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: CHEMBL3225597

Protocol: N/A

Comment: Journal: MedChemComm
Year: 2011
Volume: 2
Issue: 11
First Page: 1099
Last Page: 1103
DOI: 10.1039/C1MD00176K

Target ChEMBL ID: CHEMBL2039
ChEMBL Target Name: Monoamine oxidase B
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: HTS97080

Protocol: 96-well plates were coated with 100 microl of 0.25 mg/ml biotin conjugated-gelatin overnight and then washed twice with PBS. Endothelial cells (3 x 104) were plated in pericyte-conditioned medium and the cells were grown to confluence for 6 days followed by the treatment with compounds for 24 h. On day seven, 6 microg/ml of NeutrAvidin TM, FITC conjugate (FITC-avidin, 60 kDa) (Thermo Fisher Scientific) was added to cells for 3 min. The cells were washed twice with PBS to remove the unbound FITC-avidin. Wells were filled with 100 microl of PBS and bound FITC-avidin fluorescence was measured using plate reader with wavelengths of 492 nm. The fluorescence units of cells treated with compounds were normalized to values of cells treated with vehicle (DMSO) and presented as average with standard deviation.

Comment: Measuring the fluorescence of FITC-avidin that has penetrated the endothelial monolayer and bound to a biotinylated gelatin-coated plate can be used as a surrogate parameter for endothelial permeability. Here we show the average FITC-avidin fluorescence of drug-treated cells as fold of vehicle-treated cells. Compounds causing 0.3-fold or higher fluorescence changes are considered active, while compounds causing changes of less than 0.3-fold are considered inactive.

External ID: CHEMBL3225596

Protocol: N/A

Comment: Journal: MedChemComm
Year: 2011
Volume: 2
Issue: 11
First Page: 1099
Last Page: 1103
DOI: 10.1039/C1MD00176K

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: CHEMBL3225598

Protocol: N/A

Comment: Journal: MedChemComm
Year: 2011
Volume: 2
Issue: 11
First Page: 1099
Last Page: 1103
DOI: 10.1039/C1MD00176K

Target ChEMBL ID: CHEMBL2039
ChEMBL Target Name: Monoamine oxidase B
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned