Background: Inflammation is a critical biological response to harmful stimuli, with tumour necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) serving as key mediators of acute and chronic inflammatory diseases. The development of peptide-based therapeutics has emerged as a promising approach for targeted immunomodulation. The larvae of Culex quinquefasciatus, a mosquito species of significant public health importance, synthesize a diverse repertoire of proteins during development, including hexamerins and storage proteins, which may serve as potential sources of bioactive peptides with therapeutic properties. However, the potential of these larval peptides as anti-inflammatory agents remains largely unexplored.

Objective: This study aimed to identify, characterize, and evaluate potential bioactive peptides derived from Culex quinquefasciatus larval proteins through computational approaches, assessing their binding affinity and interaction profiles with the pro-inflammatory cytokine’s TNF-α and IL-6.

Methods: A comprehensive in silico pipeline was employed, integrating homology modelling, molecular docking, and molecular dynamics simulations. Larval storage protein sequences were retrieved from genomic databases. Homology models of TNF-α and IL-6 were constructed using Swiss-Model and validated through PROCHECK and ERRAT analyses. A library of bioactive peptides (8-25 amino acids) was generated through in silico digestion of larval proteins using Expose Peptide Cutter. Molecular docking was performed using Auto Dock Vina and HADDOCK, followed by 100 ns molecular dynamics simulations using GROMACS to assess binding stability. Peptide toxicity, allergenicity, and physicochemical properties were evaluated using Toxin Pred, Aller TOP, and Peptide Ranker.

Results: A total of 157 peptides were generated from 11 larval storage proteins identified in Cx. quinquefasciatus. From these, 23 peptides exhibited favourable drug-like properties and were selected for docking studies. Three lead peptides demonstrated significant binding affinities: LSP-1.2-derived peptide YFYPYTPY (ΔG = -9.8 kcal/mol) and LSP-2.1-derived peptide FEYWPNEF (ΔG = -9.2 kcal/mol) for TNF-α, and LSP-1.7-derived peptide WHWYFYP (ΔG = -10.1 kcal/mol) for IL-6. Molecular dynamics simulations confirmed stable binding conformations with RMSD values < 2.5 Å over 100 ns. Key binding interactions involved hydrogen bonds with TNF-α residues TYR-119, GLN-125, and SER-147, and IL-6 residues PHE-74 and ARG-179. All three peptides were predicted as non-toxic and non-allergenic.