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: VANDERBILT_HTS_GIRK2_MPD

Protocol: To screen for modulators of the G protein-gated inwardly-rectifying potassium channel subunit 2 (GIRK2) homomeric channels, HEK293 cells were engineered to overexpress GIRK2. These cells were screened using a high-throughput thallium-flux assay in a 384-well format. High-throughput thallium flux assays are based on three principles: (1) extracellular thallium can permeate through potassium channels into cells, (2) the fluorescent dye Thallos enters and stays in the cytoplasm of HEK293 cells, and (3) the fluorescence of Thallos increases dramatically when it interacts with thallium ions. Compounds that modulate the activity of an overexpressed channel, in this case GIRK2, are identified by the change in fluorescence that results from changes in thallium influx into cells through the overexpressed channel.

The screening protocol was conducted as follows. One hour prior to screening, cells were loaded with 1.5 micromolar (muM) Thallos in assay buffer (1x Hank's Balanced Salt Solution and 20 millimolar (mM) HEPES). The liquid handling and imaging was conducted using a kinetic-imaging plate reader, Panoptic (WaveFront Biosciences). Before screening, Thallos-containing buffer was removed from cells and replaced with 20 microliters (muL) per well of assay buffer. Compounds were dissolved in assay buffer at 20 muM. After 8 seconds of imaging, 20 muL of compound solution was added per well and allowed to incubate for 150 seconds. Next, 10 muL of a solution containing 2.0 mM thallium in Base Buffer (125 mM NaHCO3, 1 mM MgSO4, 1.8 mM CaSO4, 5mM Glucose, and 20 mM HEPES) was added to the wells. 30 seconds later, 12 muL of base buffer containing 2.0 mM thallium and 50 mM 2-methyl-2,4-pentanediol (MPD), a partially-activating concentration, was added to each well. MPD has is a previously-described GIRK2 channel activator. Data was collected for 60 seconds after this final addition. The positive control for this screen was 30 muM ivermectin, a natural product previously identified as a GIRK2 activator as part of a small-scale pilot screen. Microsoft Excel was utilized for data analysis. Data were normalized, F/Fo, on a well-to-well basis to account for differences in cell number. Compound activity was analyzed by comparing the sums of control-subtracted amplitudes of fluorescence intensity at 20 seconds after each thallium addition. The top 0.2% compounds corresponding to wells that demonstrated the largest increases in fluorescence were selected as "hits" for counter screening, as described below.

All 63,228 compounds screened using the method described above are listed in column OUTCOME_SCREEN_HITS. The 133 compounds selected as hits based on the above criteria are labelled with "100" in the ACTIVITY column. Fluorescent compounds were included in the inactive compounds and marked with "0" in the ACTIVITY column.

The 133 active compounds from column OUTCOME_SCREEN_HITS were tested for non-specific activity in untransfected HEK293 cells using assay conditions otherwise identical to the primary screen. Column OUTCOME_HEK_COUNTERSCREEN lists the 23 compounds that displayed an increase in fluorescence upon thallium addition with "100" in the ACTIVITY column.

The 110 compounds which were identified as hits in the primary screen but inactive when tested in untransfected HEK293 cells were tested for activity in GIRK2-overexpressing HEK293 cells that did not express NPY4 receptor. Column OUTCOME_GIRK2_COUNTERSCREEN lists the 44 compound that displayed an increase in fluorescence upon thallium addition with "100" in the ACTIVITY column.

The 44 active compounds from column OUTCOME_GIRK2_COUNTERSCREEN were tested at various concentrations to determine if compound activity was concentration dependent. Column OUTCOME_GIRK2_DOSE_RESPONSE provides the efficacy of these compounds; activity of all compounds at the provided concentrations was normalized to the activity of VU0537695 at 25 muM, which was the highest efficacy observed during this screening. For each compound concentration listed, the efficacy of that compound provided refers to the normalized, control-subtracted fluorescence intensity at 15 seconds after the addition of thallium. The 28 compounds that demonstrated >5% efficacy and concentration-dependent activity were labelled as "100" in the ACTIVITY column.

Finally, column PUBCHEM_ACTIVITY_OUTCOME combines all of the information from the above counterscreens together, indicating the 28 hits from this GIRK2 screen.

Comment: