In vitro activity: CU-CPT17e is a multi-TLR(Toll-like receptor) agonist that activates TLR3, TLR8, and TLR9. Therapies based on activation of multiple Toll-like receptors (TLRs) may offer superior therapeutic profiles than that of single TLR activation. CU-CPT17e was identified from a cell-based high-throughput screening of a small-molecule library based on TLR3-mediated NF-κB activation. Biochemical studies demonstrated that CU-CPT17e could induce a strong immune response via the production of various cytokines in human monocytic THP-1 cells. Furthermore, CU-CPT17e inhibited the proliferation of HeLa cancer cells by triggering apoptosis and arresting the cell cycle at the S phase. These results showcase potential therapeutic applications of CU-CPT17e in both vaccine adjuvants and anticancer therapies based on multi-TLR activation.

Kinase Assay: CU-CPT17e shows strong NF-κB activation in TLR3, TLR8 and TLR9 HEK293 cells with EC50 values of 4.80±0.73, 13.5±0.58 and 5.66±0.17 μM, respectively. CU-CPT17e significantly improves the activity with 13.9±0.9 fold of NF-κB activation and an EC50 value of 4.8±0.7 μM. CU-CPT17e inhibits the proliferation of HeLa cancer cells by triggering apoptosis and arresting the cell cycle at the S phase. The induction of apoptosis by CU-CPT17e in HeLa cells is investigated. HeLa cells are cultured with increasing concentrations of CU-CPT17e or poly I:C or blank control (DMSO) for 24 h. Treatment with CU-CPT17e for 24 h at different concentrations (10 to 40 μM) results in an elevation of apoptotic cell population ranging from 10% to 17%, which is more effective than poly I:C at 5 μg/mL. These results suggest that the antiproliferative activity of CU-CPT17e against HeLa cells might result from its ability to directly induce apoptosis.

Cell Assay: HeLa cells are seeded at a density of 3×105 cells/well in 6-well plates and allowed to attach for 24 h. After treatment of indicated concentrations of CU-CPT17e or poly I:C (5 μg/mL) for another 24 h, cells are harvested with 0.25% trypsin without EDTA and rinsed twice with PBS, then stained using a Annexin V-FITC apoptosis detection kit. Cells are analyzed with a BD Accuri C6 flow cytometer.