Liu Minglong, Morewood Richard, Yoshisada Ryoji, Pascha Mirte N, Hopstaken Antonius J P, Tarcoveanu Eliza, Poole David A, de Haan Cornelis A M, Nitsche Christoph, Jongkees Seino A K
Department of Chemistry and Pharmaceutical Sciences, Vrije Universiteit Amsterdam Amsterdam The Netherlands
Amsterdam Institute of Molecular and Life Sciences (AIMMS), Vrije Universiteit Amsterdam Amsterdam The Netherlands.
Chem Sci. 2023 Sep 8;14(38):10561-10569. doi: 10.1039/d3sc03117a. eCollection 2023 Oct 4.
Peptide display technologies are a powerful method for discovery of new bioactive sequences, but linear sequences are often very unstable in a biological setting. Macrocyclisation of such peptides is beneficial for target affinity, selectivity, stability, and cell permeability. However, macrocyclisation of a linear hit is unreliable and requires extensive structural knowledge. Genetically encoding macrocyclisation during the discovery process is a better approach, and so there is a need for diverse cyclisation options that can be deployed in the context of peptide display techniques such as mRNA display. In this work we show that -cyanopyridylalanine (CNP) can be ribosomally incorporated into peptides, forming a macrocycle in a spontaneous and selective reaction with an N-terminal cysteine generated from bypassing the initiation codon in translation. This reactive amino acid can also be easily incorporated into peptides during standard Fmoc solid phase peptide synthesis, which can otherwise be a bottleneck in transferring from peptide discovery to peptide testing and application. We demonstrate the potential of this new method by discovery of macrocyclic peptides targeting influenza haemagglutinin, and molecular dynamics simulation indicates the CNP cross-link stabilises a beta sheet structure in a representative of the most abundant cluster of active hits. Cyclisation by CNP is also shown to be compatible with thioether macrocyclisation at a second cysteine to form bicycles of different architectures, provided that cysteine placement reinforces selectivity, with this bicyclisation happening spontaneously and in a controlled manner during peptide translation. Our new approach generates macrocycles with a more rigid cross-link and with better control of regiochemistry when additional cysteines are present, opening these up for further exploitation in chemical modification of translated peptides, and so is a valuable addition to the peptide discovery toolbox.
肽展示技术是发现新生物活性序列的有力方法，但线性序列在生物环境中往往非常不稳定。此类肽的大环化有利于提高靶标亲和力、选择性、稳定性和细胞通透性。然而，对线性命中肽进行大环化是不可靠的，并且需要广泛的结构知识。在发现过程中对大环化进行基因编码是一种更好的方法，因此需要多种可在诸如mRNA展示等肽展示技术中应用的环化选项。在这项工作中，我们表明，β-氰基吡啶丙氨酸（CNP）可以通过核糖体掺入肽中，在与翻译过程中绕过起始密码子产生的N端半胱氨酸发生的自发且选择性反应中形成大环。这种反应性氨基酸在标准的Fmoc固相肽合成过程中也很容易掺入肽中，否则这可能是从肽发现转移到肽测试及应用过程中的一个瓶颈。我们通过发现靶向流感血凝素的大环肽证明了这种新方法的潜力，分子动力学模拟表明，CNP交联在活性命中肽最丰富簇的一个代表中稳定了β折叠结构。还表明，CNP环化与在第二个半胱氨酸处的硫醚大环化兼容，以形成不同结构的双环，前提是半胱氨酸的位置增强了选择性，这种双环化在肽翻译过程中自发且可控地发生。当存在额外的半胱氨酸时，我们的新方法会生成具有更刚性交联且区域化学控制更好的大环化合物，为翻译后肽的化学修饰进一步开发开辟了道路，因此是肽发现工具箱中的一项有价值的补充。
