External ID: DKFZ_drug_screen_chromothripsis_LFS_MB_P

Protocol: For each cell line included in the screen (UWB1.289, UWB1.289+BRCA1, MDA-MB-436, HD-MB03, HD-N33, normal human astrocytes, SaOS-2, KHOS-240S, SJSA-1, LFS_MB_P (primary tumor), LFS_MB_1R (first relapse), LFS_MB_2R (second relapse), RCMB18, BT084 and ICB984) the IC20 and IC40 values of BGB 290 (Pamiparib, MedChemExpress, HY-104044) and cisplatin (MedChemExpress, HY-17394) were determined. The 375 compounds from 2 drug libraries, namely TargetMol (Catalog No. L3900) and DiscoveryProbe (ApexBio, L1033) were diluted in 96-well plates to achieve final concentrations of 5 microM, 0.5 microM, 0.05 microM and 0.005 microM. Cells were then seeded at optimized densities. Each cell line or tumor entity was treated with its respective IC20 concentration of BGB 290 or cisplatin. Spheroids from patient-derived xenograft models were treated with both IC20 and IC40 of BGB 290. Cells were incubated at 37 degrees C for 96 hours. The metabolic activity was measured after 96 hours with the ATPlite assay (Perkin Elmer, 6016947). Values from the blank measurements were subtracted from the treatment wells and normalized to the vehicle controls. 10% DMSO treatment was used as positive control as measure of 100% metabolic inhibition whereas vehicle DMSO concentration was used as negative control as measure of 0% metabolic inhibition. The effect of single treatments was compared to the combination treatments to identify drugs that have potential additive or synergistic effects with BGB-290 or cisplatin or both. CART (https://cart.embl.de/) was used to match chemicals to Pubchem identifiers and for drug-annotation enrichment analysis. Compounds were scored as active in Pubchem if the average cell metabolic activity inhibition is at least 80% in single or combination treatments at the 0.5 uM concentration.

Comment: A synergistic interaction between HDAC- and PARP inhibitors in childhood tumors with chromothripsis
https://www.biorxiv.org/content/10.1101/2021.04.22.440879v1

External ID: SERCaMPGLuc-p1-antagonist

Protocol: PROTOCOL TABLES
SEQUENCE No. (1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE and DESCRIPTION.
1; Reagent; 5 uL; 1000 SH-SY5Y cells per wells
2; Time; 5 hour; 37C, 5% CO2
3; Compound; 23 nL; Control inhibitor / compound library
4; Time; 16 hour; 37C, 5% CO2
5; Reagent; 100 nM; Thapsigargin
6; Time; 4 hour; 37C, 5% CO2
7; Reagent; 1 uL; 0.5x coelenterazine
8; Detection; Luminescence; ViewLux imaging system

NOTES (numbers refer to sequence above)
1; SH-SY5Y human neuroblastoma cells stably expressing GLuc-SERCaMP (SH-SY5Y-GLuc-ASARTDL) cells were seeded in 1,536 well white tissue culture treated plates (Corning, Cat# 7464) in DMEM-high glucose-sodium pyruvate (ThermoFisher Scientific, Cat #10569) supplemented with 10% bovine growth serum (Hyclone), 10 U/ml penicillin (Gibco), 10 ug/ml streptomycin (Gibco), and 20 mM HEPES.
2; Assay plates were incubated for 5 hour at 37C in a humidified incubator containing 5% CO2.
3; qHTS libraries (23 nl, final concentrations of 1.53 uM, 7.67 uM, 38.3 uM) or controls (neutral control: DMSO, positive control: dantrolene) were added using a Kalypsis pin-tool Robotic System equipped with 1536 pinheads.
4; Cells were then incubated for 16 hours at 37C, 5% CO2.
5; Thapsigargin was added at 100 nM to deplete ER calcium stores.
6; Cells were incubated for 4 hour (37oC, 5% CO2)
7; Gaussia luciferase in the medium was measured by adding 1 ul of 0.5x coelenterazine (final concentration 0.07x) prepared in Gaussia Luciferase Glow Assay Buffer (Pierce), without addition of the Cell Lysis Buffer Reagent.
8; Luminescence was measured using a ViewLux high-432 throughput CCD imaging system (Perkin Elmer) equipped with clear filters. Compounds exhibiting inhibitory activity (defined as curve class -1.1, -1.2, -1.3, -1.4, -2.1, -2.2, -2.3, -2.4, -3) were identified by normalizing plate-wise to corresponding intra-plate controls (neutral control = Tg only; positive control (100% inhibition) = DMSO vehicle) with percent activity derived using in-house software (https://tripod.nih.gov/curvefit). The same controls were also used for the calculation of the Z' factor, a measure of assay quality control, as previously described (Zhang et al., 1999). For the initial validation of activity in the SERCaMP assays, hits from the primary screen were assayed again at 11-concentrations (1.3 nM - 76.6 uM). SH-SY5Y-GLuc-SERCaMP cells were assayed for ER Ca2+ depletion as outlined above.

Reference:
1. Zhang, J.H., Chung, T.D., and Oldenburg, K.R. (1999). A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J Biomol Screen 4, 67-73.

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: DKFZ_drug_screen_chromothripsis_ICB984_MB

Protocol: For each cell line included in the screen (UWB1.289, UWB1.289+BRCA1, MDA-MB-436, HD-MB03, HD-N33, normal human astrocytes, SaOS-2, KHOS-240S, SJSA-1, LFS_MB_P (primary tumor), LFS_MB_1R (first relapse), LFS_MB_2R (second relapse), RCMB18, BT084 and ICB984) the IC20 and IC40 values of BGB 290 (Pamiparib, MedChemExpress, HY-104044) and cisplatin (MedChemExpress, HY-17394) were determined. The 375 compounds from 2 drug libraries, namely TargetMol (Catalog No. L3900) and DiscoveryProbe (ApexBio, L1033) were diluted in 96-well plates to achieve final concentrations of 5 microM, 0.5 microM, 0.05 microM and 0.005 microM. Cells were then seeded at optimized densities. Each cell line or tumor entity was treated with its respective IC20 concentration of BGB 290 or cisplatin. Spheroids from patient-derived xenograft models were treated with both IC20 and IC40 of BGB 290. Cells were incubated at 37 degrees C for 96 hours. The metabolic activity was measured after 96 hours with the ATPlite assay (Perkin Elmer, 6016947). Values from the blank measurements were subtracted from the treatment wells and normalized to the vehicle controls. 10% DMSO treatment was used as positive control as measure of 100% metabolic inhibition whereas vehicle DMSO concentration was used as negative control as measure of 0% metabolic inhibition. The effect of single treatments was compared to the combination treatments to identify drugs that have potential additive or synergistic effects with BGB-290 or cisplatin or both. CART (https://cart.embl.de/) was used to match chemicals to Pubchem identifiers and for drug-annotation enrichment analysis. Compounds were scored as active in Pubchem if the average cell metabolic activity inhibition is at least 80% in single or combination treatments at the 0.5 uM concentration.

Comment: A synergistic interaction between HDAC- and PARP inhibitors in childhood tumors with chromothripsis
https://www.biorxiv.org/content/10.1101/2021.04.22.440879v1

External ID: CHEMBL4320927

Protocol: N/A

Comment: Journal: Bioorg Med Chem Lett
Year: 2019
Volume: 29
Issue: 9
First Page: 1127
Last Page: 1132
DOI: 10.1016/j.bmcl.2019.02.025

External ID: DKFZ_drug_screen_chromothripsis_HDN33

Protocol: For each cell line included in the screen (UWB1.289, UWB1.289+BRCA1, MDA-MB-436, HD-MB03, HD-N33, normal human astrocytes, SaOS-2, KHOS-240S, SJSA-1, LFS_MB_P (primary tumor), LFS_MB_1R (first relapse), LFS_MB_2R (second relapse), RCMB18, BT084 and ICB984) the IC20 and IC40 values of BGB 290 (Pamiparib, MedChemExpress, HY-104044) and cisplatin (MedChemExpress, HY-17394) were determined. The 375 compounds from 2 drug libraries, namely TargetMol (Catalog No. L3900) and DiscoveryProbe (ApexBio, L1033) were diluted in 96-well plates to achieve final concentrations of 5 microM, 0.5 microM, 0.05 microM and 0.005 microM. Cells were then seeded at optimized densities. Each cell line or tumor entity was treated with its respective IC20 concentration of BGB 290 or cisplatin. Spheroids from patient-derived xenograft models were treated with both IC20 and IC40 of BGB 290. Cells were incubated at 37 degrees C for 96 hours. The metabolic activity was measured after 96 hours with the ATPlite assay (Perkin Elmer, 6016947). Values from the blank measurements were subtracted from the treatment wells and normalized to the vehicle controls. 10% DMSO treatment was used as positive control as measure of 100% metabolic inhibition whereas vehicle DMSO concentration was used as negative control as measure of 0% metabolic inhibition. The effect of single treatments was compared to the combination treatments to identify drugs that have potential additive or synergistic effects with BGB-290 or cisplatin or both. CART (https://cart.embl.de/) was used to match chemicals to Pubchem identifiers and for drug-annotation enrichment analysis. Compounds were scored as active in Pubchem if the average cell metabolic activity inhibition is at least 80% in single or combination treatments at the 0.5 uM concentration.

Comment: A synergistic interaction between HDAC- and PARP inhibitors in childhood tumors with chromothripsis
https://www.biorxiv.org/content/10.1101/2021.04.22.440879v1

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: DKFZ_drug_screen_chromothripsis_HD-MB03

Protocol: For each cell line included in the screen (UWB1.289, UWB1.289+BRCA1, MDA-MB-436, HD-MB03, HD-N33, normal human astrocytes, SaOS-2, KHOS-240S, SJSA-1, LFS_MB_P (primary tumor), LFS_MB_1R (first relapse), LFS_MB_2R (second relapse), RCMB18, BT084 and ICB984) the IC20 and IC40 values of BGB 290 (Pamiparib, MedChemExpress, HY-104044) and cisplatin (MedChemExpress, HY-17394) were determined. The 375 compounds from 2 drug libraries, namely TargetMol (Catalog No. L3900) and DiscoveryProbe (ApexBio, L1033) were diluted in 96-well plates to achieve final concentrations of 5 microM, 0.5 microM, 0.05 microM and 0.005 microM. Cells were then seeded at optimized densities. Each cell line or tumor entity was treated with its respective IC20 concentration of BGB 290 or cisplatin. Spheroids from patient-derived xenograft models were treated with both IC20 and IC40 of BGB 290. Cells were incubated at 37 degrees C for 96 hours. The metabolic activity was measured after 96 hours with the ATPlite assay (Perkin Elmer, 6016947). Values from the blank measurements were subtracted from the treatment wells and normalized to the vehicle controls. 10% DMSO treatment was used as positive control as measure of 100% metabolic inhibition whereas vehicle DMSO concentration was used as negative control as measure of 0% metabolic inhibition. The effect of single treatments was compared to the combination treatments to identify drugs that have potential additive or synergistic effects with BGB-290 or cisplatin or both. CART (https://cart.embl.de/) was used to match chemicals to Pubchem identifiers and for drug-annotation enrichment analysis. Compounds were scored as active in Pubchem if the average cell metabolic activity inhibition is at least 80% in single or combination treatments at the 0.5 uM concentration.

Comment: A synergistic interaction between HDAC- and PARP inhibitors in childhood tumors with chromothripsis
https://www.biorxiv.org/content/10.1101/2021.04.22.440879v1

External ID: CHEMBL4513082

Protocol: N/A

Comment: Target ChEMBL ID: CHEMBL4303835
ChEMBL Target Name: SARS-CoV-2
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

Data Source: SARS-CoV-2 Screening Data

External ID: DKFZ_drug_screen_chromothripsis_CRL2098

Protocol: For each cell line included in the screen (UWB1.289, UWB1.289+BRCA1, MDA-MB-436, HD-MB03, HD-N33, normal human astrocytes, SaOS-2, KHOS-240S, SJSA-1, LFS_MB_P (primary tumor), LFS_MB_1R (first relapse), LFS_MB_2R (second relapse), RCMB18, BT084 and ICB984) the IC20 and IC40 values of BGB 290 (Pamiparib, MedChemExpress, HY-104044) and cisplatin (MedChemExpress, HY-17394) were determined. The 375 compounds from 2 drug libraries, namely TargetMol (Catalog No. L3900) and DiscoveryProbe (ApexBio, L1033) were diluted in 96-well plates to achieve final concentrations of 5 microM, 0.5 microM, 0.05 microM and 0.005 microM. Cells were then seeded at optimized densities. Each cell line or tumor entity was treated with its respective IC20 concentration of BGB 290 or cisplatin. Spheroids from patient-derived xenograft models were treated with both IC20 and IC40 of BGB 290. Cells were incubated at 37 degrees C for 96 hours. The metabolic activity was measured after 96 hours with the ATPlite assay (Perkin Elmer, 6016947). Values from the blank measurements were subtracted from the treatment wells and normalized to the vehicle controls. 10% DMSO treatment was used as positive control as measure of 100% metabolic inhibition whereas vehicle DMSO concentration was used as negative control as measure of 0% metabolic inhibition. The effect of single treatments was compared to the combination treatments to identify drugs that have potential additive or synergistic effects with BGB-290 or cisplatin or both. CART (https://cart.embl.de/) was used to match chemicals to Pubchem identifiers and for drug-annotation enrichment analysis. Compounds were scored as active in Pubchem if the average cell metabolic activity inhibition is at least 80% in single or combination treatments at the 0.5 uM concentration.

Comment: A synergistic interaction between HDAC- and PARP inhibitors in childhood tumors with chromothripsis
https://www.biorxiv.org/content/10.1101/2021.04.22.440879v1

External ID: HMS1315

Protocol: Cybrid cells were washed with PBS, trypsinized, spun down at 300g for 5 minutes, and resuspended in 10 mLs of PBS. Cells were counted, and the appropriate amount of cells were added to a vial containing DMEM (No Glucose) + 2%FBS + 1% P/S + 10mM Galactose, DMEM (No Glucose) + 2%FBS + 1% P/S + 10mM Galactose + 0.3% DMSO, DMEM No Glucose + 2% FBS + 1% P/S + 10mM Galactose + 1uM I-BET GSK 525762A, or DMEM (No Glucose) + 2%FBS + 1% P/S + 10mM Galactose + 1.25mM Glucose. Cells were then seeded in a Corning 3570 384-well plate (40 ul/well,1500 cells/well). Two replicates were prepared as described at the same time per library plate.

100 nl of compound was added to each well via pin transfer immediately after seeding. After the addition of compound, cells were incubated for 72 hours at 37 degrees C with 5% CO2.

Following the 72 hour incubation period, the 384 well plates were centrifuged for 1 minute at 1000 rpm. The media was aspirated using an aspiration wand and fresh media DMEM only, was added to the cells (40uL/ well) using a well-mate. Fifteen-microliters of Cell Titer Glow substrate was then added to each well using the well-mate. The plates were gently vortexed for 10 seconds and then centrifuged for 1 minute at 1000 rpm. The plates were then immediately read on the Envision instrument and the luminescence was detected for each individual well.

Positive control (strong): Cells seeded in DMEM (No Glucose) + 2%FBS + 1% P/S + 10mM Galactose + 1.25mM Glucose
Positive control (weak): Cells seeded in DMEM No Glucose + 2% FBS + 1% P/S + 10mM Galactose + 1uM I-BET GSK 525762A
Negative control: Cells seeded in DMEM (No Glucose) + 2%FBS + 1% P/S + 10mM Galactose + 0.3% DMSO

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

Z-scores were calculated for both replicates separately using the plate average and standard deviation of experimental well luminescence (Z = (x - mu)/sigma). Compounds were considered active if both replicate Z-scores >= 1.8. Activity scores were determined by scaling replicate average Z-scores from 0 (activity score = 0) to 4 (activity score = 100), with activity score > 45 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: CHEMBL4303805

Protocol: N/A

Comment: Target ChEMBL ID: CHEMBL4303835
ChEMBL Target Name: SARS-CoV-2
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

Data Source: SARS-CoV-2 Screening Data

External ID: CHEMBL4495582

Protocol: N/A

Comment: Target ChEMBL ID: CHEMBL4523582
ChEMBL Target Name: Replicase polyprotein 1ab
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

Data Source: SARS-CoV-2 Screening Data

External ID: IucA Pilot Assay Tocris Library

Protocol: A solution containing 55.6 mM HEPES pH 7.5, 0.11% Tween 20, 16.7 mM MgCl2, 55.6 mM hydroxylamine, 55.6 microM ATP, 55.6 microM citrate, and 0.28 U/mL IPP was dispensed (45 microL) into clear polystyrene microplates (Corning, Inc.) using a BioTek MicroFlo dispenser.

Next, 40 nL of test compounds (10 mM in DMSO, 8 microM final concentration) were transferred from deep-well blocks to the reaction solution using a stainless-steel pin tool operated by a robotic workstation (JANUS, PerkinElmer, Waltham, MA). The IucA-catalyzed reaction was initiated by adding 5 microL of 3 microM IucA in 25 mM HEPES, 75 mM NaCl, and 0.1 mM TCEP at pH 7.5.

The reactions were allowed to proceed for 30 min at room temperature before being quenched by dispensing (microFill, BioTek) 13 microL of MG developing solution, containing 1.0 mg/mL MG oxalate, 1.5% (w/v) ammonium molybdate, 0.15% (v/v) Tween 20, and 4.7 N sulfuric acid. After allowing the assay color to develop/stabilize for 30 min, the absorbance at 620 nm was measured (EnVision 2103 Multilabel Microplate Reader, PerkinElmer).

Average positive controls from 24 wells with no test compound and average negative controls from 8 wells with no enzyme were calculated. Dynamic range was calculated by difference between Avg Pos Control and Avg Neg Control. Percent inhibition was calculated by the ratio of (Avg. Pos. Ctrl - Sample OD) to Dynamic Range.

Comment: Protein Target is
IucA

EMB09144
574 aa
G057_19877
Klebsiella pneumoniae hvKP1

Active compounds were defined by <80% activity at 8 microM screening concentration.

External ID: DKFZ_drug_screen_chromothripsis_CRL1545

Protocol: For each cell line included in the screen (UWB1.289, UWB1.289+BRCA1, MDA-MB-436, HD-MB03, HD-N33, normal human astrocytes, SaOS-2, KHOS-240S, SJSA-1, LFS_MB_P (primary tumor), LFS_MB_1R (first relapse), LFS_MB_2R (second relapse), RCMB18, BT084 and ICB984) the IC20 and IC40 values of BGB 290 (Pamiparib, MedChemExpress, HY-104044) and cisplatin (MedChemExpress, HY-17394) were determined. The 375 compounds from 2 drug libraries, namely TargetMol (Catalog No. L3900) and DiscoveryProbe (ApexBio, L1033) were diluted in 96-well plates to achieve final concentrations of 5 microM, 0.5 microM, 0.05 microM and 0.005 microM. Cells were then seeded at optimized densities. Each cell line or tumor entity was treated with its respective IC20 concentration of BGB 290 or cisplatin. Spheroids from patient-derived xenograft models were treated with both IC20 and IC40 of BGB 290. Cells were incubated at 37 degrees C for 96 hours. The metabolic activity was measured after 96 hours with the ATPlite assay (Perkin Elmer, 6016947). Values from the blank measurements were subtracted from the treatment wells and normalized to the vehicle controls. 10% DMSO treatment was used as positive control as measure of 100% metabolic inhibition whereas vehicle DMSO concentration was used as negative control as measure of 0% metabolic inhibition. The effect of single treatments was compared to the combination treatments to identify drugs that have potential additive or synergistic effects with BGB-290 or cisplatin or both. CART (https://cart.embl.de/) was used to match chemicals to Pubchem identifiers and for drug-annotation enrichment analysis. Compounds were scored as active in Pubchem if the average cell metabolic activity inhibition is at least 80% in single or combination treatments at the 0.5 uM concentration.

Comment: A synergistic interaction between HDAC- and PARP inhibitors in childhood tumors with chromothripsis

https://www.biorxiv.org/content/10.1101/2021.04.22.440879v1

External ID: 7124-01_Inhibitor_SinglePoint_HTS_Activity

Protocol:
Protocol:
1) Plate 10 uL BL2-NFkB-luc cells at 12.5K cells/well, using a Multidrop Combi reagent dispenser (1,250 cells/uL).
2) Add 25 nL compound or positive control, by pin transfer; IKK inhibitor VII will be used at 50 uM.
3) Incubate at 37 masculineC for 1 hour (allowing compounds to enter cells for action).
4) Add 5 uL 192 ng/mL tCD40L per well for a final concentration of 64 ng/ml using a Thermo Multidrop Combi.
5) Incubate at 37 masculineC for 4 hrs, then leave plate at RT for 30 minutes.
6) Add 15 uL SteadyGlo luciferase substrate (Promega) per well using a Thermo Multidrop Combi.
7) Incubate at RT for 5min, then read Luminescence using LJL Analyst HT plate reader.

Comment: PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.

PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.10.0.2) was applied.

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the mean of the normalized sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at -50.

PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 55.

PUBCHEM_ACTIVITY_OUTCOME:
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T

Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T

Samples passing AT only were assigned an outcome of 1 (inactive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T

Samples passing PAR_T only were assigned an outcome of 1 (inactive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T

External ID: CHEMBL4808149

Protocol: N/A

Comment: Target ChEMBL ID: CHEMBL1865
ChEMBL Target Name: Histone deacetylase 6
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

Data Source: Fraunhofer Institute HDAC6 screening

External ID: 2125-01_Inhibitor_SinglePoint_HTS_Activity

Protocol: Protocol:
PRC2 activity was measured using Dissociation-Enhanced Lanthanide Fluoro-ImmunoAssays (DELFIA) performed on 384-well, white, streptavidin-coated plates (PerkinElmer). In short, PRC2 was diluted to 30 ng and 3 ng per well in 20 uL 2X Enzyme Buffer (50mM Tris HCl pH 8.5, 10 mM DTT, 5 mM MgCl). Compounds were pinned at 100 nL per well and reactions were initiated with 20 uL of a 5 uM SAM (NEB) solution containing 600 nM H3[21-44]-GK-biotin (AnaSpec; PRC2).

Plates were incubated at room temperature for 1 hr and then washed three times with 100 uL of Wash Buffer (50mM Tris pH 7.4, 150 mM NaCl, 0.05% Tween 20, 0.2% BSA). 50 ul of Fluoroimmunoassay (FI) Buffer (50 mM Tris HCl pH 7.8, 150 mM NaCl, 0.05% Tween 40, 25 iM DTPA, 0.2% BSA, 0.05% BGG)containing a 1:4000 dilution of anti-H3K27me2 rabbit IgG (Cell Signaling, #9728) with 691 ng/mL Eu-N1-anti-rabbit IgG (PerkinElmer)was added to each well for PRC2 reactions.

Following 1 hr incubation at room temperature, the plates were washed three times with Wash Buffer and 50 uL of Enhancement Solution (PerkinElmer) was added to each well. Plates were incubated for 30 min at room temperature and time-resolved fluorescence (TRF) was measured on Wallac Envision 2104 Multilabel Reader (400 us window, 400 us delay, 320 excitation, 615 emission).

Comment: PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.
All well activities were then multiplied by -1 to create a positive activity readout value range, to match Pubchem convention.

PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the mean of the normalized sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 50.

PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 100.

PUBCHEM_ACTIVITY_OUTCOME:
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T

Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T

Samples passing AT only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T

Samples passing PAR_T only were assigned an outcome of 1 (inactive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T

External ID: CHEMBL4808150

Protocol: N/A

Comment: Target ChEMBL ID: CHEMBL1865
ChEMBL Target Name: Histone deacetylase 6
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

Data Source: Fraunhofer Institute HDAC6 screening

External ID: StopGo1

Protocol: 3000 Hek-293T-WT cells were plated overnight before adding 23 nL of FireFly inhibitors per well using a pintool workstation (Kalypsys) followed by incubation for 4 hr. Luciferase activity was then measured using Amplite Luciferase Reagent.

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: DKFZ_drug_screen_chromothripsis_LFS_MB_2R

Protocol: For each cell line included in the screen (UWB1.289, UWB1.289+BRCA1, MDA-MB-436, HD-MB03, HD-N33, normal human astrocytes, SaOS-2, KHOS-240S, SJSA-1, LFS_MB_P (primary tumor), LFS_MB_1R (first relapse), LFS_MB_2R (second relapse), RCMB18, BT084 and ICB984) the IC20 and IC40 values of BGB 290 (Pamiparib, MedChemExpress, HY-104044) and cisplatin (MedChemExpress, HY-17394) were determined. The 375 compounds from 2 drug libraries, namely TargetMol (Catalog No. L3900) and DiscoveryProbe (ApexBio, L1033) were diluted in 96-well plates to achieve final concentrations of 5 microM, 0.5 microM, 0.05 microM and 0.005 microM. Cells were then seeded at optimized densities. Each cell line or tumor entity was treated with its respective IC20 concentration of BGB 290 or cisplatin. Spheroids from patient-derived xenograft models were treated with both IC20 and IC40 of BGB 290. Cells were incubated at 37 degrees C for 96 hours. The metabolic activity was measured after 96 hours with the ATPlite assay (Perkin Elmer, 6016947). Values from the blank measurements were subtracted from the treatment wells and normalized to the vehicle controls. 10% DMSO treatment was used as positive control as measure of 100% metabolic inhibition whereas vehicle DMSO concentration was used as negative control as measure of 0% metabolic inhibition. The effect of single treatments was compared to the combination treatments to identify drugs that have potential additive or synergistic effects with BGB-290 or cisplatin or both. CART (https://cart.embl.de/) was used to match chemicals to Pubchem identifiers and for drug-annotation enrichment analysis. Compounds were scored as active in Pubchem if the average cell metabolic activity inhibition is at least 80% in single or combination treatments at the 0.5 uM concentration.

Comment: A synergistic interaction between HDAC- and PARP inhibitors in childhood tumors with chromothripsis
https://www.biorxiv.org/content/10.1101/2021.04.22.440879v1

External ID: DKFZ_drug_screen_chromothripsis_UWB1.289

Protocol: For each cell line included in the screen (UWB1.289, UWB1.289+BRCA1, MDA-MB-436, HD-MB03, HD-N33, normal human astrocytes, SaOS-2, KHOS-240S, SJSA-1, LFS_MB_P (primary tumor), LFS_MB_1R (first relapse), LFS_MB_2R (second relapse), RCMB18, BT084 and ICB984) the IC20 and IC40 values of BGB 290 (Pamiparib, MedChemExpress, HY-104044) and cisplatin (MedChemExpress, HY-17394) were determined. The 375 compounds from 2 drug libraries, namely TargetMol (Catalog No. L3900) and DiscoveryProbe (ApexBio, L1033) were diluted in 96-well plates to achieve final concentrations of 5 microM, 0.5 microM, 0.05 microM and 0.005 microM. Cells were then seeded at optimized densities. Each cell line or tumor entity was treated with its respective IC20 concentration of BGB 290 or cisplatin. Spheroids from patient-derived xenograft models were treated with both IC20 and IC40 of BGB 290. Cells were incubated at 37 degrees C for 96 hours. The metabolic activity was measured after 96 hours with the ATPlite assay (Perkin Elmer, 6016947). Values from the blank measurements were subtracted from the treatment wells and normalized to the vehicle controls. 10% DMSO treatment was used as positive control as measure of 100% metabolic inhibition whereas vehicle DMSO concentration was used as negative control as measure of 0% metabolic inhibition. The effect of single treatments was compared to the combination treatments to identify drugs that have potential additive or synergistic effects with BGB-290 or cisplatin or both. CART (https://cart.embl.de/) was used to match chemicals to Pubchem identifiers and for drug-annotation enrichment analysis. Data from the primary screen was analyzed using a shiny app developed for this purpose. Compounds were scored as active in Pubchem if the average cell metabolic activity inhibition is at least 80% in single or combination treatments for the 0.5 uM concentration.

Comment: A synergistic interaction between HDAC- and PARP inhibitors in childhood tumors with chromothripsis
https://www.biorxiv.org/content/10.1101/2021.04.22.440879v1

External ID: DKFZ_drug_screen_chromothripsis_UWB1.289_BRCA1

Protocol: For each cell line included in the screen (UWB1.289, UWB1.289+BRCA1, MDA-MB-436, HD-MB03, HD-N33, normal human astrocytes, SaOS-2, KHOS-240S, SJSA-1, LFS_MB_P (primary tumor), LFS_MB_1R (first relapse), LFS_MB_2R (second relapse), RCMB18, BT084 and ICB984) the IC20 and IC40 values of BGB 290 (Pamiparib, MedChemExpress, HY-104044) and cisplatin (MedChemExpress, HY-17394) were determined. The 375 compounds from 2 drug libraries, namely TargetMol (Catalog No. L3900) and DiscoveryProbe (ApexBio, L1033) were diluted in 96-well plates to achieve final concentrations of 5 microM, 0.5 microM, 0.05 microM and 0.005 microM. Cells were then seeded at optimized densities. Each cell line or tumor entity was treated with its respective IC20 concentration of BGB 290 or cisplatin. Spheroids from patient-derived xenograft models were treated with both IC20 and IC40 of BGB 290. Cells were incubated at 37 degrees C for 96 hours. The metabolic activity was measured after 96 hours with the ATPlite assay (Perkin Elmer, 6016947). Values from the blank measurements were subtracted from the treatment wells and normalized to the vehicle controls. 10% DMSO treatment was used as positive control as measure of 100% metabolic inhibition whereas vehicle DMSO concentration was used as negative control as measure of 0% metabolic inhibition. The effect of single treatments was compared to the combination treatments to identify drugs that have potential additive or synergistic effects with BGB-290 or cisplatin or both. CART (https://cart.embl.de/) was used to match chemicals to Pubchem identifiers and for drug-annotation enrichment analysis. Compounds were scored as active in Pubchem if the average cell metabolic activity inhibition is at least 80% in single or combination treatments at the 0.5 uM concentration.

Comment: A synergistic interaction between HDAC- and PARP inhibitors in childhood tumors with chromothripsis
https://www.biorxiv.org/content/10.1101/2021.04.22.440879v1

External ID: DKFZ_drug_screen_chromothripsis_BT084_ICGC_MB145

Protocol: For each cell line included in the screen (UWB1.289, UWB1.289+BRCA1, MDA-MB-436, HD-MB03, HD-N33, normal human astrocytes, SaOS-2, KHOS-240S, SJSA-1, LFS_MB_P (primary tumor), LFS_MB_1R (first relapse), LFS_MB_2R (second relapse), RCMB18, BT084 and ICB984) the IC20 and IC40 values of BGB 290 (Pamiparib, MedChemExpress, HY-104044) and cisplatin (MedChemExpress, HY-17394) were determined. The 375 compounds from 2 drug libraries, namely TargetMol (Catalog No. L3900) and DiscoveryProbe (ApexBio, L1033) were diluted in 96-well plates to achieve final concentrations of 5 microM, 0.5 microM, 0.05 microM and 0.005 microM. Cells were then seeded at optimized densities. Each cell line or tumor entity was treated with its respective IC20 concentration of BGB 290 or cisplatin. Spheroids from patient-derived xenograft models were treated with both IC20 and IC40 of BGB 290. Cells were incubated at 37 degrees C for 96 hours. The metabolic activity was measured after 96 hours with the ATPlite assay (Perkin Elmer, 6016947). Values from the blank measurements were subtracted from the treatment wells and normalized to the vehicle controls. 10% DMSO treatment was used as positive control as measure of 100% metabolic inhibition whereas vehicle DMSO concentration was used as negative control as measure of 0% metabolic inhibition. The effect of single treatments was compared to the combination treatments to identify drugs that have potential additive or synergistic effects with BGB-290 or cisplatin or both. CART (https://cart.embl.de/) was used to match chemicals to Pubchem identifiers and for drug-annotation enrichment analysis. Compounds were scored as active in Pubchem if the average cell metabolic activity inhibition is at least 80% in single or combination treatments at the 0.5 uM concentration.

Comment: A synergistic interaction between HDAC- and PARP inhibitors in childhood tumors with chromothripsis
https://www.biorxiv.org/content/10.1101/2021.04.22.440879v1