Targeted protein degradation is a novel pharmacology established by drugs that recruit target proteins to E3 ubiquitin ligases. Based on the structure of the degrader and the target, different E3 interfaces are critically involved, thus forming defined ‘functional hotspots’. Understanding disruptive mutations in functional hotspots informs on the architecture of the assembly, and highlights residues susceptible to acquire resistance phenotypes. Here we employ haploid genetics to show that hotspot mutations cluster in substrate receptors of hijacked ligases, where mutation type and frequency correlate with gene essentiality. Intersection with deep mutational scanning revealed hotspots that are conserved or specific for chemically distinct degraders and targets. Biophysical and structural validation suggests that hotspot mutations frequently converge on altered ternary complex assembly. Moreover, we validated hotspots mutated in patients that relapse from degrader treatment. In sum, we present a fast and widely accessible methodology to characterize small-molecule degraders and associated resistance mechanisms.

靶向蛋白质降解是一种新的药理学，由将靶蛋白招募到 E3 泛素连接酶的药物建立。根据降解器和目标的结构，关键涉及不同的E3接口，从而形成定义的“功能热点”。了解功能热点中的破坏性突变可以了解组装的结构，并突出显示容易获得抗性表型的残基。在这里，我们利用单倍体遗传学来证明热点突变聚集在被劫持的连接酶的底物受体中，其中突变类型和频率与基因必要性相关。与深度突变扫描的交叉揭示了化学上不同的降解剂和靶标的保守或特异性热点。生物物理和结构验证表明热点突变经常集中在改变的三元复合物组装上。此外，我们还验证了降解剂治疗复发的患者中的热点突变。总之，我们提出了一种快速且广泛使用的方法来表征小分子降解剂和相关的耐药机制。