Cell experiment:

Cells are harvested, and an equal volume of first compound (NCT-505 or paclitaxel (Taxol)) at the indicated concentration or vehicle DMSO (final DMSO concentration is the same in all conditions) is added to the cell suspension before dispensing. Cells are dispensed into 384-well, white, TC-treated plates at a density of 3000 cells/well in a volume of 30 μL of growth media/well using a Multidrop Combi dispenser. Immediately after dispensing, the second compound (ALDH1A1 inhibitor or paclitaxel) and control solutions (92 nL) are transferred using a pintool. Plates are covered with a breathable seal and incubated for 4 days at 37℃, 5% CO2, 85% RH followed by addition of 20 μL of CellTiter-Glo. After a ~30 min incubation at rt, samples are analyzed for luminescence intensity using a ViewLux high-throughput CCD imager equipped with clear filters. Pinned compounds are tested as 16-point dilution series, with concentrations ranging from 30.7 μM to 70.1 nM for ALDH1A1 inhibitors (NCT-505, etc.) or 31.7 μM to 0.034 nM for paclitaxel, in triplicate. Data are normalized to positive control bortezomib (1 μM final) and neutral control DMSO[1].

NCT-505 is a potent and selective aldehyde dehydrogenase (ALDH1A1) inhibitor, with an IC50 of 7 nM, and weakly inhibits hALDH1A2, hALDH1A3, hALDH2, hALDH3A1 (IC50s, >57, 22.8, 20.1, >57 μM).

NCT-505 (Compound 86) is a potent and selective aldehyde dehydrogenase (ALDH1A1) inhibitor, with an IC50 of 7 nM, and weakly inhibits hALDH1A2, hALDH1A3, hALDH2, hALDH3A1 (IC50s, >57, 22.8, 20.1, >57 μM). NCT-505 has no obvious inhibitory effec on 5-hydroxyprostaglandin dehydrogenase (HPGD) and type-4 hydroxysteroid dehydrogenase (HSD17β4) (IC50, >57 μM). Moreover, NCT-505 shows potent cellular activities, reducing the viability of OV-90 cells with an EC50 of 2.10-3.92 μM. NCT-505 is also cytotoxic to SKOV-3-TR cells, with IC50s of 1, 3, 10, 20, 30 μM, respectively, in the titration assay[1].

[1]. Yang SM, et al. Discovery of Orally Bioavailable, Quinoline-Based Aldehyde Dehydrogenase 1A1 (ALDH1A1) Inhibitors with Potent Cellular Activity. J Med Chem. 2018 Jun 14;61(11):4883-4903.