title: Engineering an Affinity-Enhanced Peptide through Optimization of Cyclization Chemistry.
abstract: Peptide cyclization is a strategy used to improve stability and activity of peptides. The most commonly used cyclization method is disulfide bridge formation of cysteine-containing peptides, as is typically found in nature. Over the years, an increasing number of alternative chemistries for peptide cyclization with improved efficiency, kinetics, orthogonality, and stability have been reported. However, there has been less appreciation for the opportunity to fine-tune peptide activity via the diverse chemical entities introduced at the site of linkage by different cyclization strategies. Here, we demonstrate how cyclization optimization of an M2 "anti-inflammatory" macrophage-binding peptide (M2pep) resulted in a significant increase in binding affinity of the optimized analog to M2 macrophages while maintaining binding selectivity compared to M1 "pro-inflammatory" macrophages. In this study, we report synthesis and evaluation of four cyclic M2pep(RY) analogs with diverse cyclization strategies: (1) Asp-[amide]-Lys, (2) azido-Lys-[triazole(copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC))]-propargyl-Gly, (3) Cys-[decafluorobiphenyl (DFBP)]-Cys, and (4) Cys-[decafluorobiphenyl sulfone (DFS)]-Cys, whereby the chemical entity or linker at the linkage site is shown in the square bracket and is between the residues involved in cyclization. These peptides are compared to a disulfide-cyclized M2pep(RY) that we previously reported as a serum-stable, affinity-enhanced analog to the original linear M2pep. DFBP-cyclized M2pep(RY) exhibits the highest binding activity to M2 macrophages with apparent dissociation constant (KD) about 2.03 μM compared to 36.3 μM for the original disulfide-cyclized M2pep(RY) and 220 μM for the original linear peptide. DFS-cyclized M2pep(RY) also binds more strongly than the original cyclized analog, whereas amide- and triazole-cyclized M2pep(RY) analogs bind less strongly. We verified that DFBP alone has negligible binding to M2 macrophages and the incorporation of diphenylalanine to the original sequence improves binding activity at the expense of solubility and increased toxicity. In conclusion, we report development of cyclic M2pep(RY) analogs with diverse cyclization strategies leading to the discovery of DFBP-cyclized M2pep(RY) with enhanced M2 macrophage-binding activity.
authors: Department of Bioengineering and Molecular Engineering and Sciences Institute, University of Washington , Seattle, Washington 98195, United States.
authors: Chayanon
authors: C
authors: Ngambenjawong
authors: Department of Bioengineering and Molecular Engineering and Sciences Institute, University of Washington , Seattle, Washington 98195, United States.
authors: Julio Marco B
authors: JM
authors: Pineda
authors: Department of Bioengineering and Molecular Engineering and Sciences Institute, University of Washington , Seattle, Washington 98195, United States.
authors: Suzie H
authors: SH
authors: Pun
mesh_terms: D000818:Animals; D001386:Azides / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D060326:Chemistry Techniques, Synthetic; D002942:Circular Dichroism; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D010455:Peptides / Q000097:blood / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods; D055550:Protein Stability
chemical_list: D001386:Azides; D010455:Peptides; D001710:Biotin