In silico peptide-based therapeutics against human colorectal cancer by the activation of TLR5 signaling pathways

Objective Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in both men and women. Toll-like receptor 5 (TLR5), an autoimmune signaling receptor that plays a role in cancer, can be exploited for the suppression of human colon cancer. Salmonella flagellin protein, a novel agonist of TLR5 activating downstream signaling, could be a basis for designing anticancer peptides. Methods The three-dimensional crystal structure of TLR5 (PDB ID: 3J0A, Resolution = 26.0 Å) was optimized using the AMBER force field in the YASARA suit. In silico enzymatic digestion tool, PeptideCutter, was used to identify peptides from Salmonella flagellin, an agonist against human TLR5. The 3D structure of the peptides was generated using PEP-FOLD3. These peptides were screened against human TLR5 using shape complementarity principles based on the binding affinity and interactions with the active residue of TLR5 monomer, and the selected peptides were further validated by molecular dynamic (MD) simulation. Results In this study, we generated 42 peptides from Salmonella flagellin protein by in silico protein digestion. Then, based on a new hidden Markov model sub-optimal conformation sampling approach as well as the size of the fragments, we select 38 effective peptides from these 42 cleavages. These peptides were screened against the monomeric Xray structure of human TLR5 using shape complementarity principles. Based on the binding affinity and interactions with the active residue of TLR5 monomer (residues 294 and 366 of TLR5), nine top-scored peptides were selected for the initial molecular dynamic (MD) simulation. Among these peptides, Clv10, Clv17, and Clv28 showed high stability and less flexibility during MD simulation. A 1 μs MD simulation was performed on TLR5-Clv10, TLR-Clv17, and TLR5-Clv28 complexes to further analyze the stability, conformational changes, and binding mode (Clv10, Clv17, and Clv28). During this MD study, the peptides showed high salt bridges and ionic interactions with residue ASP294 and residue ASP366 throughout the simulation and remained in the concave of the human TLR5 monomer. The RMSD and Rg values showed that the peptide-protein complexes become stable after 200 ns of contraction and extraction. Conclusion These findings can facilitate the rational design of selected peptides as an agonist of TLR5, which have antitumor activity, suppress colorectal cancer tumors, and can be used as promising candidates and novel agonists of TLR5. Graphical Abstract