External ID: cmt-p4-fda-celltiter_regid

Protocol: Native S16 cells were seeded in white solid-bottom 1536 well plates at between 500-750 cells/6uL in DMEM medium containing 10% FBS, w/o phenol red. After overnight incubation at 37 degree Celsius/5% C02, 23 nL of compounds or DMSO were delivered to each well using a pin tool, followed by 24h-incubation at 37 degree Celsius/5% C02. Then one volume of CellTiter-Glo assay reagent (Promega) was added to each well for cell viability measured by a ViewLux plate reader (PerkinElmer). The % activity was determined by normalizing to the average readings from wells containing no cells relative to DMSO-treated cells (0% Activity). The concentration-effect curves were then classified based on curve quality (r2), response magnitude and degree of measured activity.

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, toxic compounds are ranked higher than non-toxic compounds.
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. Toxic compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.

External ID: HMS1158_MLP

Protocol: A 5 mL overnight pre-culture of E. coli is started by inoculating a tube of lysogeny broth with a single colony from an agar plate. The tube is incubated at 37 degrees C overnight with shaking. The resulting saturated culture is removed from the shaker and diluted into fresh lysogeny broth at 1:100 proportion and incubated at 37 degrees C with shaking for two hours. The culture is removed from the shaker and absorbance is measured at 600 nm. The culture is diluted into fresh lysogeny broth to an estimated absorbance of 0.0002. For each library plate to be screened, two 384-well assay plates are filled with 30 microL per well of fresh lysogeny broth, except for the positive control columns, which are left empty. The multiple of two is necessary to expose the strain in duplicate to the same compounds from a given library plate.

300 nL of experimental compounds are transferred into the assay plates from library plates using a robotically manipulated stainless steel pin array, exposing the strain to the same compounds at the same concentration. The positive control columns are filled with 30 microL of the positive control compound in LB. Thirty microL of E. coli culture at absorbance of 0.0002 are then added to the assay plates, producing a total volume of 60 microL in each well. Plates are incubated at 37 degrees C without shaking and with humidification for 23 hours. The plates are removed from the incubator, and absorbance of the cultures is measured at 600 nm using a PerkinElmer EnVision plate reader. The absorbance is used as the readout.

Positive control: (R)-4-(4-(2-fluoro-4-methoxyphenyl)-2-oxopyridin-1(2H)-yl)-N-hydroxy-2-methyl-2-(methylsulfonyl)butanamide was present at 0.0754 microM in all wells of column 24.

Negative control: No treatment in remaining empty wells of the assay plate (minimally, wells in column 23).

Comment: A threshold constant was calculated by subtracting 3.6 X experimental well standard deviation for absorbance from the experimental well average absorbance, combining all plates in a screening run. Well absorbance was then subtracted from this threshold value. Wells were considered active if the resulting value after subtracting the threshold constant > 0 for at least one replicate. Activity scores were calculated by subtracting well absorbance from negative control plate average absorbance, dividing by the difference between plate negative and positive control plate average absorbance, and multiplying by 100. Resulting values > 100 were set to 100 and < 0 were set to 0, where 100 = 100% activity.

External ID: HMS1482

Protocol: Prior to screening, cells were passaged in high glucose DMEM supplemented with 10% FBS, 1% P/S, 1 mM pyruvate, and HEPES. Media was changed every 1-2 days to prevent starvation and maintain cellular health.

On the day of screening, 384-well plates (Corning 3765) were filled with 30 uL of control cybrid cell suspension (33.3 cells/ul). Media for resuspension was DMEM supplemented with 10% FBS, 1% P/S, 1 mM pyruvate, and HEPES. Positive control columns were 23 and 24 for most plate setups. Initially, tunicamycin (15 nM) and galactose media (10 mM + 1 mM glucosamine) were used in columns 23 and 24, respectively. Over the course of the screen, galactose was substituted with Vidofludimus (11 uM). Next, 33 nL of each compound were pin-transferred to each plate in duplicate. Final assay well volume was 30 uL. Plates were stacked 2 high, covered with lids, and incubated at 37 degrees C for 2 days.

After 2 days of incubation, Hoescht (10 ul/well) was added to assay plates and fluorescence was read using the Acumen laser scanning cytometer. After, the bottom of plates were sealed with two layers of Brightmax Sealing Films. Then Promega NanoGlo Live cell reagent (20 ul/well) was added to each well and luminescence was read on a PerkinElmer EnVision plate reader after 5 minute of shaking.

Comment: Data analysis method and criteria for scoring active compounds:

Normalized luminescence values (luminescence / cell number) for each replicate were calculated (based on average of 2 luminescence reads per replicate). Z-scores were calculated using the plate average and standard deviation of experimental well normalized values. Each plate replicate was individually analyzed to measure reproducibility across plates. Z-scores were then averaged across replicates and a Z-score threshold of 1.96 was selected for positive hits, indicating increased mitochondrial supercomplex formation. Wells were excluded from further consideration based on low cell number (1 or both replicates with number of objects count < 1500). Activity scores were determined by scaling Z-scores from 0 (activity score = 0) to 4 (activity score = 100), with activity score > 50 being considered active. Z-scores < 0 were set to activity score = 0; Z-scores > 4 were set to activity score = 100 (100% activity).

External ID: MCF7_OneDose

Protocol: NCI-60 Human Tumor Cell Lines Screen was performed using the standard one-dose NCI protocol, which is described in more detail at https://dtp.cancer.gov/discovery_development/nci-60/default.htm.

Compounds with GIPRCNT values below 10 were considered active. Activity score was based on GIPRCNT using the following formula:
max(-GIPRCNT/2 + 50, 0)
Score is 0 for GIPRCNT values of 100 or above, 50 for GIPRCNT values of 0, and 100 for GIPRCNT values of -100.

Comment: These data are a subset of the data from the NCI human tumor cell line screen. Compounds are identified by the NCI National Service Center (NSC) number, which is contained in the
PUBCHEM_EXT_DATASOURCE_REGID field. In the NCI cell line identification system, MCF7 is panel number 5, cell number 1. PANELCODE or PANELNAME identify the NCI-60 cell panel. PANELNBR does NOT identify the NCI-60 panel.

EXPID is the NCI internal tracking number for the experiment identifier, and PREFIX = 'S' for the NSCs submitted through the NCI Chemical Agents repository.

Substance concentration unit is indicated in the data table, using the following abbreviations: M = molar (M), u = micrograms/milliliter (microg/mL), V = volumetric fraction.

M_GIPRCNT represents the mean growth percent for that experiment, and N_GIPRCNT indicates the number of values for that NSC within an experiment, while STDEV_GIPRCNT is the standard deviation. Most NSCs are tested once per experiment, resulting in EXP_COUNT = 1.

External ID: adst-fluc-km-fda-o1_regid

Protocol: Three microliters/well of substrate- buffer solution (0.01 mM D-Luciferin, 0.01 mM ATP, 50 mM Tris Acetate, 10 mM Mg Acetate, 0.01% Tween-20 and 0.038% BSA final concentrations) were dispensed into white solid-bottom 1536 well plates. Compounds were transferred to the plates in 23nl, and one microliter/well luciferase enzyme-buffer solution (10 nM P. pyralis luciferase and 50 mM Tris Acetate final concentrations) was dispensed yielding a final reaction volume of 4 ul/well. Luciferase luminescence was measured by a ViewLux plate reader within 5 minutes of enzyme-buffer addition. The % activity was determined by normalizing to the average readings of PTC124 (a positive control) relative to DMSO (0% Activity). The concentration-effect curves were then classified based on curve quality (r2), response magnitude and degree of measured activity.

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: IGROV1_OneDose

Protocol: NCI-60 Human Tumor Cell Lines Screen was performed using the standard one-dose NCI protocol, which is described in more detail at https://dtp.cancer.gov/discovery_development/nci-60/default.htm.

Compounds with GIPRCNT values below 10 were considered active. Activity score was based on GIPRCNT using the following formula:
max(-GIPRCNT/2 + 50, 0)
Score is 0 for GIPRCNT values of 100 or above, 50 for GIPRCNT values of 0, and 100 for GIPRCNT values of -100.

Comment: These data are a subset of the data from the NCI human tumor cell line screen. Compounds are identified by the NCI National Service Center (NSC) number, which is contained in the
PUBCHEM_EXT_DATASOURCE_REGID field. In the NCI cell line identification system, IGROV1 is panel number 6, cell number 10. PANELCODE or PANELNAME identify the NCI-60 cell panel. PANELNBR does NOT identify the NCI-60 panel.

EXPID is the NCI internal tracking number for the experiment identifier, and PREFIX = 'S' for the NSCs submitted through the NCI Chemical Agents repository.

Substance concentration unit is indicated in the data table, using the following abbreviations: M = molar (M), u = micrograms/milliliter (microg/mL), V = volumetric fraction.

M_GIPRCNT represents the mean growth percent for that experiment, and N_GIPRCNT indicates the number of values for that NSC within an experiment, while STDEV_GIPRCNT is the standard deviation. Most NSCs are tested once per experiment, resulting in EXP_COUNT = 1.

External ID: cmt-p4-fluc-fda_regid

Protocol: S16 Schwann cell PMP22 intronic element firefly luciferase assay
The FLuc-expressing S16 cells were seeded in white solid 1536-well plates at between 500-750 cells/4uL in DMEM medium containing 10% FBS, w/o phenol red. After overnight incubation at 37 degree Celsius/5% C02, 23 nL of compounds or DMSO were delivered to each well using a pin tool, followed by 24h-incubation at 37 degree Celsius/5% C02. Then 4 uL ONE-Glo luminescent substrate mix (Promega) was added to each well. Luminescence was measured on a ViewLux plate reader (Perkin Elmer). The % activity was determined by normalizing to the average readings of PTC124 (a positive control) - treated cells relative to DMSO-treated cells (0% Activity). The concentration-effect curves were then classified based on curve quality (r2), response magnitude and degree of measured activity.

Comment: Compound Ranking:

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

External ID: PolK100

Protocol: Three microliters of reagents (buffer in column 3 and 4 as negative control and 10 nM Pol kappa in columns 1, 2, and 5-48) were dispensed into a 1536-well black solid-bottom plate. Compounds (23 nL) were transferred via Kalypsys pin tool equipped with 1536-pin array. The plates were then incubated for 15 min at room temperature, and 1 uL substrate (50 nM final concentration) were then added to start the reaction and kinetically read twice at 0 min and 10 min on the Viewlux reader

Comment: Compound Ranking:

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

External ID: HMS1364-MLP_WT

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

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

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

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

External ID: HMS1485

Protocol: Prior to screening, HeLa cells were transduced with a lentivirus carrying the vector PHAGE-N CMVt N-RIG3 p53(R273C) encoding constitutively expressed SGFP2 fused to histone 2B and mRuby-p53(R273C). Following transduction, the cells were selected using neomycin and fluorescent activated cell sorting. The day before the compound treatment, cells were plated (750 cells/well) in a final volume of 30 L/well of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum using a Thermo Combi. Plates were spun for 45 s at 500 rpm and incubated overnight (~24 h) at 37 degrees C, 5% CO2.

On the day of the screening, 33 nL of each compound was transferred to assay wells via a custom pin-transfer workstation. Additionally, 33 nL of positive (Velcade 300 M) and negative (DMSO 100%) controls were transferred to each plate into the corresponding columns that did not contain experimental compounds. For every compound library plate, there were two daughter plates (A and B). Plates were stacked 5 high, covered with lids, and incubated at 37 degrees C for 24h and 48h.

Assay plates were read using an Acumen Laser Scanning Cytometer 24h and 48h after compound treatment, using the following settings: For SGFP2 (channel 2), the 488 nm laser was used, with 6 mW output, 600 voltage gain and sensitivity threshold at 1; and for mRuby (channel 3), the 561 nm laser was used, with 8 mW output, 600 voltage gain and sensitivity threshold at 1.

Comment: Total nuclei and %p53 positive was calculated for all wells at 24 and 48h using the Cellista software. Percentage of p53 positive cells values were normalized to the positive and negative controls to determine a normalized percent of p53 activation. This was done on a per plate basis. The proteasome inhibitor Velcade (Adipogen, AG-CR1-3602-M005) at 330 nM was used as positive control while only the vehicle (DMSO, Sigma, D8418, 0.1% final concentration) was used as negative control. From these values the following normalized values were calculated (for both 24h and 48h): Normalized total nuclei = (total nuclei in experimental well / plate average negative control total nuclei) * 100. Normalized %p53 positive = (%p53 positive in experimental well - plate average negative control %p53 positive cells) / (plate average positive control %p53 positive cells - plate average negative control %p53 positive cells) * 100.

Based on the normalized percent p53 positive values, a compound was considered active when replicate average normalized % p53 positive cells > 50% for either or both time points. Activity scores were based on the replicate average normalized %p53 positive cells at 24hr. Values less than 0 were set to 0, and values > 100 were set to 100 (100% activity). Compounds with an activity score > 50 were considered active; some compounds were scored active despite having an activity score < 50 since % p53 positive cells was > 50% only at 48hr.

The average normalized total nuclei values were categorized as:
Very Low: x < 25%
Low: 25% < X > 50%
Medium: 50% < X > 75%
High: x < 75%

The average normalized % of p53 positive cells values were categorized as:
Low: x < 25%
Medium: 25% < X > 50%
Strong: 50% < X > 75%
Very Strong: x < 75%

Compounds that scored as strong or very strong in this classification (at either time point) were selected for follow up.

External ID: HMS1364-MLP_ugtP

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

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

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

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

External ID: HMS1149-MLP

Protocol: A 558 bp region upstream of hspX was PCR amplified and cloned upstream of the GFP-mut2 (Cormack et al., 1996). This construct was cloned into a modified version of the replicating plasmid pSE100 and transformed into CDC1551 to generate CDC1551(hspX'::GFP).

The M. tuberculosis CDC 1551 (hspX'::GFP) fluorescent biosensor was grown in pH 7.0 Middlebrook 7H9 medium to mid- to late-log phase. Black-walled, clear-bottom, tissue-culture treated 384-well microtiter plates (Corning #3712) were filled with 25 uL of buffered pH 7.0 7H9 medium using a Matrix Combi. 100 nL of compound was then transferred to each well via stainless steel pin array. Plates were manually sealed with DMSO-resistant aluminum seals and frozen at -80C until they were ready to be screened. The cultures were re-suspended in pH 7.0 7H9 and 25 uL was dispensed into each well of the 384-well assay plates already containing the perturbator utilizing a Cy-Bio Selma liquid handler robot to an OD595 of 0.05. The plates were then placed in a Ziploc bag with a moistened paper towel (to limit evaporation) and incubated for 6 days at 37C. Fluorescence and optical density (OD) readings were made on an EnSpire plate reader (Perkin Elmer, Inc.) at excitation and emission wavelengths of 488 and 509 nm as a top read, with the OD being taken at 595 nm as a bottom read.

The ~328,633 compound library was arrayed into black-walled, clear-bottom tissue-culture treated 384-well microtiter plates (Corning #3712) by pin transfer (100 nL), containing 25 uL of buffered pH 7.0 7H9 medium. In a previous screen utilizing the same compound library and screening method, four random compound plates were screened in duplicate to observe variation in hit robustness across plates and method. The result was that the duplicate plates did not vary in a statistically significant manner and due to the high volume of compounds to be screened (~328,633), replicates were not utilized.

Negative control: DMSO, 0.1% final concentration in buffered pH 7.0 7H9 in column 2 or column 23 of each plate

Positive control: 0.3 uM Rifampicin final concentration in buffered pH 7.0 7H9 in column 1 or column 24 of each plate

Comment: Analysis methods:
To calculate normalized fluorescence percent inhibition, plate average negative control fluorescence intensity (based on all plates within a screening run) was subtracted from well fluorescence intensity, divided by the difference between plate negative and positive control average fluorescence intensity (based on all plates within a screening run), and multiplied by 100. To calculate normalized growth inhibition, plate average negative control absorbance (based on all plates within a screening run) was subtracted from well absorbance, divided by the difference between plate negative and positive control average absorbance (based on all plates within a screening run), and multiplied by 100. Normalized fluorescence percent inhibition was divided by normalized growth inhibition to calculate a ratio. Wells were considered active if growth was not greatly decreased and showed either a 3-fold selectivity fluorescence to growth inhibition ratio (with 35-45% fluorescence inhibition) or a 1.5-fold selectivity fluorescence to growth inhibition ratio (with > 45% fluorescence inhibition). These compounds may be directly or indirectly inhibiting DosRS signaling. Wells were considered general inhibitors of M. tuberculosis growth if both fluorescence and growth inhibition > 50%. Activity scores were scaled from 100 to 0 and derived from normalized fluorescence percent inhibition, with 100 (or > 100) = 100% inhibition and 0 (or < 0) = no inhibition.