三元络合物
化学
等温滴定量热法
合作性
生物物理学
受体-配体动力学
表面等离子共振
血浆蛋白结合
动力学
合作约束
蛋白质水解
生物化学
结合位点
纳米技术
受体
生物
材料科学
物理
量子力学
纳米颗粒
酶
作者
Wei Jiang,Holly H. Soutter
摘要
E3 ligases and proteins targeted for degradation can be induced to form complexes by heterobifunctional molecules in a multi-step process. The kinetics and thermodynamics of the interactions involved contribute to efficiency of ubiquitination and resulting degradation of the protein. Biophysical techniques such as surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC) provide valuable information that can be used in the optimization of those interactions. Using two model systems, a biophysical assay tool kit for understanding the cooperativity of ternary complex formation and the impact of the 'hook effect' on binding kinetics was established. In one case, a proteolysis targeting chimera (PROTAC) molecule that induced ternary complex formation between Brd4BD2 and VHL was evaluated. The heterobifunctional molecule, MZ1, has nM affinities for both the Brd4BD2 protein (SPR KD = 1 nM, ITC KD = 4 nM) and the VHL complex (SPR KD = 29 nM, ITC KD = 66 nM). For this system, robust SPR, BLI, and ITC assays were developed that reproduced published results demonstrating the cooperativity of ternary complex formation. In the other case, a molecule that induced ternary complexes between a 46.0 kDa protein, PPM1D, and cereblon [CRBN (319-442)] was studied. The heterobifunctional molecule, BRD-5110, has an SPR KD = 1 nM for PPM1D but much weaker binding against the truncated CRBN (319-442) complex (SPR KD= ~ 3 µM). In that case, the binding for CRBN in SPR was not saturable, resulting in a "hook-effect". Throughput and reagent requirements for SPR, BLI, and ITC were evaluated, and general recommendations for their application to PROTAC projects were provided.
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