Redirecting the specificity of tripartite motif containing-21 scaffolds using a novel discovery and design approach

作者：Derek VanDyke, Linda Xu, Paul R. Sargunas, Ryan Gilbreth, Manuel Baca, Changshou Gao, James Hunt, Jamie B. Spangler · 发表于：Journal of Biological Chemistry · 年份：2023 · DOI：10.1016/j.jbc.2023.105381 · 被引用次数：7 · 研究领域：Protein Degradation and Inhibitors、Ubiquitin and proteasome pathways、Multiple Myeloma Research and Treatments

Hijacking the ubiquitin proteasome system to elicit targeted protein degradation (TPD) has emerged as a promising therapeutic strategy to target and destroy intracellular proteins at the post-translational level. Small molecule-based TPD approaches, such as proteolysis-targeting chimeras (PROTACs) and molecular glues, have shown potential, with several agents currently in clinical trials. Biological PROTACs (bioPROTACs), which are engineered fusion proteins comprised of a target-binding domain and an E3 ubiquitin ligase, have emerged as a complementary approach for TPD. Here, we describe a new method for the evolution and design of bioPROTACs. Specifically, engineered binding scaffolds based on the third fibronectin type III domain of human tenascin-C (Tn3) were installed into the E3 ligase tripartite motif containing-21 (TRIM21) to redirect its degradation specificity. This was achieved via selection of naïve yeast-displayed Tn3 libraries against two different oncogenic proteins associated with B-cell lymphomas, mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) and embryonic ectoderm development protein (EED), and replacing the native substrate-binding domain of TRIM21 with our evolved Tn3 domains. The resulting TRIM21-Tn3 fusion proteins retained the binding properties of the Tn3 as well as the E3 ligase activity of TRIM21. Moreover, we demonstrated that TRIM21-Tn3 fusion proteins efficiently degraded their respective target proteins through the ubi...