Kristin Richling, Promega, USA

Targeted protein degradation is a promising new therapeutic strategy consisting of small molecules, most commonly molecular glues or Proteolysis Targeting Chimeras (PROTACs), which elicit degradation of a target protein.  Significant challenges persist to characterize the cellular mechanism of action and the highly dynamic interactions required for degradation. One of the keys steps in the pathway is the formation of an induced ternary complex consisting of the target protein:degrader:E3 ligase component. Here we will present several research stories investigating the role and types of formation of cellular ternary complexes, and how this is correlated with degradation outcome for several different targets and E3 ligase combinations. We will also demonstrate how ternary complex ensemble modeling can predict target lysine positioning and whether mutations to these residues in the cellular environment impacts PROTAC-mediated target ubiquitination.  The approaches highlighted will further understanding of cellular mechanism of action and can be used to advance discoveries in the area of targeted protein degradation and induced proximity.

Martin Schwalm, Inst. F. Biochemie, Uni Frankfurt

PROteolysis TArgeting Chimeras (PROTACs) are chimeric molecules to induce the multistep degradation process of a protein of interest through the ubiquitin proteasomal system (UPS). Despite PROTACs are from rising interest in the drug-development field and entering the clinic, the rate limiting steps to determine their efficiency are largely unknown. Additionally, the major analysis of PROTAC-induced degradation is marked by endpoint assays like Western blotting or proteomics, which throughput and dynamic range limit the analysis of PROTAC SAR series. To tackle this, we developed an assay pipeline based on NanoLuciferase and fluorescently labelled HaloTag which allows measurement of ternary complex and degradation kinetics to test the influence of ternary complex formation and stability on degradation efficiency in live cells. Using this pipeline, we analyzed a set of chemically divers VHL recruiting WDR5 degraders to find a correlation between the steps in the degradation cascade where we were able to reveal a key role of ternary complex formation and stability over the binary PROTAC-WDR5 and PROTAC-E3 complexes. The developed pipeline outlines a strategy for rational PROTAC optimization in live cells for the early PROTAC induced degradation pathway.

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

Felix Hausch, Technical University, Darmstadt

Molecular Glues and bifunctional degraders have generated substantial interest in the drug development community. However, the identification molecular glues and the optimization of bifunctional degraders (PROTACs) is far from trivial. Here, I will present the development of the first FKBP51 PROTACs and the discovery of the first FKBP12-degrading molecular glues.

Hedeby Hasse, Fido Biosystems

Applying a „first principle“ biophysical method for Targeted Protein degradation”

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