Modulation of the Inflammatory Process through the Control of Cyclooxygenase Using Peptides Obtained from Lithobates Catesbeianus Identified by Molecular Docking

Inflammatory diseases encompass a wide range of disorders that affect different systems of the body, such as the skin, joints, and gastrointestinal tract. Notable examples of such disorders include psoriasis, rheumatoid arthritis, and Crohn’s disease. Research highlights the therapeutic potential of animal-derived peptides under these conditions. Advances in discovering promising drugs involve advanced bioinformatics techniques, such as molecular docking combined with in vitro studies, which have shown consistent results. This study aimed to explore and characterize peptides derived from the enzymatic hydrolysis of bullfrog skin proteins with the potential to modulate multiple inflammatory pathways, particularly through cyclooxygenase (COX) inhibition and IL-6 downregulation. Bullfrog skin was lyophilized, and peptides were extracted through enzymatic hydrolysis. Peptide fractionation was performed using solid-phase extraction, and mass spectrometry was performed on the target fraction. A molecular docking analysis was subsequently utilized to predict the interactions of the peptides with the COX active site. The peptide sequences were investigated for their potential to inhibit the COX enzymes through a colorimetric inhibition assay. The IL-6 expression was evaluated on the selected peptide sequence using a Murine Mini ABTS IL-6 enzyme-linked immunosorbent assay (ELISA) development kit. Based on the results, the bullfrog skin hydrolysates, especially those obtained through trypsin digestion, exhibited a significant dose-dependent enhancement in the cell viability of RAW 264.7 macrophages. Notably, the F4 fraction, isolated by solid-phase extraction, demonstrated the most pronounced effect. Mass spectrometry analysis of the F4 fraction identified 71 low-molecular-weight peptide sequences corresponding to different bullfrog proteins. Four peptides (P1, P2, P3, and P4) were selected for synthesis based on molecular docking results, which predicted a high binding affinity and favorable interactions with the COX active site, especially for peptide GPSGPAGARGDK (P3). Despite the strong binding affinity of P3, biological proof-of-concept studies revealed that SGHPGAMGPVGPR (P1) exhibited the most significant results, effectively inhibiting total COX activity and downregulating IL-6 expression in RAW 264.7 macrophages at a concentration of 1 mM. The P1 peptide exhibited structural stability and demonstrated a superior ability to modulate the inflammatory response. Although some discrepancies between molecular docking and in vitro results were observed, this study highlights the importance of integrative analyses in enhancing success rates for identifying viable therapeutic candidates. Overall, the findings indicate that peptide P1, identified within the F4 fraction, is a promising candidate for further optimization as a cyclooxygenase inhibitor, with potential applications in the development of biopharmaceuticals for the treatment of inflammatory skin diseases. Further controlled studies are necessary to elucidate additional mechanisms underlying its anti-inflammatory properties and to refine its therapeutic potential.