Programmable protein stabilization with language model-derived peptide guides

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Dysregulated protein degradation via the ubiquitin-proteasomal pathway is implicated in diseases such as cancer, neurodegeneration, and diabetes.
• Current targeted protein stabilization (TPS) and targeted protein degradation (TPD) platforms require structured binding pockets, limiting their application to "undruggable" targets.
• There is a need for novel therapeutic strategies to address protein dysregulation in diseases with limited treatment options.

Purpose of the Study:

• To expand the scope of targeted protein stabilization (TPS) by developing a novel platform for stabilizing proteins lacking traditional binding pockets.
• To engineer deubiquibodies (duAbs) by fusing computationally designed peptides to the OTUB1 deubiquitinase catalytic domain.
• To demonstrate the efficacy of duAbs in stabilizing diverse protein targets, including tumor suppressors and fusion oncoproteins, and their potential for therapeutic applications.

Main Methods:

• Computational design of peptides targeting specific proteins using protein language models.
• Fusion of designed peptides to the catalytic domain of OTUB1 to create deubiquibodies (duAbs).
• Validation of duAb-mediated protein stabilization in human cells, assessing deubiquitinase (DUB) dependence.
• Engineering duAbs against conformationally diverse targets like p53, WEE1, and PAX3::FOXO1.
• Encapsulation of p53-targeting duAb mRNA in lipid nanoparticles for intracellular delivery.

Main Results:

• Engineered duAbs effectively stabilize exogenous and endogenous proteins in a DUB-dependent manner.
• DuAbs were successfully engineered to target conformationally diverse proteins, including tumor suppressors (p53, WEE1) and disordered fusion oncoproteins (PAX3::FOXO1).
• mRNA-encapsulated p53-targeting duAbs demonstrated effective intracellular delivery, p53 stabilization, and apoptosis activation in cellular models.

Conclusions:

• Deubiquibodies (duAbs) represent a novel platform for targeted protein stabilization (TPS), overcoming limitations of traditional drug design.
• This approach expands the druggable proteome, offering potential therapeutic strategies for diseases driven by protein dysregulation.
• The successful in vitro demonstration of mRNA-delivered duAbs warrants further investigation for in vivo translation and clinical development.