Profiling the Landscape of Drug Resistance Mutations in Neosubstrates to Molecular Glue Degraders

Targeted protein degradation (TPD) holds immense promise for drug discovery, but mechanisms of acquired resistance to degraders remain to be fully identified. Here, we used clustered regularly interspaced short palindromic repeats (CRISPR)-suppressor scanning to identify mechanistic classes of drug resistance mutations to molecular glue degraders in GSPT1 and RBM39, neosubstrates targeted by E3 ligase substrate receptors cereblon and DCAF15, respectively. While many mutations directly alter the ternary complex heterodimerization surface, distal resistance sites were also identified. Several distal mutations in RBM39 led to modest decreases in degradation, yet can enable cell survival, underscoring how small differences in degradation can lead to resistance. Integrative analysis of resistance sites across GSPT1 and RBM39 revealed varying levels of sequence conservation and mutational constraint that control the emergence of different resistance mechanisms, highlighting that many regions co-opted by TPD are nonessential. Altogether, our study identifies common resistance mechanisms for molecular glue degraders and outlines a general approach to survey neosubstrate requirements necessary for effective degradation.靶向蛋白质降解（Targeted protein degradation, TPD）在药物研发领域具有巨大潜力，但降解剂的获得性耐药机制仍有待全面阐明。本研究采用成簇规律间隔短回文重复序列（CRISPR）抑制扫描技术，对分别由E3泛素连接酶底物受体cereblon与DCAF15所靶向的新底物GSPT1和RBM39，鉴定出其中针对分子胶降解剂的耐药突变机制类别。尽管多数突变直接改变三元复合物异二聚化界面，研究团队同时也鉴定到了远端耐药位点。RBM39中存在若干远端突变，此类突变可使降解效率出现适度降低，却能帮助细胞存活，这凸显了降解效率的细微差异即可介导耐药的过程。对GSPT1与RBM39的耐药位点进行整合分析后发现，不同水平的序列保守性与突变约束，调控了不同耐药机制的出现，同时也表明靶向蛋白质降解（TPD）所利用的诸多区域并非必需。综上，本研究鉴定出了分子胶降解剂的常见耐药机制，并提出了一种通用方法，用于探究实现高效降解所需的新底物需求。