In vitro activity: ML239 was originally identified as a potent and selective inhibitor of breast cancer stem cells with an IC50 of 1.16 μM, but recent studies (2016 Nature 12(2):109-16.) suggested that it most likely acts through activation of fatty acid desaturase 2 (FADS2). ML239 was discovered from high-throughput screen (HTS) with the National Institute of Health–Molecular Libraries Small Molecule Repository (NIH–MLSMR) compound collection which identified a class of acyl hydrazones to be selectively lethal to breast cancer stem cell (CSC) enriched populations. Medicinal chemistry efforts were undertaken to optimize potency and selectivity of this class of compounds. The optimized compound was declared as a probe (ML239) with the NIH Molecular Libraries Program and displayed greater than 20-fold selective inhibition of the breast CSC-like cell line (HMLE_sh_Ecad) over the isogenic control line (HMLE_sh_GFP).

Kinase Assay: ML239 is a potent and selective inhibitor of breast cancer stem cells, with an IC50 of 1.16 μM, with ~24-fold selectivity against the control cell line. ML239 inhibits breast cancer stem-like cells, most likely through activation of fatty acid desaturase 2 (FADS2). ML239 is cytotoxic to NCIH661 cells, and FADS2 knockdown reduces ML239 cytotoxicity, and furthermore, FADS2 inhibitor SC-26196 also reduces ML239 cytotoxicity in cancer cell lines (CCLs).

Cell Assay: Small molecules were selected individually to interrogate important targets and/or cellular processes in cancer with high reported selectivity, and collectively to target diverse nodes in cancer cell circuitry, from sources including FDA-approved drugs, clinical candidates, previous screening and sensitivity profiling experiments, scientific literature and patents, bioactives, and collaborator contributions. CCLs were plated at a density of 500 cells/well in white opaque tissue-culture-treated Aurora 1536-well MaKO plates (Brooks Automation) in the provider-recommended growth media using a highly automated platform. Compounds were added by acoustic transfer using a Labcyte Echo 555 (Labcyte Inc.) 24 hours after plating. The effects of small molecules were measured over a 16-point concentration range (two-fold dilution) in duplicate. DMSO was used at a constant concentration of 0.33%, including vehicle-only control wells. As a surrogate for viability, cellular ATP levels were assessed 72 hours after compound transfer by addition of CellTiterGlo (Promega) followed by luminescence measurement using a ViewLux Microplate Imager (PerkinElmer). Duplicates were averaged and luminescence values normalized to vehicle (DMSO) treatment and background (media-only) wells.