Inhibition of protein kinases by anticancer DNA intercalator, 4-butylaminopyrimido[4',5':4,5]thieno(2,3- b)quinoline

Abstract

Targeting protein kinases (PKs) has been a promising strategy in treating cancer, as PKs are key regulators of cell survival and proliferation. Here in this study, we studied the ability of pyrimido[4',5':4,5]thieno(2,3-b)quinolines (PTQ) to inhibit different PKs by performing computational docking and in vitro screening. Docking studies revealed that 4-butylaminopyrimido[4',5':4,5]thieno(2,3-b)quinoline (BPTQ) has a higher order of interaction with the kinase receptors than other PTQ derivatives. In vitro screening confirms that BPTQ inhibits VEGFR1 and CHK2, with the IC₅₀ values of 0.54 and 1.70 µmol/L, respectively. Further, cytotoxicity of BPTQ was measured by trypan blue assay. Treatment with BPTQ decreased the proliferation of HL-60 cells with an IC₅₀ value of 12 µmol/L and induces apoptosis, as explicated by the fall in the mitochondrial membrane potential, annexin V labeling and increased expression of caspase-3. Taken together, these data suggest that BPTQ possess ability to inhibit PKs and to induce cell death in human promyelocytic leukemia cells.

Keywords: Anticancer drugs; Apoptosis; Chemotherapy; DNA intercalator; Kinase inhibitor; Molecular docking.

Graphical abstract

Figure 1

Structures of test compounds used for the study.

Figure 2

Molecular docking of BPTQ and staurosporine with CHK2 and VEGFR1 receptor. Molecular interaction of BPTQ with CHK2 (A and B) and VEGFR1 (C and D); molecular interaction of staurosporine with CHK2 (E) and VEGFR1 (F).

Figure 3

In vitro primary kinase profiling and dose–response curve of BPTQ on CHK2 and VEGFR1. (A) Bar diagram showing the kinase activity (%) of a panel of protein kinases tested with 1 μmol/L BPTQ using an in vitro assay; (B) dose response curve of BPTQ on CHK2 and VEGFR1.

Figure 4

Effect of BPTQ on the proliferation of HL-60 cells. (A) Line diagram showing cytotoxicity of BPTQ on HL-60 cell line. HL-60 cells were incubated with different concentrations of BPTQ for different time points and number of viable cells was determined by trypan blue assay. (B) Accumulation of BPTQ to HL-60 cells. FIU, fluorescence intensity unit. (C) Cytotoxicity measured as LDH release upon BPTQ treatment. In all panels, error bars indicates±SD (n=3). ^**P<0.01, a significant difference from the controls.

Figure 5

Effect of BPTQ on the cell cycle distribution of HL-60 cells analyzed using flow cytometry. The experiment was repeated three times and representative histograms are shown. (A) Control; (B) BPTQ 10 µmol/L; (C) BPTQ 50 µmol/L; (D) BPTQ 100 µmol/L.

Figure 6

Evaluation of cell death mode induced by BPTQ in HL-60 cells. (A) Cells were visualized by confocal microscopy after staining with fluorescein isothiocyanate-conjugated annexin V and propidium iodide. Left panel, untreated control cells; Middle panel, cells treated with 5 μmol/L BPTQ; Right panel, cells treated with 10 μmol/L BPTQ. (B) Effect of BPTQ on mitochondrial membrane potential. (C) Effect of BPTQ on caspase-3 expression. Values were expressed as the concentration of pNA formed per min per mL of cell lysate. Error bars indicate±SD (n=3). *P<0.05, **P<0.01; significance is compared with the control group.