In vitro activity: HH-GV-678 can predominantly inhibit the autophosphorylation of Bcr-Abl in K562 cell. In higher concentration, HH-GV-678 can inhibit the phosphorylation of c-Kit in Mo7e cell and the phosphorylation of PDGFR in Swiss3T3 cell, however, HH-GV-678 has no or little effect on other tyrosine kinase including EGFR, KDR, c-Src and HER2. Flumatinib effectively overcame the drug resistance of certain KIT mutants with activation loop mutations (i.e., D820G, N822K, Y823D, and A829P).

Kinase Assay: Murine stem cell virus-based retroviral constructs carrying murine–human hybrid WT KIT cDNA or activating mutant D816V (816 Asp→Val) KIT cDNA were generously provided by Michael H. Tomasson (Washington University School of Medicine, St. Louis, MO, USA). Hybrid KIT alleles were generated by fusing in-frame the extracellular and transmembrane regions of murine KIT with the intracellular region of human KIT. It has been shown that replacement of the human extracellular and transmembrane domains of KIT with homologous murine sequences can improve the expression efficiency and rescue the transforming potential of certain KIT mutants in murine cells. Owing to a downstream internal ribosomal entry site–enhanced GFP cassette, KIT alleles would coexpress with enhanced GFP. The KIT point mutations were generated following Protocol 3 of mutagenesis in Molecular Cloning (3rd edition). For deletion and insertion mutagenesis, mutagenic primers were designed to avoid the deleted sequence or harbor the inserted sequence, respectively. All the PCRs above used the high-fidelity Primestar Hot Start DNA Polymerase (Takara, Dalian, China). Other enzymes used in above experiments were also purchased from Takara. The sequences of all mutants in this study were verified by direct sequencing.

Cell Assay: Cells (5 × 103) in 200 μL medium with or without IL-3 were incubated with various concentrations of imatinib, flumatinib, or sunitinib in 96-well plates for 72 h in triplicate. We added MTT, and cells were incubated for 4 h. A solubilization solution (a solution of the detergent SDS in diluted hydrochloric acid) was added to dissolve the insoluble purple formazan product into a colored solution. The absorbance of this colored solution was quantified by measuring at 570 nm with a reference filter of 650 nm by a spectrophotometer. Growth inhibition was plotted as the ratio of the average absorbance in drug-treated wells relative to no-drug controls. The IC50 values were calculated by the curve-fitting software GraphPad Prism version 5.