percent inhibition at 58uM (Max_Response) was obtained and used for compound ranking.
2. For all inactive compounds, with Max_Response >= -25.00, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds (Max_Response < -25.00, more than 25% inhibition) a score range was given between 25 and 100. The activity score is based on the absolute value of the Max_Response.

External ID: SNCA-p-activity-luciferase

Protocol: PROTOCOL TABLE
SEQUENCE No. (e.g., 1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE; DESCRIPTION

1; Cells; 4 uL; Dispense 1500 HEK-293-SNCA-luc cells/well into Greiner 1536-well white / solid bottom tissue culture treated plate. The plate was covered with metal lids with gas-exchange holes.
2; Incubate; 24 hours; Incubate at 37C, 5% CO2, 95% RH.
3; Compounds; 23 nL; Compounds and controls were transferred via a Kalypsys Pin Tool (Wako USA) equipped with a 1536-slotted pin array. The plate was covered with metal lids with gas-exchange holes.
4; Incubate; 24 hours; Incubate at 37C, 5% CO2, 95% RH.
5; Dispense; 1 uL; Dispense Gly-Phe-7-amino-4-trifluoromethylcoumarin (GF-AFC, prepared at 125 uM in PBS) was added. The plate was covered with metal lids with gas-exchange holes.
6; Incubate; 30 min; Incubate at 37C, 5% CO2.
7; Detector; Fluorescence; Measure fluorescence with ViewLux microplate reader (PerkinElmer) equipped with 405/10 excitation and 540/25 emission filters.
8; Dispense; 3 uL; Dispense ONE-Glo (PerkinElmer) lucifase detection reagent was added to each well. Plates were covered with metal lids with gas-exchange holes.
9; Incubate; 15 min; Incubate at room temperature.
10; Detector; Luminescence; Measure luminescence with ViewLux microplate reader (PerkinElmer) equipped with clear filters.

NOTES (numbers refer to sequence above)
1; HEK-293-SNCA-luc were cultured and suspended in phenol-red free DMEM (4.5 g/L glucose, 25 mM HEPES, cat #21063 (Thermo)).
3; Compounds were added to the assay plate in an 11-point intra plate dose response, 1:3 titration in DMSO with a final concentration range of xxx - yyy uM. Vehicle-only plates, with DMSO being pin-transferred to every well, were inserted at the beginning of screening runs to confirm expected assay performance. Activity was normalized to wells containing medium only (-100% activity, full inhibition) and SNCA-luc cells treated with DMSO vehicle control (0% activity), contained on the same plate as test samples.
10; Signals were analyzed, and dose-response curves were fit using the Hill equation. Compounds in curve classes -1.1, -1.2, -2.1, -2.2 in the SNCA-luc assay were considered active. Compounds were eliminated from further consideration if also active (curve class -1.1, -1.2, -1.3, -1.4, -2.1, -2.2, -2.3, -2.4) in the GF-AFC cytotoxicity assay.

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

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

External ID: CPF004

Protocol: 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 60uM 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).

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: ST2_IL33_Inhibitors_Primary_Screening_77700

Protocol: High-throughput screening was performed at Indiana University screening facility (Indiana University, Indianapolis, IN).

In summary, a mixture of ST2 and IL-33 was prepared by adding 0.30 microL of 14.1 microM recombinant human ST2-Fc Chimera and 0.30 microL of 55.25 biotinylated recombinant human IL-33 to 1.4 mL of assay buffer. In the HTS, each well of a 384-well Proxi-plate was first blocked by 100 microL of assay buffer for 1 h at room temperature. After removing the blocking buffer, 20 microL of ST2/IL-33 mixture was pipetted into each well and incubated at room temperature for 1 h followed by addition of compounds at 17 microM (0.8 microL) to each well and incubated at room temperature for 1 h. Ten microliters of 60 microg/mL anti-6xHis-conjugated AlphaLISA acceptor beads were added to each well and incubated at room temperature for 1 h before addition of 10 microL of 60 microg/mL of streptavidin-labeled AlphaLISA donor beads for incubation at room temperature for 30 min. Incubation of acceptor and receptor beads was conducted in darkness. The well with 3 nM ST2 alone was used as a negative control, whereas the ST2/IL-33 mixture with a human ST2 antibody added at 0.45 ng/microL was used as the positive control. Plates were read using the Envision Plate Reader. The buffer used in the assay contains 14.37 mL PBS, 30 microL Tween 20, and 600 microL of 5% bovine serum albumin in PBS.

Comment: PubChem active indicates >= 30% inhibition of ST2/IL-33 at 17 uM of the compound. Inconclusive: >=10% and < 30% inhibition.

External ID: KUHTS-Muma KU-CaM-Htt INH-01

Protocol: 1; Dispense 45 nl compounds (10 mM stocks) using ECHO 555 to Alpha 384 well assay plates. Dispense 45 nl DMSO to control columns 1 and 2 of 384 well plates.
2; Incubate 5 ul of 6XHis-mHTT (Final, 13 nM) with the compounds for 40 mins at room temperature in buffer containing 10 mM Tris.HCl pH 7.4, 1 mM calcium chloride, 150 mM sodium chloride, 0.1% BSA and 20% glycerol.
3; Dispense 5 ul of 6XGST-CaM (Final 13 nM) in buffer A.
4; Incubation; 1 hour (dark at 25C)
5; Dispense 20 ul of Nickel chelate acceptor beads (Final, 20 ugs/ml) and Glutathione donor beads (Final, 30 ugs/ml). Incubate for 2h, room temperature.
6; Detector: Perkin Elmer Enspire, Alphascreen Module (Excitation 680nm/Emission 570nm).

NOTES (numbers refer to Sequence numbers above)
1. Alphascreen bead incubations were performed in green light, TiterTek setting 400rpm.
2. All incubation and addition steps were followed by mixing and centrifugation at 400g,1 min.
3. The percent inhibition for each compound was calculated as follows:
100- [100 *((Test Compound-Median Low Control) / (Median High Control - Median Low Control))]

Where:
Test_Compound is defined as wells containing His mHTT + GST CaM in the presence of test compound

High_Control is defined as wells containing His mHTT + GST CaM and DMSO.

Low_Control is defined as wells containing His mHTT and DMSO.

Comment: All percent inhibition data reported were normalized to high and low controls on a per-plate basis. The results of primary screening data include compounds contributing to assay signal interference, interference with tags binding to Alpha beads, chelators, process related artifacts etc.. The actives were defined as compounds that inhibited Alphascreen reads to greater than 50%.

External ID: hNprA-p1

Protocol: PROTOCOL TABLES (as described by Inglese J, Shamu CE and Guy RK. 2007)

SEQUENCE No. (e.g., 1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE and DESCRIPTION.
GLOSENSOR PROTOCOL

1. Cells. Seed 1500 HEK293 cGMP-sensor cells expressing hNpr1 per well in 4uL into Greiner 1536-well white/solid bottom high base, tissue culture-treated plates.
2. Incubation. Incubate plated cells at 37C in 5% CO2, 95% RH overnight.
3. Reagent. Add 1uL per well GloSensor reagent from Promega (2% final concentration) or CO2 independent media vehicle control to respective columns of Greiner 1536-well plates.
4. Incubation. Incubate plated cells at room temperature for 2 h, protect from light.
5. Compounds. Library compounds and A-71915 control added at 23 nL per well, transferred by Pin Tool.
6. Incubation. Incubate plated cells for 30 min. at room temperature, protect from light.
7. Detector. GloSensor baseline pre-read luminescence measured using CCD-based camera (Viewlux).
8. Compound. Add 1uL of hNppA peptide to 0.1 nM final concentration to stimulate hNpr1 or CO2 independent media as vehicle control.
9. Incubation. Incubate treated cells at room temperature for 20 min., protect from light.
10. Detector. GloSensor post-hNppA luminescence measured using CCD-based camera (Viewlux).

NOTES (numbers refer to Sequence numbers above)

1. HEK293 cells expressing hNpr1 and GIP/pGS-40F grown to 85-95% confluence (48-72 h) in T175 culture flasks (CO2 independent media supplemented with 10% FBS, 1% Pen/Strep antibiotics and 1 ug/mL puromycin). Trypsinize and harvest cells from T175 culture flasks and filter through 40 um cell strainer. Cells counted with Invitrogen Countess (30,000,000 - 50,000,000 cells/flask). Cells diluted to a final concentration of 375 cells/uL in CO2 independent media and a Millipore multidrop used to dispense 1500 cells per well (4uL/well) into 403 x 1536-well Greiner White/Solid bottom, high base tissue-culture treated (TC) plates and one Aurora 1536-well Black/Clear bottom, low base, TC plate (for visual inspection of cell health throughout the duration of the assay).

2. Cells incubated overnight at ambient temperature, 24 h. at 37C in 95% RH incubator protected from light, covered with metal lid with gas-exchange holes.

3. Prepare GloSensor Reagent (Promega): Reconstitute GloSensor luciferase substrate by adding 8.2mL Hepes buffer to 250mg substrate, mix well, protect from light and stored at -80C. Prepare a fresh 12% GloSensor solution in CO2 independent supplemented plating media (see above). Mix well and store at ambient temperature, protected from light. 1uL 12% GloSensor Luciferase Reagent added (2% final concentration) to respective columns of 403 x 1536-well Greiner plate with BioRaptr, 1uL CO2 independent plating media added to respective columns as vehicle control with BioRaptr.

4. Cells incubated for 2 h. at ambient temperature, protected from light with metal lid with gas-exchange holes.

5. A-71915 stock solution prepared (10 mM in DMSO). Working dilution for dispense = 10 mM stock dilution (38.3 uM final concentration; 16-pt 1:2 titration in duplicate = final concentration range of 38.3 uM - 1.17 nM). 23nl of GENESIS library compounds, A-71915 antagonist control or DMSO vehicle control transferred to the 403 cell-containing Greiner white/solid bottom high base, tissue culture-treated plates in 5-, 6- or 7-pt. interplate titrations where concentrations range 10 mM to 640 uM stock concentrations (see Yasgar, A. et al. 2008) with Pintool. 27 of the 403 plates are DMSO-only in compound field interspersed approximately every 20 plates between 376 library-treated plates.

6. Cells incubated for 30-50 min. at ambient temperature protected from light with metal lid with gas-exchange holes.

7. Read cellular baseline GloSensor luminescence in presence of A-71915 antagonist or test library compounds on the ViewLux [Exposure = 10 s.; Gain = High; Speed = Slow; and Binning = 2X.]

8. hNppA peptide agonist solution prepared at 250 uM (in H2O) stock concentration. From this a working dilution was prepared at 0.6 nM in CO2 Independent media. 1uL of 0.6 nM hNppA agonist (0.1 nM final concentration) or CO2 Independent media vehicle control dispensed into respective columns of 403 x Greiner 1536-well white/solid bottom, tissue culture plates with BioRaptr.

9. Cells incubated with agonist for 20 min. at ambient temperature, protected from light, covered with metal lid with gas-exchange holes.

10. Read final Luminescence on the ViewLux. [Exposure = 10 s.; Gain = High; Speed = Slow; and Binning = 2X.]

REFERENCES:

Inglese J, Shamu CE and Guy RK, Reporting data from high throughput screening of small molecule libraries, Nature Chemical Biology, 2007, 3(8): 438-441. doi.org/10.1038/nchembio0807-438

Yasgar A, Shinn P, Jadhav A, Auld DS, Michael S, Zheng W, Austin CP, Inglese J and Simeonov A, Compound Management for Quantitative High-Throughput Screening. J. Assoc. Lab. Auto., 2008, 13: 79-89. doi: 10.1016/j.jala.2007.12.004

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, with an activity curve class of 4, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. For activity curve class = -1.1, score = 80+abs((log_ac50+4.5)*inf_activity/20). For activity.curve_class == -1.2 && abs(activity.max_response) > 6*10, score = 60+abs((log_ac50+4.5)*inf_activity/20). For activity.curve_class == -2.1 || ( activity.curve_class==-2.2 && abs(activity.max_response) > 6*10), score = 40+abs((log_ac50+4.5)*inf_activity/20). For activity.curve_class == -1.2 || activity.curve_class == -2.2, score = 20+abs((log_ac50+4.5)*inf_activity/20). Inconclusive compounds, with a preread curve class other than 4, have PUBCHEM_ACTIVITY_SCORE of 10.

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: CPF003

Protocol: NIH 3T3 cells were seeded into white 1536-well plates using a Multidrop Combi peristaltic dispenser (ThermoFisher, Waltham, MA) at a density of 400 cells/well in 5 uL of medium respectively. A pintool (Kalypsys) was used to transfer 23 nL of compound solution to the 1536-well assay plates. After 48 or 72 hr incubation at 37 degree celcius, 5% CO and 95% humidity, 2.5 uL of CellTiter-Glo (Promega) was dispensed into each well using a dispenser (Aspect Automation, St. Paul, MN) with solenoid valves (Lee Valves, Westbrook CT). Plates were left at room temperature for 10 min before imaging the ATP-coupled luminescence using a ViewLux microplate imager (PerkinElmer, Waltham, MA).

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: CPF002

Protocol: HEK 293 cells were seeded into white 1536-well plates using a Multidrop Combi peristaltic dispenser (ThermoFisher, Waltham, MA) at a density of 250 cells/well in 5 uL of medium respectively. A pintool (Kalypsys) was used to transfer 23 nL of compound solution to the 1536-well assay plates. After 48 or 72 hr incubation at 37 degree celcius, 5% CO and 95% humidity, 2.5 uL of CellTiter-Glo (Promega) was dispensed into each well using a dispenser (Aspect Automation, St. Paul, MN) with solenoid valves (Lee Valves, Westbrook CT). Plates were left at room temperature for 10 min before imaging the ATP-coupled luminescence using a ViewLux microplate imager (PerkinElmer, Waltham, MA).

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.