Montero Fernando, Parra-López Marisa, Rodríguez-Martínez Alejandro, Murciano-Calles Javier, Buzon Pedro, Han Ziying, Lin L-Y, Ramos Maria C, Ruiz-Sanz Javier, Martinez Jose C, Radi Marco, Moog Christiane, Diederich Sandra, Harty Ronald N, Pérez-Sánchez Horacio, Vicente Francisca, Castillo Francisco, Luque Irene
Department of Physical Chemistry, Institute of Biotechnology, and Unit of Excellence in Chemistry Applied to Biomedicine and Environment, School of Sciences, University of Granada, Granada, Spain.
Structural Bioinformatics and High-Performance Computing (BIO-HPC) Research Group, Universidad Católica de Murcia (UCAM), Guadalupe, Spain.
Protein Sci. 2025 Jan;34(1):e70005. doi: 10.1002/pro.70005.
The ubiquitin E2 variant domain of TSG101 (TSG101-UEV) plays a pivotal role in protein sorting and virus budding by recognizing PTAP motifs within ubiquitinated proteins. Disruption of TSG101-UEV/PTAP interactions has emerged as a promising strategy for the development of host-oriented broad-spectrum antivirals with low susceptibility to resistance. TSG101 is a challenging target characterized by an extended and flat binding interface, low affinity for PTAP ligands, and complex binding energetics. Here, we assess the druggability of the TSG101-UEV/PTAP binding interface by searching for drug-like inhibitors and evaluating their ability to block PTAP recognition, impair budding, and inhibit viral proliferation. A discovery workflow was established by combining in vitro miniaturized HTS assays and a set of cell-based activity assays including high-content bimolecular complementation, virus-like particle release measurement, and antiviral testing in live virus infection. This approach has allowed us to identify a set of chemically diverse molecules that block TSG101-UEV/PTAP binding with IC50s in the low μM range and are able to disrupt the interaction between full-length TSG101 and viral proteins in human cells and inhibit viral replication. State-of-the-art molecular docking studies reveal that the active compounds exploit binding hotspots at the PTAP binding site, unlocking the full binding potential of the TSG101-UEV binding pockets. These inhibitors represent promising hits for the development of novel broad-spectrum antivirals through targeted optimization and are also valuable tools for investigating the involvement of ESCRT in the proliferation of different virus families and study the secondary effects induced by the disruption of ESCRT/virus interactions.
肿瘤易感基因101（TSG101）的泛素E2变异结构域（TSG101-UEV）通过识别泛素化蛋白中的PTAP基序，在蛋白质分选和病毒出芽过程中发挥关键作用。破坏TSG101-UEV/PTAP相互作用已成为一种有前景的策略，用于开发对耐药性低敏感性的宿主导向型广谱抗病毒药物。TSG101是一个具有挑战性的靶点，其特征在于结合界面延伸且平坦、对PTAP配体亲和力低以及结合能量复杂。在此，我们通过寻找类药物抑制剂并评估它们阻断PTAP识别、损害出芽以及抑制病毒增殖的能力，来评估TSG101-UEV/PTAP结合界面的成药潜力。通过结合体外小型化高通量筛选试验和一系列基于细胞的活性试验，包括高内涵双分子互补、病毒样颗粒释放测量以及活病毒感染中的抗病毒测试，建立了一个发现流程。这种方法使我们能够鉴定出一组化学结构多样的分子，它们以低 microM范围内的IC50阻断TSG101-UEV/PTAP结合，并且能够破坏全长TSG101与人类细胞中病毒蛋白之间的相互作用并抑制病毒复制。最先进的分子对接研究表明，活性化合物利用PTAP结合位点的结合热点，释放TSG101-UEV结合口袋的全部结合潜力。这些抑制剂是通过靶向优化开发新型广谱抗病毒药物的有前景的命中物，也是研究内体分选转运复合体（ESCRT）参与不同病毒家族增殖以及研究ESCRT/病毒相互作用破坏所诱导的次级效应的有价值工具。
