Choudhury Asha Rani, Gaikwad Vikram, Maity Atanu, Chakrabarti Rajarshi
Department of Chemistry, Indian Institute of Technology Bombay, Mumbai, India.
Bioinfomatics Center, Department of Bioscience and Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, India.
Proteins. 2025 Jun;93(6):1090-1106. doi: 10.1002/prot.26793. Epub 2025 Jan 3.
Short-length peptides are used as therapeutics due to their high target specificity and low toxicity; for example, peptides are designed for targeting the interaction between oncogenic protein p53 and E3 ubiquitin ligase MDM2. These peptide therapeutics form a class of successful inhibitors. To design such peptide-based inhibitors, stapling is one of the methods in which amino acid side chains are stitched together to get conformationally rigid peptides, ensuring effective binding to their partners. In the current work, we use computer simulations to investigate p53 peptides stapled with hydrocarbon chains of different lengths and positions of attachment to the peptide. We subsequently analyze their binding efficiency with MDM2. The introduction of stapling agents restricts the conformational dynamics of peptides, resulting in higher persistence of helicity. The efficiency of the stapling agents has also been verified imposing these stapled peptides to adverse conditions viz. thermal and chemical denaturation. In addition, the conformational exploration of peptides has been investigated using temperature replica exchange molecular dynamics (T-REMD) simulations. From both the unbiased and T-REMD simulations, p53 with a long hydrocarbon cross-linker shows a more conformationally rigid structure having high helicity compared to other stapled peptides. The rigidity gained due to cross-linking reduces the entropy of the peptide in the free state and thereby facilitates the complexation process. From the binding studies, we have shown that the peptide having multiple short staples has a larger enthalpy change during binding, resulting from its orientation and interactions with residues in the binding interface. On the other hand, a peptide with a single long stapling agent shows less entropic penalty than other systems. Our study suggests a plausible rationale for the relation between the length and the position of attachment of cross-linkers to peptides and their binding affinity for target partners.
短肽因其高靶向特异性和低毒性而被用作治疗剂；例如，设计肽以靶向致癌蛋白p53与E3泛素连接酶MDM2之间的相互作用。这些肽治疗剂构成了一类成功的抑制剂。为了设计这种基于肽的抑制剂，订书钉法是其中一种方法，即将氨基酸侧链缝合在一起以获得构象刚性的肽，确保与它们的结合伴侣有效结合。在当前的工作中，我们使用计算机模拟来研究用不同长度和连接到肽上的位置的烃链订书钉化的p53肽。我们随后分析它们与MDM2的结合效率。订书钉剂的引入限制了肽的构象动力学，导致更高的螺旋持续性。订书钉剂的效率也通过将这些订书钉化的肽置于不利条件下（即热变性和化学变性）得到了验证。此外，使用温度复制交换分子动力学（T-REMD）模拟研究了肽的构象探索。从无偏模拟和T-REMD模拟中，与其他订书钉化肽相比，具有长烃交联剂的p53显示出具有更高螺旋度的更构象刚性的结构。由于交联获得的刚性降低了游离状态下肽的熵，从而促进了络合过程。从结合研究中，我们已经表明，具有多个短订书钉的肽在结合过程中具有更大的焓变，这是由于其取向以及与结合界面中残基的相互作用所致。另一方面，具有单个长订书钉剂的肽比其他系统显示出更小的熵罚。我们的研究为交联剂与肽的连接长度和位置与其对靶标伴侣的结合亲和力之间的关系提出了一个合理的原理。
