Design, Synthesis and Biological Activity Evaluation of S-Substituted 1 H-5-Mercapto-1,2,4-Triazole Derivatives as Antiproliferative Agents in Colorectal Cancer

Abstract

Colon cancer is a widespread pathology with complex biochemical etiology based on a significant number of intracellular signaling pathways that play important roles in carcinogenesis, tumor proliferation and metastasis. These pathways function due to the action of key enzymes that can be used as targets for new anticancer drug development. Herein we report the synthesis and biological antiproliferative evaluation of a series of novel S-substituted 1H-3-R-5-mercapto-1,2,4-triazoles, on a colorectal cancer cell line, HT-29. Synthesized compounds were designed by docking based virtual screening (DBVS) of a previous constructed compound library against protein targets, known for their important role in colorectal cancer signaling: MEK1, ERK2, PDK1, VEGFR2. Among all synthesized structures, TZ55.7, which was retained as a possible PDK1 (phospholipid-dependent kinase 1) inhibitor, exhibited the most significant cytotoxic activity against HT-29 tumor cell line. The same compound alongside other two, TZ53.7 and TZ3a.7, led to a significant cell cycle arrest in both sub G0/G1 and G0/G1 phase. This study provides future perspectives for the development of new agents containing the 1,2,4-mercapto triazole scaffold with antiproliferative activities in colorectal cancer.

Keywords: 1; 2; 4-triazole; antiproliferative; cell cycle; colon cancer; docking.

Figure 1

Signaling pathways in colorectal cancer (simplified scheme). AKT, protein kinase B; RAF, serin/treonin-protein kinase proto-oncogenă; RAS, retrovirus-associated DNA sequences; EGFR, epidermal growth factor receptor; ERK, extracellular regulated kinase; FZD, frizzled receptor; IGF-1R, insulin growth factor receptor 1; IRS, insulin receptor substrate; MAPK, mitogen activated protein kinase Mtorc, mammalian target of rapamycin complex; NFAT, factorul nuclear de activare a celulelor T; PDK1, phospholipid-dependent kinase 1; PI3K, phosphatidylinositol 3-kinaze; PIP2, fosfatidil-inozitol-difosfat; PIP3, fosfatidil-inozitol-trifosfat; PTEN, Phosphatase and tensin homolog; Rheb, Ras homolog enriched in brain; TSC, tuberous sclerosis protein; Shc, SHC-transforming proteins, VEGFR, vascular endothelial growth factor receptor; Vrap, VEGF Receptor-Associated Protein.

Figure 2

Compound library construction scheme.

Figure 3

Synthesis pathway of S-functionalized 1H-3-R-5-mercapto-1,2,4-triazoles.

Figure 4

Compound TZ53.3 docked in the ATP active site of the ERK2 protein, PDB ID: 4G6N (A); ERK2 protein co-crystalized with ATP molecule, PDB ID: 4GT6 (B); superimposed structures of compound TZ53.3 and the co-crystalized ligand of ERK2, PDB ID: 4G6N (C); HBs are depicted as green dotted lines.

Figure 5

Compounds TZ53.7 (A) and TZ55.7 (B) docked in the active site of PDK1, PDB ID: 2PE1; HBs are depicted as green dotted lines.

Figure 6

Cell viability recorded on HT-29 cell line, for the tested compounds TZ53.3, TZ53.7, TZ55.7, TZ3a.7, TZ53.11, TZ55.11, at concentrations of: 50, 150, 200, 250, and 350 μM; (*p < 0.05; **p < 0.01).

Figure 7

ELISA determination of VEGF-R2 secreted by HT-29 cells into culture medium after stimulation with test compounds (TZ55.7 - 50 and 100 μM, TZ53.7 - 150 and 250 μM) and TZ3a.7 - 150 and 250 μM) for 24 h. The values are expressed as mean ± standard deviation. The statistical significance was determined with One-way ANOVA using GraphPad Prism 6 software (**p < 0.01, ***p < 0.001).

Figure 8

Immunostaining of PDK1 expression in HT-29 cells stimulated with TZ55.7 (50 and 100 μM) (A), TZ53.7 (150 and 250 μM) (B), TZ3a.7 (150 and 250 μM) (C), for 24 h compared to control cells (unstimulated cells). Quantification of the fluorescence intensity was assessed for five randomly selected areas using ImageJ software and is presented in graphical representation. The statistical significance was performed with One-way ANOVA using GraphPad Prism 6 software (****p < 0.0001).

Figure 9

Representative dotplots for the flow cytometric analysis of HT-29 cell apoptosis.

Figure 10

Representative histograms for the flow cytometric analysis of HT-29 cell cycle stimulated with test compounds.