Chemical Biology Approach to Study Unprecedented Effectors of TGFbeta Receptor-II Dynamics

• Applicant: Professor Dr. Dennis Schade
• Subject Area: Biochemistry ; Pharmacy
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 449159634

Members of the transforming growth factor-beta (TGFb) family modulate a plethora of cellular processes such as proliferation, differentiation, migration, adhesion and apoptosis in a tissue context-specific manner. Disruption of these pathways is associated with a number of pathologies including cancer, autoimmune diseases and fibrosis.We have discovered "new-in-class" TGFb signaling inhibitors (i.e., annelated 1,4-dihydropyridines, DHPs) in a phenotypic stem cell screen. These compounds induce the selective proteasomal degradation of type II TGFb receptors (TGFBR-II). Interfering with TGFBR-II abrogates both canonical and non-canonical pathways, and thus, provides an efficient means of TGFb shutdown.TGFb responses are regulated by alterations of receptor compartmental localization and concentration through partitioning in membrane domains and by distinct internalization, trafficking, degradation and recycling processes. There is a great need to explore TGFBR interaction partners and elucidate their roles for receptor fates and dynamics. We believe that our DHPs interfere with at least one of such (yet unknown) interaction partners, thereby promoting TGFBR-II degradation specifically. Global proteome analyses and ligand-based target prediction suggested several plausible targets and effectors for the DHPs that we plan to verify. Our working hypothesis is that receptor trafficking is perturbed and cellular pools of receptors degraded. The identity and localization of these pools and their regulation remain unclear.We developed DHPs as high-quality "chemical probes" to identify their cellular target(s) and to study TGFb signaling, specifically by illuminating the dynamics of TGFBR-II. Our chemical biology toolbox will be expanded with photoaffinity labeling probes. They enable bioanalytical methods such as pulldown-proteomics and image-based (co)localization in the context of TGFBR-II degradation. We will ask whether TGFBR-II production, processing and trafficking during biosynthesis is affected by the DHPs. We also anticipate clarifying whether the DHPs induce receptor shifts to non-raft compartments and alter trafficking of TGFBR-II towards proteasomal degradation at the expense of the signaling endosomes (or lysosomes). In this context, the role of suggested DHP effectors and targets from our previous work as well as the newly identified targets from pulldown-proteomics will be verified. Our convergent efforts of target identification and cellular studies should allow us to construct a plausible mechanism-of-action for these DHPs as TGFBR-II degraders.Results from this work is expected to provide the community with (bio)chemical tools to study TGFb, add to the fundamental understanding of how compartmentalization and receptor fates are regulated and may provide novel "druggable" targets of therapeutic value.

• DFG Programme: Research Grants
• International Connection: Canada
• Cooperation Partner: Professor Dr. Gianni M. Di Guglielmo, Ph.D.