Integrated <i>In Silico</i> and Experimental Insights into Immunomodulatory Toll-Like Receptor 7/8 Agonists with Anticancer Potential

Abstract Toll-like receptors (TLRs) are key pattern recognition receptors that play a central role in regulating and bridging both innate and adaptive immune responses. Notably, TLR7 and TLR8 agonists have gained significant attention as promising therapeutic candidates because of their ability to activate immune responses and modulate immune signaling pathways for treating viral infections, autoimmune disorders, and cancer. Despite the growing interest in the area of immunotherapy, there remains a substantial gap in the development of new immunomodulators with improved efficacy and safety profiles. The primary objective of the study was to identify and evaluate newly synthesized TLR7/8 agonists using integrated in silico and in vivo strategies. Computational analyses, including molecular docking, molecular dynamics (MD) simulations, pharmacokinetic-pharmacodynamic profiling, and network pharmacology analysis, revealed favorable binding interactions, stability, drug-like properties, and potential targeting of immune- and cancer-related pathways, specifically those relevant to colorectal cancer. In vitro cytotoxicity assays showed that the TLR7/8 agonists exhibit dose-dependent effects in both colorectal cancer cells and RAW 264.7 macrophages. Additionally, the compounds exhibit minimal hemolytic activity, indicating a low potential for systemic toxicity. Furthermore, the cell migration assay demonstrated that several compounds effectively inhibited cancer cell migration, particularly when combined with 5-fluorouracil (5-FU), suggesting potential anticancer and antimetastatic activity. Overall, these findings integrate molecular-level, system-level, and experimental validation approaches to comprehensively evaluate TLR7/8 agonists as potential immunotherapeutic agents. However, further comprehensive in vitro and in vivo investigation studies are necessary to establish the potential of TLR7/8 agonists for use in cancer immunotherapy.