Computational design, chemical synthesis, and biological evaluation of a novel ERK inhibitor (BL-EI001) with apoptosis-inducing mechanisms in breast cancer

Abstract

Extracellular signal-regulated kinase1/2 (ERK1/2) plays a crucial role in the resistance of apoptosis in carcinogenesis; however, its targeted small-molecule inhibitors still remain to be discovered. Thus, in this study, we computationally and experimentally screened a series of small-molecule inhibitors targeting ERK toward different types of human breast cancer cells. Subsequently, we synthesized some candidate ERK inhibitors, identified a novel ERK inhibitor (BL-EI001) with anti-proliferative activities, and analyzed the BL-EI001/ERK complex. Moreover, we found that BL-EI001 induced breast cancer cell apoptosis via mitochondrial pathway but independent on Ras/Raf/MEK pathway. In addition, we carried out proteomics analyses for exploring some possible BL-EI001-induced apoptotic pathways, and further found that BL-EI001-induced apoptosis affected ERK phosphorylation in breast cancer. Further, we found that BL-EI001 bear anti-tumor activities without remarkable toxicities, and also induced mitochondrial apoptosis by targeting ERK in vivo. Taken together, these results demonstrate that in silico design and experimental discovery of a synthesized small-molecule ERK inhibitor (BL-EI001)as a potential novel apoptosis-inducing drug in the treatment of breast cancer.

Keywords: ERK inhibitor (BL-EI001); apoptosis; breast cancer; extracellular signal-regulated kinase 1/2 (ERK1/2); mitochondrial pathway.

Figure 1. Molecular modeling, docking and anti-proliferative screening of candidate small-molecule compounds targeting ERK1/2

(A) The molecular structure of ERK1/2; (B) The top ten candidate small molecule compounds targeting ERK1/2 from Drugbank and ZINC, respectively; (C) Anti-proliferative activities of candidate compounds (E1-E11) toward MCF-7 cells, MDA-MB468 cells and MDA-MB231 cells, respectively.

Figure 2. Chemical synthesis of BL-EI001

Chemical structure modification strategies of compound E1, and synthesis of targeted compound BL-EI001 as a novel ERK1/2 inhibitor.

Figure 3. Molecular dynamics (MD) simulation and anti-proliferative activities of E1 and BL-EI001

(A) Comparison of molecular dynamics simulation between E1 and BL-EI001 binding to ERK1/2; (B) Comparison of anti-proliferative effects between E1 and BL-EI001 in CF-7 cells, MDA-MB468 cells and MDA-MB231 cells, respectively.

Figure 4. BL-EI001 induces MCF-7 cell apoptosis

(A) The cellular morphology was observed without or with BL-EI001 under the inverted, fluorescent microscopy and electron microscopy, respectively; (B) Cells were consecutively treated with indicated concentrations of BL-EI001 for two weeks. Cell proliferation was examined by colony formation assay; (C) The population of SubG1 cells was measured by flow cytometry after collection; (D) Apoptosis was detected via Annexin V-FITC/PI staining in MCF-7 cells treated with BL-EI001 for 12, 24, 36 h by flow cytometry analysis.

Figure 5. BL-EI001-induced apoptotic mechanism is the mitochondrial pathway but not the Ras-Raf-MEK1/2 pathway

(A) BL-EI001 treatment resulted in decrease of mitochondrial membrane potential; (B) BL-EI001-induced apoptosis activated the mitochondrial pathway; (C) BL-EI001-induced apoptosis activated ERK1/2 but not the Ras-Raf-MEK1/2 pathway.

Figure 6. Proteomics analyses of BL-EI001-induced apoptosis in breast cancer cells

(A) Different protein levels (upregulation or downregulation) between control and BL-EI001-treated MCF-7 and MDA-MB231 cells; (B) Apoptosis-related enrichment analyses in BL-EI001-treated MCF-7 and MDA-MB231 cells; (C) Proteomics-based identification of novel ERK apoptotic pathways in BL-EI001-treated MCF-7 and MDA-MB231 cells.

Figure 7. BL-EI001-induced apoptosis is regulated by novel ERK-mediated pathways

(A) BL-EI001-induced apoptosis is associated with BRIC6, HMGB1 and AIFM2; (B) BL-EI001-induced apoptosis is regulated by ERK1/2-HMGB1 pathway; (C) BL-EI001-induced apoptosis is affecting ERK1/2.

Figure 8. Anti-tumor effects of BL-EI001 in vivo

(A) The toxicity of BL-EI001 in breast cancer xenograft mouse model; (B) Illustration shows the tumor excised from each treatment group and data represent the mean tumor weight for each group; (C) Immunohistochemistry of proliferative marker KI67 and TUNEL assay.

Figure 9. BL-EI001 induces apoptosis in vivo

(A) Immunohistochemistry of p-ERK1/2, Bax, Bcl-2 and cleaved caspase 3 between model- and BL-EI001 groups; (B) Some apoptotic marker expressions between control- and different dose-groups; (a) Bax, Bcl-2 and caspase 9/3 and PARP1 expressions; (b) ERK1 and p-ERK1 expressions.