In vitro activity: LMK-235 causes HDAC inhibition with IC50 of<1 μM in human cancer cell lines with different sensitivity towards cisplatin. In breast cancer cell line MDA-MB-231, tongue cancer cell line Cal27, and esophagus cell line Kyse510 cell line, LMK-235 displays a high cytotoxicity, and markedly enhances the cytotoxicity of cisplatin. In addition, LMK-235 also shows nanomolar activity against multiple malaria parasite life cycle stages.

Kinase Assay: The in vitro inhibitory activity of compounds against seven human HDAC isoforms (1, 2, 4 C2A, 5 C2A, 6, 8, and 11) are performed with a fluorescent based assay according to the company’s standard operating procedure. The IC50 values are determined using 10 different concentrations with 3-fold serial dilution starting at 10 μM. TSA and vorinostat are used as reference compounds.

Cell Assay: The rate of cell survival under the action of test substances is evaluated by an improved MTT assay. The assay is based on the ability of viable cells to metabolize yellow MTT to violet formazan that can be detected spectrophotometrically. In brief, A2780, Cal27, Kyse510, and MDA-MB-231 cell lines are seeded at a density of 5000, 7000, 8000, and 10 000 cells/well in 96-well plates. After 24 h, cells are exposed to increased concentrations of the test compounds. Incubation is ended after 72 h, and cell survival is determined by addition of MTT solution (5 mg/mL in phosphate buffered saline). The formazan precipitate is dissolved in DMSO. Absorbance s measured at 544 and 690 nm in a FLUOstar microplate reader.