Ngambenjawong Chayanon, Pineda Julio Marco B, Pun Suzie H
Department of Bioengineering and Molecular Engineering and Sciences Institute, University of Washington , Seattle, Washington 98195, United States.
Bioconjug Chem. 2016 Dec 21;27(12):2854-2862. doi: 10.1021/acs.bioconjchem.6b00502. Epub 2016 Nov 10.
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 (K) 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.
肽环化是一种用于提高肽稳定性和活性的策略。最常用的环化方法是含半胱氨酸肽形成二硫键桥，这在自然界中很常见。多年来，已报道了越来越多用于肽环化的替代化学方法，这些方法具有更高的效率、动力学、正交性和稳定性。然而，通过不同环化策略在连接位点引入的各种化学实体来微调肽活性的机会却较少受到关注。在此，我们展示了M2“抗炎”巨噬细胞结合肽（M2pep）的环化优化如何导致优化类似物与M2巨噬细胞的结合亲和力显著增加，同时与M1“促炎”巨噬细胞相比保持结合选择性。在本研究中，我们报告了四种采用不同环化策略的环状M2pep（RY）类似物的合成与评估：（1）天冬氨酸-[酰胺]-赖氨酸，（2）叠氮基赖氨酸-[三唑（铜（I）催化的炔烃-叠氮环加成反应（CuAAC））]-炔丙基甘氨酸，（3）半胱氨酸-[十氟联苯（DFBP）]-半胱氨酸，以及（4）半胱氨酸-[十氟联苯砜（DFS）]-半胱氨酸，其中连接位点处的化学实体或连接子显示在方括号中，位于参与环化的残基之间。将这些肽与我们之前报道的作为血清稳定、亲和力增强的原始线性M2pep类似物的二硫键环化M2pep（RY）进行比较。与原始二硫键环化M2pep（RY）的36.3 μM和解离常数（K）相比，DFBP环化的M2pep（RY）对M2巨噬细胞表现出最高的结合活性，表观解离常数约为2.03 μM，原始线性肽的解离常数为220 μM。DFS环化的M2pep（RY）也比原始环化类似物结合更强，而酰胺和三唑环化的M2pep（RY）类似物结合较弱。我们证实单独的DFBP与M2巨噬细胞的结合可忽略不计，并且在原始序列中引入二苯丙氨酸会以溶解性降低和毒性增加为代价提高结合活性。总之，我们报告了采用不同环化策略的环状M2pep（RY）类似物的开发，从而发现了具有增强的M2巨噬细胞结合活性的DFBP环化M2pep（RY）。
