Cell experiment:

Cells are plated in 96-well plates with a density of 4000 to 8000 cells per well and incubated for 24 h at 37℃ with 5% CO2. Then cells are treated with increasing drug (including ML349) concentrations and their combinations. Cell viability is measured with the cell viability assay according to the manufacturer's protocol[3].

ML349 is a potent and specific acyl protein thioesterase 2 (APT-2) inhibitor with a Ki of 120 nM. ML349 is also an inhibitor of LYPLA2 with an IC50 of 144 nM.

ML349 is an inhibitor of acyl protein thioesterase 1 and 2 (APT-1 and APT-2) with Kis of >10000 and 120±20 nM, respectively[1]. ML349 is also an inhibitor of both LYPLA1 and LYPLA2 with IC50s of >3000 and 144 nM, respectivley[2]. ML348 and ML349 do not decrease cell viability, but they lead to a slight activation of AKT in NRAS mutant cells[3].

References:
[1]. Won SJ, et al. Molecular Mechanism for Isoform-Selective Inhibition of Acyl Protein Thioesterases 1 and 2 (APT1 and APT2). ACS Chem Biol. 2016 Dec 16;11(12):3374-3382.
[2]. Adibekian A, et al. Characterization of a Selective, Reversible Inhibitor of Lysophospholipase 2 (LYPLA2).
[3]. Vujic I, et al. Acyl protein thioesterase 1 and 2 (APT-1, APT-2) inhibitors palmostatin B, ML348 and ML349 have different effects on NRAS mutant melanoma cells. Oncotarget. 2016 Feb 9;7(6):7297-306.