Kurt Han, Akyol Ali, Son Cagdas Devrim, Zheng Chen, Gado Irene, Meli Massimiliano, Ferrandi Erica Elisa, Bassanini Ivan, Vasile Francesca, Gurevich Vsevolod V, Nebol Aylin, Cagavi Esra, Morra Giulia, Sensoy Ozge
Istanbul Medipol University, Graduate School of Engineering and Natural Sciences, 34810, Istanbul, Turkey.
present address: University of Cagliari, Department of Physics, Cittadella Universitaria, I-09042 Monserrato (CA), Italy.
bioRxiv. 2024 Dec 13:2024.12.12.628161. doi: 10.1101/2024.12.12.628161.
G protein-coupled receptor (GPCR) signaling is terminated by arrestin binding to a phosphorylated receptor. Binding propensity has been shown to be modulated by stabilizing the pre-activated state of arrestin through point mutations or C-tail truncation. Here, we hypothesize that pre-activated rotated states can be stabilized by small molecules, and this can promote binding to phosphorylation-deficient receptors, which underly a variety of human disorders. We performed virtual screening on druggable pockets identified on pre-activated conformations in Molecular Dynamics trajectories of arrestin-3, and found a compound targeting an activation switch, the back loop at the inter-domain interface. According to our model, consistent with available biochemical and structural data, the compound destabilized the ionic lock between the finger and the back loop, and enabled transition of the gate loop towards the pre-activated state, which stabilizes pre-activated inter-domain rotation. The predicted binding pocket is consistent with saturation-transfer difference NMR data indicating close contact between the piperazine moiety of the compound and C/finger loops. The compound increases in-cell arrestin-3 binding to phosphorylation-deficient and wild-type β-adrenergic receptor, but not to muscarinic M2 receptor, as verified by FRET and NanoBiT. This study demonstrates that the back loop can be targeted to modulate interaction of arrestin with phosphorylation-deficient GPCRs in a receptor-specific manner.
G蛋白偶联受体（GPCR）信号传导通过抑制蛋白与磷酸化受体的结合而终止。通过点突变或C末端截短来稳定抑制蛋白的预激活状态，已显示结合倾向会受到调节。在此，我们假设小分子可以稳定预激活的旋转状态，这可以促进与磷酸化缺陷型受体的结合，而磷酸化缺陷型受体是多种人类疾病的基础。我们对在抑制蛋白-3的分子动力学轨迹中的预激活构象上确定的可成药口袋进行了虚拟筛选，发现了一种靶向激活开关（结构域间界面处的后环）的化合物。根据我们的模型，与现有的生化和结构数据一致，该化合物破坏了手指结构与后环之间的离子锁，并使“门环”向预激活状态转变，从而稳定了预激活的结构域间旋转。预测的结合口袋与饱和转移差异核磁共振数据一致，表示该化合物的哌嗪部分与C/手指环之间紧密接触。如通过FRET和NanoBiT所验证的那样，该化合物增加了细胞内抑制蛋白-3与磷酸化缺陷型和野生型β-肾上腺素能受体的结合，但不增加与毒蕈碱M2受体的结合。这项研究表明，可以靶向作用后环，以受体特异性方式调节抑制蛋白与磷酸化缺陷型GPCR的相互作用。
