Compound (SMILES): C[C@@H]1COCCN1c1cc(C2CCN(C(=O)C3CC3)CC2)nc(-c2ccnc3[nH]ccc23)n1
InChIKey: LRPJIBQLWYZMLY-MRXNPFEDSA-N
Target: Serine/threonine-protein kinase Chk1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14757
Assay: pCHK1 Cellular Assay: Inhibitors of ATR kinase are effective at inhibiting the ATR-driven phosphorylation of the downstream target Chk1 kinase at Serine 345, following the addition of 4-nitroquinoline N-oxide, a chemical used to induce DNA damage. Cellular IC50 for the inhibitors of ATR described herein were measured in HT-29 colorectal adenocarcinoma cells. HT-29 cells were routinely maintained in McCoy's 5A media (ATCC Catalog #30-2007) supplemented with 10% fetal bovine serum (Sigma Catalog # F2442) and 1× Penicillin-Streptomycin (Gibco Catalog #15140-122) using a humidified incubator (37° C., 5% CO2, and ambient 02).In preparation for the CHK1 (p-Ser345) ALPHASCREEN SUREFIRE assay, cells were harvested and resuspended in McCoy's 5A media supplemented with 10% fetal bovine serum and 1× Penicillin-Streptomycin. Cells were seeded onto a 384-well black CELLSTAR Tissue Culture Plate (VWR Catalog #89085-314) at a density of 13,000 cells/well in a volume of 40 uL. The microplate was incubated overnight (approximately 20 hours) at 37° C. with 5% CO2 and ambient 02. Stock solutions of the test compounds were prepared in 100% DMSO (Sigma, Catalog # D2650) and serially diluted 1:3 using 100% DMSO. Compounds were additionally diluted 1:33 in culture medium, and 10 ul/well were transferred to the tissue culture plate. Following the compound addition the microplate was incubated at 37° C. for 90 minutes. 10 uL of 4-nitroquinoline N-oxide (Sigma Aldrich Catalog # N8141-1G) diluted in media (final concentration 12 uM) were added to the tissue culture plate followed by a 120 minute incubation at 37° C. The cells were then washed with PBS and lysed using 10 uL/well SUREFIRE® Kit lysis buffer diluted to 1× in water (PerkinElmer Catalog # TGRCHK1S50K), with mixing on an orbital shaker at 500 rpm for 20 min at RT. Lysates were frozen at −20° C. overnight.4 uL/well of lysate was then transferred from the tissue culture plate to a 384-well, white, low volume, PROXIPLATE (PerkinElmer Catalog #600828). 5 uL/well of the acceptor bead solution, prepared by diluting SUREFIRE Kit activation buffer (PerkinElmer Catalog # TGRCHK1S50K) and ALPHASCREEN Protein A acceptor beads (PerkinElmer Catalog #6760617R) in SUREFIRE Kit reaction buffer (PerkinElmer Catalog # TGRCHK1S50K), were added to the lysates under subdued light and incubated at room temperature for 120 min. 2 uL/well of the donor bead solution, prepared by diluting ALPHASCREEN Streptavidin donor beads (PerkinElmer Catalog #6760617R) in SUREFIRE Kit dilution buffer (PerkinElmer Catalog # TGRCHK1S50K), were added under subdued light and incubated at room temperature for an addition 120 minutes. The pCHK1 ALPHASCREEN signal was measured using an ENVISION® plate reader (PerkinElmer). IC50 values were calculated using a four-parameter logistic curve fit using Genedata Screener software.
Measurement: IC50 = 82 nM (pChEMBL 7.090000152587891)
Descriptors: MW 446.56, ALogP 3.36, HBA 6, HBD 1, PSA 87.24, QED 0.66