External ID: CBX7-Alpha-primary

Protocol: The protocol for the CBX7 AlphaScreen that we use is as follows: We create a standard alpha buffer (50mM HEPES, 150mM NaCl, 0.1% w/v BSA, 0.01% w/v Tween20) at pH 8.0 and make up two stock solutions at 2x final concentration in the alpha buffer. Solution A contains Human His6-CBX7 is used with biotinylated-H3K27me3 (residues 20-34), at 100nM and 50nM respectively. Solution B contains 20nM streptavidin donor beads and 20nM nickel acceptor beads. For 384 well assay formats, 10uL of solution A is added to each well of the assay plate and the plate is spun at 1000 rpm for 30 seconds. 100 nL of experimental ccompounds from stock plates are delivered by robotic pin transfer using a Janus Workstation (PerkinElmer), allowing the compounds to interact with CBX7 binding prior to assay measurement, followed by another spin and an incubation room temperature. Finally, 10uL of solution B is added to each well, the plate is spun again and then incubated at room temperature. AlphaScreen measurements are performed on an Envision 2104 (PerkinElmer) utilizing the manufacturer's protocol that has the correct excitation and emission wavelengths, cutoff filters, delay time, etc. We also do a crosstalk calculation through the Envision software to correct for luminescence for adjacent wells while reading the plate. The signal is then normalized to DMSO control wells on the compound plate prior to creation of the IC50 curves.

Comment:

External ID: LDHA

Protocol: The assay was optimized to 1536-well plates. Briefly, 3 muL of human lactate dehydrogenase 5 (#A38558H, Meridian Life Science, Inc., Memphis, TN) in LDH assay buffer (200 mM Tris HCl pH 7.4, 100 microM EDTA and 0.01% Tween-20) was added to a black solid bottom 1536-well assay plate (Greiner Bio-One) using a BioRAPTR FRD dispenser (Beckman Coulter, Brea, CA). A 1536-well pintool dispenser outfitted with 20 nL pins (Wako Automation, San Diego, CA) was used to transfer 23 nL of DMSO-solubilized compound (both library and vehicle controls) to each 1536-well assay plate. Following compound transfer, 1 muL of substrate solution containing NADH and sodium pyruvate (Sigma-Aldrich, St. Louis, MO) in LDH assay buffer was dispensed via BioRAPTR FRD to initiate the reaction. Final concentrations in the 4 microL reaction volume were 2 nM LDHA enzyme, 0.06 mM NADH and 0.2 mM sodium pyruvate. Following a 5 minute incubation period at room temperature, 1 muL of detection reagent (C. kluyveri diaphorase (Sigma-Aldrich) and resazurin sodium salt (Sigma-Aldrich) in LDH assay buffer) was added to a total volume of 5 muL. Final concentrations of detection reagents were 0.133 mg/mL diaphorase and 37 muM resazurin. Plates were immediately transferred to a ViewLux microplate imager (PerkinElmer, Waltham, MA), and any resulting resorufin fluorescence was measured (ex540, em590 nm) at 0 and 20 minutes. Fluorescence was normalized using enzyme-free and DMSO-treated control wells on each plate.

Comment: PubChem score indicates % inhibition at 229 uM or 114 uM of the compound (Max_response). Active compounds with Max_response greater than -100%, PUBCHEM_ACTIVITY_SCORE is 100. Active compounds with Max_response between -80% and -100%, PUBCHEM_ACTIVITY_SCORE is 80. Active compounds with Max_response between -60% and -80%, PUBCHEM_ACTIVITY_SCORE is 60. Active compounds with Max_response between -30% and -60%, PUBCHEM_ACTIVITY_SCORE is 40. Inconclusive Compounds with Max_response between -25% and -30% have PUBCHEM_ACTIVITY_SCORE 20. For all inactive compounds with Max_response greater than -25%, PUBCHEM_ACTIVITY_SCORE is 0

External ID: stopgo-p2-SytraCBC-dual-FF

Protocol: Reagents:
StopGo2A HEK-NPG-Luc cells and other necessary media and controls were provided by Dr. Atkins's lab. Compound PTC124 was used as a control. Dose response was assessed using a 1:2 dilution of control compound, with the top dose of 50uM final concentration. Background signal was normalized to wells treated with DMSO only.

Assay Protocol:
In brief, four microliters (2400 cells/well) of StopGo2A HEK-NPG-Luc cells were plated and incubated overnight at 37C. Twenty-three nanoliters of compounds and control were added to the wells using a 1536 array Kalypsys pintool workstation. After an over night incubation (at 37C) with compounds / control, 2.5 uL of Dual/Glo Firefly reagent was added. Firefly luciferase signal was obtained using the PerkinElmer ViewLux plate reader after 20min of room temperature incubation. Dual/Glo Renilla Lucifrase reagents were then added followed by additional 20 min room temperature incubation. Finally, the renilla luciferase signal was obtained using the PerkinElmer ViewLux reader.

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: 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: stopgo-p2-SytraCBC-dual-Ren

Protocol: Reagents:
StopGo2A HEK-NPG-Luc cells and other necessary media and controls were provided by Dr. Atkins's lab. Compound PTC124 was used as a control. Dose response was assessed using a 1:2 dilution of control compound, with the top dose of 50uM final concentration. Background signal was normalized to wells treated with DMSO only.

Assay Protocol:
In brief, four microliters (2400 cells/well) of StopGo2A HEK-NPG-Luc cells were plated and incubated overnight at 37C. Twenty-three nanoliters of compounds and control were added to the wells using a 1536 array Kalypsys pintool workstation. After an overnight incubation (at 37C) with compounds / control, 2.5 uL of Dual/Glo Firefly reagent was added. Firefly luciferase signal was obtained using the PerkinElmer ViewLux plate reader after 20min of room temperature incubation. Dual/Glo Renilla Lucifrase reagents were then added followed by additional 20 min room temperature incubation. Finally, the renilla luciferase signal was obtained using the PerkinElmer ViewLux reader.

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: 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: 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.