Department of Biochemistry & Biophysics and Center for RNA Biology, University of Rochester School of Medicine & Dentistry, Rochester, New York, USA.
Department of Chemistry, University of Rochester, Rochester, New York, USA.
J Biol Chem. 2021 Dec;297(6):101390. doi: 10.1016/j.jbc.2021.101390. Epub 2021 Nov 9.
RNA represents a potential target for new antiviral therapies, which are urgently needed to address public health threats such as the human immunodeficiency virus (HIV). We showed previously that the interaction between the viral Tat protein and the HIV-1 trans-activation response (TAR) RNA was blocked by TB-CP-6.9a. This cyclic peptide was derived from a TAR-binding loop that emerged during lab evolution of a TAR-binding protein (TBP) family. Here we synthesized and characterized a next-generation, cyclic-peptide library based on the TBP scaffold. We sought to identify conserved RNA-binding interactions and the influence of cyclization linkers on RNA binding and antiviral activity. A diverse group of cyclization linkers, encompassing disulfide bonds to bicyclic aromatic staples, was used to restrain the cyclic peptide geometry. Thermodynamic profiling revealed specific arginine-rich sequences with low to submicromolar affinity driven by enthalpic and entropic contributions. The best compounds exhibited no appreciable off-target binding to related molecules, such as BIV TAR and human 7SK RNAs. A specific arginine-to-lysine change in the highest affinity cyclic peptide reduced TAR binding by tenfold, suggesting that TBP-derived cyclic peptides use an arginine-fork motif to recognize the TAR major groove while differentiating the mode of binding from other TAR-targeting molecules. Finally, we showed that HIV infectivity in cell culture was reduced in the presence of cyclic peptides constrained by methylene or naphthalene-based linkers. Our findings provide insight into the molecular determinants required for HIV-1 TAR recognition and antiviral activity. These findings are broadly relevant to the development of antivirals that target RNA molecules.
RNA 是新抗病毒疗法的潜在靶点，对于应对人类免疫缺陷病毒 (HIV) 等公共卫生威胁，这些疗法是急需的。我们之前已经表明，病毒 Tat 蛋白与 HIV-1 反式激活反应 (TAR) RNA 之间的相互作用被 TB-CP-6.9a 阻断。这种环肽来源于 TAR 结合环，它是在 TAR 结合蛋白 (TBP) 家族的实验室进化过程中出现的。在这里，我们基于 TBP 支架合成并表征了下一代环肽文库。我们试图确定保守的 RNA 结合相互作用以及环化接头对 RNA 结合和抗病毒活性的影响。使用了多种环化接头，包括二硫键到双环芳族支架，以限制环肽的几何形状。热力学分析揭示了具有低至亚毫摩尔亲和力的特定富含精氨酸的序列，这是由焓和熵贡献驱动的。最好的化合物对相关分子（如 BIV TAR 和人 7SK RNA）没有明显的非靶标结合。最高亲和力环肽中的特定精氨酸到赖氨酸变化使 TAR 结合减少了十倍，这表明 TBP 衍生的环肽使用精氨酸叉基序来识别 TAR 主槽，同时区分与其他 TAR 靶向分子的结合模式。最后，我们表明，在存在由亚甲基或萘基接头约束的环肽的情况下，细胞培养中的 HIV 感染性降低。我们的研究结果为 HIV-1 TAR 识别和抗病毒活性所需的分子决定因素提供了深入了解。这些发现与开发针对 RNA 分子的抗病毒药物具有广泛的相关性。
