Henrik Daub, NEOsphere Biotechnologies GmbH, Martinsried, Germany
Targeted protein degradation (TPD) is a breakthrough therapeutic approach to eliminate disease-relevant proteins previously considered undruggable. Established degrader drugs that reprogram E3 ligases come mainly in two varieties: heterobifunctional molecules also known as PROTACs (PROteolysis TArgeting Chimeras) or molecular glue compounds. Systematic discovery of novel molecular glues and their cellular degradation targets has so far been challenging. Here, we introduce our deep proteomics screening and validation platform to initiate and advance TPD drug discovery programs. Deep proteomic screening is a mass spectrometry (MS)-based technology to screen libraries of degrader against cellular proteomes at unprecedented throughput, coverage, and sensitivity. Deep proteomic screening identifies and quantifies up to 11,000 proteins per sample from cell lines treated with molecular glues or other degrader compounds, enabling comprehensive proteomics-based drug and drug target discovery. To extract likely neosubstrates from such large datasets, we have developed tailored scoring and reviewing concepts to classify significant hits according to their likelihood of being primary degrader targets. Potential neosubstrates are further investigated by unbiased, MS-based validation approaches such as global ubiquitinomics up to 50,000 sites as well as interactomics based on affinity-enrichment MS or cellular proximity labeling. Thus, we determine the ubiquitination of neosubstrate candidate proteins upon drug treatment as well as ternary complex formation both in cell lysates and directly in cells. In summary, our innovative proteomics platforms connect degrader libraries to their targets, enabling systematic neosubstrate identification for molecular glue compounds as well as in-depth characterization of cellular degrader selectivity and mechanisms.
Uli Ohmayer, Anastasia Bednarz, Sophie Machata, Jutta Fritz, Bjoern Schwalb and Martin Steger
