Chen Hongxue, Katoh Takayuki, Suga Hiroaki
Department of Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
ACS Bio Med Chem Au. 2023 Aug 13;3(5):429-437. doi: 10.1021/acsbiomedchemau.3c00027. eCollection 2023 Oct 18.
Membrane permeability is an important factor that determines the virtue of peptides targeting intracellular molecules. By introducing a membrane penetration motif, some peptides exhibit better membrane permeabilities. Previous choices for such motifs have usually been polycationic sequences, but their protease vulnerabilities and modest endosome escapability remain challenging. Here, we report a strategy for macrocyclization of peptides closed by a hydrophobic bipyridyl (BPy) unit, which grants an improvement of their membrane permeability and proteolytic stability compared with the conventional polycationic peptides. We chemically prepared model macrocyclic peptides closed by a thioether-BPy unit and determined their cell membrane permeability, giving 200 nM CP (an indicative value of membrane permeability), which is 40-fold better than that of the ordinary thioether macrocycle consisting of the same sequence composition. To discover potent target binders consisting of the BPy unit, we reprogrammed the initiator with chloromethyl-BPy (BPy) for the peptide library synthesis with a downstream Cys residue(s) and executed RaPID (Random nonstandard Peptide Integrated Discovery) against the bromodomains of BRD4. One of the obtained sequences exhibited a single-digit nanomolar dissociation constant against BRD4 in vitro and showed approximately 2-fold and 10-fold better membrane permeability than positive controls, R9 and Tat peptides, respectively. Moreover, we observed an intracellular activity of the BPy macrocycle tagged with a proteasome target peptide motif (RRRG), resulting in modest but detectable degradation of BRD4. The present demonstration indicates that the combination of the RaPID system with an appropriate hydrophobic unit, such as BPy, would provide a potential approach for devising cell penetrating macrocycles targeting various intracellular proteins.
膜通透性是决定靶向细胞内分子的肽类性质的一个重要因素。通过引入膜穿透基序，一些肽表现出更好的膜通透性。以往此类基序的选择通常是聚阳离子序列，但其蛋白酶敏感性和有限的内体逃逸能力仍然是具有挑战性的问题。在此，我们报道了一种通过疏水性联吡啶（BPy）单元封闭来实现肽大环化的策略，与传统的聚阳离子肽相比，这提高了它们的膜通透性和蛋白水解稳定性。我们化学合成了由硫醚-BPy单元封闭的模型大环肽，并测定了它们的细胞膜通透性，得到200 nM的CP（膜通透性的指示值），这比由相同序列组成的普通硫醚大环肽的通透性好40倍。为了发现由BPy单元组成的有效靶标结合物，我们用氯甲基-BPy（BPy）重新编程引发剂，用于合成带有下游半胱氨酸残基的肽库，并针对BRD4的溴结构域进行了RaPID（随机非标准肽整合发现）。所获得的序列之一在体外对BRD4表现出个位数纳摩尔的解离常数，并且分别比阳性对照R9和Tat肽的膜通透性高约2倍和10倍。此外，我们观察到带有蛋白酶体靶标肽基序（RRRG）的BPy大环肽具有细胞内活性，导致BRD4有适度但可检测到的降解。目前的证明表明，将RaPID系统与合适的疏水单元（如BPy）相结合，将为设计靶向各种细胞内蛋白质的细胞穿透大环肽提供一种潜在方法。
