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: 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: 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: 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: 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: 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.