Center for the Development of Therapeutics, The Broad Institute of MIT and Harvard.
Center for the Development of Therapeutics, The Broad Institute of MIT and Harvard;
J Vis Exp. 2024 Jan 12(203). doi: 10.3791/65718.
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 Brd4 and VHL was evaluated. The heterobifunctional molecule, MZ1, has nM affinities for both the Brd4 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.
E3 连接酶和靶向降解的蛋白质可以通过多步过程中异双功能分子诱导形成复合物。涉及的相互作用的动力学和热力学有助于泛素化的效率和导致蛋白质的降解。生物物理技术，如表面等离子体共振（SPR）、生物层干涉（BLI）和等温热量滴定（ITC）提供了有价值的信息，可用于优化这些相互作用。使用两个模型系统，建立了一种用于理解三元复合物形成协同作用和“钩效应”对结合动力学影响的生物物理测定工具包。在一种情况下，评估了一种诱导 Brd4 和 VHL 之间形成三元复合物的蛋白水解靶向嵌合体（PROTAC）分子。该异双功能分子 MZ1 对 Brd4 蛋白（SPR KD = 1 nM，ITC KD = 4 nM）和 VHL 复合物（SPR KD = 29 nM，ITC KD = 66 nM）均具有纳摩尔亲和力。对于该系统，开发了强大的 SPR、BLI 和 ITC 测定法，重现了表明三元复合物形成协同作用的已发表结果。在另一种情况下，研究了一种诱导 46.0 kDa 蛋白 PPM1D 和 cereblon [CRBN（319-442）]之间形成三元复合物的分子。该异双功能分子 BRD-5110 对 PPM1D 的 SPR KD = 1 nM，但对截短的 CRBN（319-442）复合物的结合较弱（SPR KD = ~ 3 µM）。在这种情况下，CRBN 在 SPR 中的结合不是饱和的，导致“钩效应”。评估了 SPR、BLI 和 ITC 的通量和试剂要求，并提供了将其应用于 PROTAC 项目的一般建议。
