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

Oct 23, 2023

Contributors: Derek VanDyke,Linda Xu,Paul R Sargunas,Ryan N Gilbreth,Manuel Baca,Changshou Gao,James Hunt,Jamie B Spangler

Hijacking the ubiquitin proteasome system (UPS) to elicit targeted protein degradation (TPD) has emerged as a promising therapeutic strategy to target and destroy intracellular proteins at the posttranslational 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 (EED) protein, 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 UPS in cellular models. We explored the effects of binding domain avidity and E3 ligase utilization to gain insight into the requirements for effective bioPROTAC design. Overall, this study presents a versatile engineering approach that could be used to design and engineer TRIM21-based bioPROTACs against therapeutic targets.
Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.

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ABOUT THE CONTRIBUTORS

Derek VanDyke,Linda Xu,Paul R Sargunas,Ryan N Gilbreth,Manuel Baca,Changshou Gao,James Hunt,Jamie B Spangler
Derek VanDyke

Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA; Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA; Biologics Engineering, R&D, AstraZeneca, Gaithersburg, MD, USA.

Linda Xu

Biologics Engineering, R&D, AstraZeneca, Gaithersburg, MD, USA.

Paul R Sargunas

Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA; Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA.

Ryan N Gilbreth

Biologics Engineering, R&D, AstraZeneca, Gaithersburg, MD, USA.

Manuel Baca

Biologics Engineering, R&D, AstraZeneca, Gaithersburg, MD, USA.

Changshou Gao

Biologics Engineering, R&D, AstraZeneca, Gaithersburg, MD, USA.

James Hunt

Biologics Engineering, R&D, AstraZeneca, Cambridge, UK.

Jamie B Spangler

Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA; Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA; Bloomberg∼Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University, Baltimore, MD 21231, USA; Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD 21231, USA; Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA; Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, MD 21287 USA. Electronic address: jamie.spangler@jhu.edu.