External ID: Sarm1 TIR NADase inhibitors

Protocol: Sarm1 "Sam-Tir" (Sarm1 without N-terminus, including residues 410-721 of human Sarm1 protein) was expressed in NRK1-HEK/293T cells by transient transfection: 15 million cells were transfected with 15microg plasmid DNA (Kow et al., 2018. Current Biology, 28:421), supplementing with 1mM nicotinamide riboside (NR). After 48 hours, cells were harvested and lysed. Reaction mixtures were prepared by mixing SARM1 SAM-TIR cell lysate (0.13 microg total protein), compound stock (5 microM final concentration, NCI diversity IV), and PBS (pH 7.4) to a final volume of 12 microl. NAD (5 microM final concentration) was then added for a final reaction volume of 20 microl. The reaction was incubated at 37 degrees C for 60 min, and then stopped by addition of 180 microl of 0.55 M perchloric acid (HClO4) and placed on ice. After 10 min on ice, the reaction plates were centrifuged for 10 min at 4,000 rpm (Sorvall ST 16R centrifuge). The supernatant (120 microl) was transferred to a new plate and 10 microl of 3M K2CO3 was added to neutralize the solution. Precipitated salts were removed by centrifugation 10 min at 4,000 rpm (Sorvall ST 16R centrifuge). The supernatant was transferred and analyzed by HPLC (Shimadzu Nexera X2) to quantify the amounts of NAD and the enzymatically cleaved product ADPR in each sample. From these values, the NAD/ADPR ratio was calculated for each compound as a measure of NAD cleavage activity (Note: there is a residual but detectable ADPR in control samples). This ratio was compared to the ratio generated in the absence of compound inhibitors. A reduction of NADase activity (NAD/ADPR ratio > 4) was used to identify compounds that inhibited the SAM-TIR catalyzed NAD cleavage.

Comment: The ratio of NAD/ADPR at time 0 is 15-16. The compounds are listed with their NSC numbers.

External ID: CHEMBL4296192

Protocol: N/A

Comment: Target ChEMBL ID: CHEMBL352
ChEMBL Target Name: Staphylococcus aureus
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

Data Source: CO-ADD Antimicrobial Screening

External ID: CHEMBL4296783

Protocol: N/A

Comment: Target ChEMBL ID: CHEMBL354
ChEMBL Target Name: Escherichia coli
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

Data Source: CO-ADD Antimicrobial Screening

External ID: CHEMBL4296193

Protocol: N/A

Comment: Target ChEMBL ID: CHEMBL614425
ChEMBL Target Name: Acinetobacter baumannii
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

Data Source: CO-ADD Antimicrobial Screening

External ID: CHEMBL4296187

Protocol: N/A

Comment: Compounds with activity <= 10uM or explicitly reported as active by ChEMBL are flagged as active in this PubChem assay presentation.

Target ChEMBL ID: CHEMBL348
ChEMBL Target Name: Pseudomonas aeruginosa
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

Data Source: CO-ADD Antimicrobial Screening

External ID: CHEMBL4296190

Protocol: N/A

Comment: Compounds with activity <= 10uM or explicitly reported as active by ChEMBL are flagged as active in this PubChem assay presentation.

Target ChEMBL ID: CHEMBL365
ChEMBL Target Name: Cryptococcus neoformans
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

Data Source: CO-ADD Antimicrobial Screening

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: CHEMBL4296189

Protocol: N/A

Comment: Compounds with activity <= 10uM or explicitly reported as active by ChEMBL are flagged as active in this PubChem assay presentation.

Target ChEMBL ID: CHEMBL366
ChEMBL Target Name: Candida albicans
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

Data Source: CO-ADD Antimicrobial Screening

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: CHEMBL4296186

Protocol: N/A

Comment: Compounds with activity <= 10uM or explicitly reported as active by ChEMBL are flagged as active in this PubChem assay presentation.

Target ChEMBL ID: CHEMBL350
ChEMBL Target Name: Klebsiella pneumoniae
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

Data Source: CO-ADD Antimicrobial Screening