Integrated computational design, synthesis, and evaluation of dipeptide-coumarin conjugates as anti-inflammatory...

Development of safer and more effective anti-inflammatory agents remains a significant challenge. Peptide-based conjugates have attracted increasing attention as alternative therapeutic strategies, owing to their enhanced selectivity and potentially improved safety profiles. In the present study, a combined computational and experimental approach was employed to investigate a series of novel dipeptide-coumarin conjugates as candidate anti-inflammatory agents. A series of rationally designed dipeptides conjugated with coumarin-3-carboxylic acid was first evaluated through molecular docking to estimate their binding affinity toward cyclooxygenase-2 (COX-2). Their pharmacokinetic and toxicity properties were further predicted using ADMETLab 3.0. Compounds showing favorable profiles were synthesized via solid-phase peptide synthesis (SPPS) and characterized by ¹H NMR, ¹³C NMR, and ESI-MS analyses. Anti-inflammatory activity was initially assessed using the albumin denaturation assay. Based on these findings, the most promising compound (C011) was selected for further investigation. Its cytotoxicity was evaluated using an MTT assay on normal human cell lines prior to in vivo studies. Subsequently, C011 was tested in a formaldehyde-induced paw edema model in rats, followed by hematological, histopathological, and behavioral evaluations. Docking analysis revealed consistently favorable binding affinities among the designed compounds, with C011 (VA-coumarin) exhibiting the strongest interaction (ΔG = -9.73 kcal mol^-1), exceeding that of the reference drug ketoprofen. In vitro screening confirmed its notable anti-inflammatory activity, supporting its selection for further studies. The MTT assay demonstrated acceptable cytocompatibility at the tested concentrations, supporting the continued evaluation of C011 in subsequent in vivo studies. In vivo evaluation further validated these findings, as C011 produced a significant and dose-dependent reduction in paw edema and inflammatory responses. Notably, treatment with the higher dose resulted in a marked decrease in white blood cell count and key inflammatory indices, including the neutrophil-to-lymphocyte ratio (NLR) and systemic immune-inflammation index (SII). These findings were further supported by histopathological observations and behavioral assessments, which demonstrated anti-inflammatory and analgesic effects while indicating no severe treatment-related tissue injury under the experimental conditions employed. Collectively, these findings identify C011 as a promising peptide-based scaffold for anti-inflammatory drug development. The study highlights the effectiveness of a stepwise evaluation strategy that integrates computational design with in vitro screening, preliminary safety assessment, and in vivo validation. Further studies are required to clarify the molecular mechanisms underlying the anti-inflammatory activity and to establish the long-term safety profile of C011.