Carola Sparn/Birgitta Leuthner, Merck, Darmstadt

Targeted protein degradation can remove disease-causing proteins from the organism by hijacking the cells own proteasomal degradation mechanism. Thus, it is an attractive therapeutic option whenever mere inhibition of the activity of a target is not sufficient or achievable. Therefore, also targets can be addressed, which as yet have been classified undruggable. It is no surprise that this new modality is evolving fast across many therapeutic areas. At Merck Healthcare KGaA targeted protein degradation is an integral part of our drug discovery pipeline activities to enable us to bring more drugs to more patients. Due to the complex interplay of binding, enzymatic and functional events in the degrader mechanism there is a demand for a tailored assay suite to identify, characterize and optimize active compounds. Here, we are focusing on two different modalities, the bifunctional degraders and the molecular glue degraders. This talk will give an insight into how biochemical as well as cell-based assays are utilized to screen in-house libraries for the identification and characterization of new degraders and the optimization of our early bifunctional PROTACs to effective eliminators of drug targets.

Johanna Huchting, Fraunhofer ITMP, Hamburg

Targeted protein degradation (TPD) is a highly promising modality for novel therapeutics where, in contrast to traditional drugs that stoichiometrically inhibit protein function, molecular degraders catalytically deplete proteins of interest. Although hugely challenging for development, advanced TPD drug candidates mainly follow the modular design of the bRo5 hetero-bivalent PROTAC-prototype. Rational approaches for the discovery of monovalent molecular glue (MG)-type degraders are beginning to emerge; yet, clinical success for non-serendipitous MG degraders is still lagging behind.

At Fraunhofer ITMP, and with our partners in the PROXIDRUGS consortium, we are developing a versatile and multi-domain platform to identify molecular degraders for different targets in cell-based, screening-compatible assay formats. In addition, we are building informatics tools for integrated knowledge representation in the TPD field.

Here, we present our integrated platform approach PRISKA (PRoxidrugs Integrated Screening- and Knowledge-plAtform) through the lens of the androgen receptor as a validated anti-cancer target for TPD therapeutics. Modules of PRISKA comprise dedicated chemical libraries as well as assays that enable automated screening for protein degraders in 384-well plate format at high throughput. These include no-wash immunoassays highly specific for quantifying endogenous target protein, high-content imaging and positive selection setups. PRISKA integrated informatics module facilitates rational compound selection, screening analysis and refinement through different algorithms. Among them a knowledge graph that maps TPD actors (PROTACs, MGs, etc.), annotated through links to other public chemical biology data, as well as chemoinformatics tools aimed at identifying MG physicochemical features, thus allowing a fast filtering of virtual small molecule libraries for MG candidate probability.

Funding: This work is funded by the German Federal Ministry of Education and Research (BMBF) within the Cluster4Future PROXIDRUGS Grants No. 03ZU1109AA, 03ZU1109JA, 03ZU1109KB, 03ZU1109GB, 03ZU1109DB.

Emilia Hietala, CCD Vault, UK 

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.

Grasilda Zenkeviciute, Proxygen, Wien

Targeted protein degradation (TPD) is emerging as an important approach in pharmaceutical research and holds potential for changing the scope of drugs by enabling the targeting of proteins previously deemed undruggable. Molecular Glue Degraders (MGDs) induce a ternary complex through protein–protein interactions between a ubiquitin ligase and a target protein, thereby resulting in ubiquitination and subsequent degradation of the target protein.

Proxygen’s rational molecular glue discovery platform enables the identification and optimization of novel MGDs at scale. Using this platform, a novel class of MGDs were identified. Chemical optimization of structurally distinct hits and characterization by cellular and biophysical methods revealed two distinct mechanisms of action. In particular, it emerged how modulation of assembly and dissociation dynamics of the ternary complex can affect the target degradation. This leads to a better understanding of how to rationally modulate degradation kinetics, maximal degradation levels (Dmax), and potency (DC50) to guide the design of MGDs towards the optimal drug profile.


Ralf Strasser, Dynamic Biosensors, München

Proteolysis targeting chimeras (PROTACs) are essential bifunctional small molecules that engage the formation of a ternary complex consisting of an E3 ubiquitin ligase, a target protein of interest and the PROTAC itself.

Using switchSENSE® technology and the novel DNA Y-structure, an E3 ligase as well as a target protein can be functionalized on each separate end of two FRET pair color-coded Y-arms. The Y-structure closes upon PROTAC binding and the subsequent ternary complex formation bringing together the green donor and the red acceptor dye into a closer, FRET sensitive, distance. The change in red fluorescence signal intensity directly correlates with ternary complex formation kinetics.

Here, we show that the Y-structure is an extremely versatile tool for studying any type of protein-protein complex formations. With the switchSENSE® technology and the highly sensitive FRET read-out, it is possible to perform high-throughput PROTAC screening and to characterize their kinetics (PROTACs ranking), gaining information on binary and ternary binding at the same time.
Alice Soldá, Irene Ponzo and Ralf Strasser