External ID: CHEMBL748379

Protocol: N/A

Comment: Journal: J Med Chem
Year: 1982
Volume: 25
Issue: 9
First Page: 1103
Last Page: 1106
DOI: 10.1021/jm00351a020

Target ChEMBL ID: CHEMBL2094109
ChEMBL Target Name: Muscarinic acetylcholine receptor
ChEMBL Target Type: PROTEIN FAMILY - Target is a group of closely related proteins
Relationship Type: H - Homologous protein target assigned
Confidence: Multiple homologous protein targets may be assigned

External ID: TargetID_659_CEMA

Protocol: Black, standard capacity streptavidin-coated 384-well plates (Pierce 15407) were first washed with 50 L of phosphate buffer (100 mM, pH 7.0; PB7) three times using a Biotek 405 ELX plate washer. Subsequently, 5 L of biotinylated pre-miRNA substrate (500 nM final) was dispensed into the plate using a Multidrop Combi Reagent Dispenser (Thermo Scientific). Plates were then centrifuged for 1 min at 1,000 RPM (223 g), sealed with plate tape, and incubated overnight at 4 C. The following morning, plates were washed three times with 50 L of PB7, followed by the addition of 5 L of Dicer digest buffer (20 mM Tris, 12 mM NaCl, 2.5 mM MgCl2, 1 mM fresh DTT, and 4.5% DMSO) and centrifugation. Compounds (50 nL of 5 mM DMSO stock, 25 M final) were then added into the sample wells using a Sciclone (Caliper) liquid handler with V&P pintool; the same volume of DMSO was added to the control wells. The plates were incubated at 25 C for 15 min before addition of 5 L of digest buffer containing 217 g/nL Dicer (108 g/mL Dicer, 5% glycerol and 0.01% Triton X-100 final, excess with respect to pre-miRNA). For the positive control wells, digest buffer without Dicer was added. The plates were centrifuged again and resealed before being placed in a 37 C incubator for 5 h. After Dicer cleavage, plates were washed three times with 50 L of PB7. mTet-HRP in PB7 (10 L, 750 nM final) was then dispensed into each well. The plates were subsequently centrifuged, sealed, and incubated at 25 C for 2 h. Plates were then washed three times with 50 L of wash buffer (2 mM imidazole, 260 mM NaCl, 0.5 mM EDTA, 0.1% Tween-20, pH 7.0), followed by washing three additional times with 50 L of PB7. Finally, SuperSignal West Pico (25 L; Pierce) was added, the plates were incubated at 25 C for 5 min, and chemiluminescence signal was detected using a PHERAstar plate reader using LUM plus module (BMG Labtech).

Comment: The activity outcome is based on a Z-score (number of standard deviations from the negative control mean) of 3 or higher on at least 50% times that the sample was screened.

For instance, if the sample was screened in n=4 runs, it would be considered active only if it had a Z-score of 3 or above in at least 2 runs.

External ID: RMI-FANCM-MM2

Protocol: FP HTS. Screening took place at the University of Wisconsin Small Molecule Screening and Synthesis Facility. A master mix of RMI core complex and F-MM2 (30 microL per well) was plated in black 384 shallow well plates (ThermoFisher, Waltham, MA), using a BioTek "MicroFlo Select" reagent dispenser. Compounds were added using a Beckman FX liquid handler; 0.33 microL of 10 mM stock was added for a final compound concentration of 33 microM. MM2 and cMM2 were each added to 4 wells of master mix per plate to a final concentration of 10 muM to serve as controls. Following compound addition, plates were covered, centrifuged briefly, and incubated for 20 minutes at room temperature. FP measurements were taking using a Tecan "Safire 2" microplate reader. Instrument settings were as follows: top read, EX 470, EM 525/20, G-factor 0.89947. A suitable gain was calculated from the first plate of each day. Z' scores for were calculated for each plate; plates with Z' scores <0.5 were rerun prior to analysis.

Comment:

External ID: CHEMBL5133515

Protocol: N/A

Comment: Journal: Bioorg Med Chem Lett
Year: 2022
Volume: 58
First Page: 128524
Last Page: 128524
DOI: 10.1016/j.bmcl.2021.128524

Target ChEMBL ID: CHEMBL614069
ChEMBL Target Name: A-431
ChEMBL Target Type: CELL-LINE - Target is a specific cell-line
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: MScreen:TargetID_600

Protocol: C-terminally 6xHis tagged CDK2/Cyclin A complex and N-terminally Flag tagged CDC25B C473S (372-566) were expressed and purified. Proteins were incubated together at a final concentration of 125 nM each for 1 hr prior to incubation with compound for 1 hr, followed by addition of anti-6xHis europium cryptate donor beads (Cisbio) and anti-Flag XL-665 acceptor beads (Cisbio) at a final dilution of 1:350 for 1 hr. 20mM potassium fluoride was added 10 minute prior to plate reading. Assay reagents were dispensed using a multidrop liquid dispenser (Thermo Scientific) onto uncoated, black, low-volume, 384-well plates (Corning). Assay plates were quantified using an Envision plate reader (Perkin-Elmer) with excitation of the europium crytate donor at 337 nm wavelength and emission of the donor at 620 nm and emission of the XL-665 acceptor at 665 nm in 18 uL volumes. Assays were performed in a buffer containing 50 mM Tris (pH 7.5), 50 mM NaCl, 10mM MgCl2, 1mM TCEP, with addition of and 1mM ATP, 0.05% BSA, and 0.05% Tween-20 immediately prior to the start of the assay.

Comment: The activity outcome is based on a Z-score (number of standard deviations from the negative control mean) of 3 or higher on at least 50% times that the sample was screened.
For instance, if the sample was screened in n=4 runs, it would be considered active only if it had a Z-score of 3 or above in at least 2 runs.

This screen was funded by NIH grant number: R01CA181185

External ID: CHEMBL843757

Protocol: N/A

Comment: Journal: J Med Chem
Year: 1982
Volume: 25
Issue: 9
First Page: 1103
Last Page: 1106
DOI: 10.1021/jm00351a020

Target ChEMBL ID: CHEMBL2094109
ChEMBL Target Name: Muscarinic acetylcholine receptor
ChEMBL Target Type: PROTEIN FAMILY - Target is a group of closely related proteins
Relationship Type: H - Homologous protein target assigned
Confidence: Multiple homologous protein targets may be assigned

External ID: CGM data for Cell Systems paper Dec 2015

Protocol: Yeast strains and Media:
All S. cerevisiae deletion strains were obtained from the Euroscarf deletion collection. The wild type parental strain for this collection is BY 4741 MATa his31 leu20 met150 ura30. An isogenic pdr1pdr3 strain (MT2481) was created using PDR1::nat and PDR::URA3 deletion cassettes. All strains were grown and screened in synthetic complete (SC) medium with 2% glucose.

Handling:
Strains were seeded at 50,000 cells per well in a volume of 100 L in 96 well plates followed by addition of 2 L of 1 mM compound stock for final concentration of 20 M. Screens were conducted in technical duplicate using a Biomek FX liquid handling workstation with an integrated stacker carousel. DMSO solvent only controls and 10 uM cycloheximide positive controls were seeded in columns 1 and 12 of each assay plate. Plates were incubated at 30 C without shaking for approximately 18 h or until culture saturation was achieved for the solvent controls. Cultures were resuspended by shaking on the robotic platform prior to reading OD600 values on either Tecan M1000 or Tecan Sunrise plate readers.

Analysis procedure:
All data was subjected to the following workflow:
1. Consistent spatial effects on growth across plates were corrected by Lowess regression using an empirically estimated sliding window of 1/3 and normalized based on plate median.
2. If more than 30% of compounds were active within a plate, data was normalized to DMSO controls.
3. Median normalization was applied to all plates and experiments.
4. Z-factors for growth inhibition were calculated using the median and the interquartile range (IQR) by fitting a normal distribution with N(1,IQR) to the experimental data. In addition, we calculated the Z-factor, percent inhibition and normalized OD values for manual validation.
5. Data points with high variation between replicates (> 3 MAD) were removed as inconsistent outliers.

BioAssay submission file header:

Pubchem_ext_datasource_regid: compound identifier used in on chemgrid.org/cgm
Orf yeast gene deletion (open reading frame)
Sym# yeast gene deletion gene symbol
Raw OD read 1 first replicate
Raw OD read 2 second replicate
Normalized OD average normalized read based on workflow described above
Z_score Z-Score calculated based on kernel density distribution from normalized average reads per screen
P_value p-value calculated based on kernel density distribution calculated from average reads per screen
Non replicate test for non-replicates between first and second replicate
Pubchem_activity_outcome pubchem activity outcome
Cryptagen if compound identifier fulfilled cryptagen rule of activity in at least 4 and not more than 2/3rds of screens per library
Bioactivity sensitive if compound decreases fitness or resistant if compound increases fitness compared to negative control

Comment: