Human cell-free system for targeted protein degradation

Diseases such as cancer, diabetes, or certain cardiovascular disorders are caused by a combination of genetic, environmental, and behavioral factors. Endogenous proteins generally play an important role in the onset and clinical manifestation of these diseases and are therefore potential targets for pharmacological treatment. Many conventional drugs act as inhibitors that bind to target proteins and block their catalytic activities or molecular interactions.

A relatively new strategy is the degradation of target proteins via the cell’s own ubiquitin–proteasome system (UPS). To achieve this, the E3 ligase complex — which under normal cellular metabolism is responsible for the specific tagging of proteins with ubiquitin — must be “redirected” to modify proteins that it would not normally recognize as substrates.

This can be accomplished either with bifunctional molecules known as PROteolysis TArgeting Chimeras (PROTACs) or with molecular glues. PROTACs contain a ligand for the E3 ligase, which is connected via a linker to a ligand for the target protein. In contrast, molecular glues induce conformational changes in the E3 ligase or the target protein, leading to the formation of a ternary complex, ubiquitination of the target protein, and ultimately its degradation.

In this way, disease-associated proteins can be targeted for which no effective drugs have yet been developed. However, the development of such molecules remains challenging, and there is currently a major bottleneck in screening technologies. While increasingly large candidate libraries can be generated — including with the help of artificial intelligence (AI) — there is still a significant need for fast, flexible, and cost-effective screening systems.

The goal of the project is to develop a novel human cell-free in vitro transcription–translation (IVTT) system with an active ubiquitin–proteasome system (UPS) for high-throughput screening of new compounds for targeted protein degradation, and to validate it in terms of time, cost, and degradation efficiency using commercial PROTACs and molecular glues.

Cell-free systems offer significant advantages compared to living cell cultures: no need for sterile culture conditions, no requirement for cell line development, ready-to-use setup, shorter design–build–test cycles, open system allowing precise adjustment of reaction conditions, no cloning required, no loss of non–cell-permeable compounds during screening, and overall lower costs.

Because of these advantages, research-based pharmaceutical companies gain entirely new possibilities to rapidly and efficiently identify candidate molecules for the treatment of diseases for which no effective drugs currently exist.

Given the current market landscape, research in the field of rare diseases is often driven by small and medium-sized enterprises (SMEs), which are therefore expected to benefit particularly from this novel human cell-free IVTT system.