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Entropy of stapled peptide inhibitors in free state is the major contributor to the improvement of binding affinity with the GK domain
RSC Chemical Biology Pub Date : 2021-06-25 , DOI: 10.1039/d1cb00087j
Ilona Christy Unarta 1, 2 , Jianchao Xu 3 , Yuan Shang 2, 4 , Carina Hey Pui Cheung 3 , Ruichi Zhu 2, 4 , Xudong Chen 2, 4 , Siqin Cao 2, 5 , Peter Pak-Hang Cheung 2, 3 , Donald Bierer 6 , Mingjie Zhang 2, 4 , Xuhui Huang 1, 2, 5, 7 , Xuechen Li 3
Affiliation  

Stapled peptides are promising protein–protein interaction (PPI) inhibitors that can increase the binding potency. Different from small-molecule inhibitors in which the binding mainly depends on energetic interactions with their protein targets, the binding of stapled peptides has long been suggested to be benefited from entropy. However, it remains challenging to reveal the molecular features that lead to this entropy gain, which could originate from the stabilization of the stapled peptide in solution or from the increased flexibility of the complex upon binding. This hinders the rational design of stapled peptides as PPI inhibitors. Using the guanylate kinase (GK) domain of the postsynaptic density protein 95 (PSD-95) as the target, we quantified the enthalpic and entropic contributions by combining isothermal titration calorimetry (ITC), X-ray crystallography, and free energy calculations based on all-atom molecular dynamics (MD) simulations. We successfully designed a stapled peptide inhibitor (staple 1) of the PSD-95 GK domain that led to a 25-fold increase in the binding affinity (from tens of μMs to 1.36 μM) with high cell permeability. We showed that entropy indeed greatly enhanced the binding affinity and the entropy gain was mainly due to the constrained-helix structure of the stapled peptide in solution (free state). Based on staple 1, we further designed two other stapled peptides (staple 2 and 3), which exerted even larger entropy gains compared to staple 1 because of their more flexible bound complexes (bound state). However, for staple 2 and 3, the overall binding affinities were not improved, as the loose binding in their bound states led to an enthalpic loss that largely compensated the excess entropy gain. Our work suggests that increasing the stability of the stapled peptide in free solution is an effective strategy for the rational design of stapled peptides as PPI inhibitors.

中文翻译:

游离状态的订书肽抑制剂的熵是提高与 GK 结构域结合亲和力的主要因素

订书钉肽是有前途的蛋白质-蛋白质相互作用 (PPI) 抑制剂,可以增加结合效力。与结合主要依赖于与其蛋白质靶标的能量相互作用的小分子抑制剂不同,长期以来人们认为订书肽的结合受益于熵。然而,揭示导致这种熵增加的分子特征仍然具有挑战性,这可能源于溶液中钉合肽的稳定性或复合物在结合时增加的灵活性。这阻碍了订书肽作为 PPI 抑制剂的合理设计。使用突触后密度蛋白 95 (PSD-95) 的鸟苷酸激酶 (GK) 域作为目标,我们通过结合等温滴定量热法 (ITC) 量化了焓和熵的贡献,X 射线晶体学和基于全原子分子动力学 (MD) 模拟的自由能计算。我们成功设计了 PSD-95 GK 结构域的订书肽抑制剂(staple 1),使结合亲和力增加了 25 倍(从几十 μM 到 1.36 μM),并具有高细胞渗透性。我们表明熵确实大大增强了结合亲和力,熵增加主要是由于溶液中钉合肽的约束螺旋结构(游离状态)。基于主食 1,我们进一步设计了另外两种装订肽(主食 2 和 3),由于它们更灵活的结合复合物(结合状态),与主食 1 相比,它们产生了更大的熵增益。然而,对于主食 2 和 3,整体结合亲和力没有提高,因为它们束缚态的松散束缚导致了焓损失,这在很大程度上补偿了过量的熵增益。我们的工作表明,增加订书肽在游离溶液中的稳定性是合理设计订书肽作为 PPI 抑制剂的有效策略。
更新日期:2021-07-15
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