Entwicklung neuer molekularer Werkzeuge für FZD-Rezeptoren

• Antragsteller: Dr. Lukas Grätz
• Fachliche Zuordnung: Pharmakologie ; Pharmazie
• Förderung: Förderung von 2022 bis 2024
• Projektkennung: Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 504098926

• Erstellungsjahr: 2024

Zusammenfassung der Projektergebnisse

G protein-coupled receptors (GPCRs) form the largest class of membrane proteins in the human genome and are generally viewed as attractive drug targets. However, our knowledge about many of them, especially the smaller receptor families, is still very limited leaving a lot of potential untapped. One example are the ten members of the Frizzled receptor family (FZDs), which are part of the class F of GPCRs and are not exploited therapeutically. FZDs are located on the surface of cells. They are binding and relaying the signals of the lipoglycoproteins of the Wingless/Int-1 family (WNT, 19 members in mammals), which makes them essential contributors during embryonic development but are equally important in adults by regulating stem cell function and tissue homeostasis. Dysregulation of these pathways, in contrast, was found to be responsible or involved in the development of malignancies, most prominently different types of cancer, making WNT signalling and FZDs in particular attractive targets to battle these diseases. Aim of this project, which was performed at the Karolinska Institute in the lab of Prof. Gunnar Schulte, was the development of new molecular tools to help us get a better understanding of the molecular pharmacology of these enigmatic receptors (signal initiation, signal specification,…). The first main goal was to develop new small molecules binding to the transmembrane core of FZDs. At the moment of this report, only a handful are available making investigations on these receptors generally cumbersome. We built upon previous efforts of the host institution and were able to identify a derivative of the Smoothened agonist/FZD6 partial agonist SAG1.3 as a non-paralog-selective negative allosteric modulator of WNT activity. Besides acting on HEK293 cells, the new compound was able to interfere with WNT signalling in primary human hepatocyte spheroids and suppress the viability of RNF43-negative pancreatic ductal adenocarcinoma cells. While still being far from being used in an actual therapeutic setting, this study will serve as a proof-of-concept that allosteric modulation on the receptor level can be an attractive strategy to target the WNT signalling pathway. The screen of a smaller compound library resulted in another hit compound and the in-depth characterization (incl. pharmacological and structural characterization) is ongoing. In the second part of the project, new test systems could be developed to characterize different processes occurring in WNT signalling cascades. For example, the analysis of conserved state-stabilizing residues, which combined computational and pharmacological techniques, allowed us to determine that different receptor conformations lead to different signalling outcomes. Focussing on the intracellular side, FZDs can interact with a set of different transducers, such as the phosphoprotein Dishevelled (DVL), yet until recently the dynamics of this interaction were hard to follow due to the lack of sufficiently sensitive and specific tools. Using different experimental paradigms incl. newly generated unimolecular BRET-based biosensors, we were able to show that the FZD-DVL complex is dynamic and is undergoing conformational rearrangements upon WNT binding. These findings suggest the existence of an allosteric interplay between the extracellular agonist and the intracellular transducer. While the aforementioned concepts have been widely accepted in class A/B GPCR research for decades, their validity is still heavily debated in FZD pharmacology. In summary, the findings obtained in course of this project will be valuable contributions in our quest to understand how the FZD family of receptors works on a molecular level. This will be fundamental so that, in the future, we will be able to target them confidently and with reduced risk for unwanted side effects, e.g., in the context of cancer therapy.

Projektbezogene Publikationen (Auswahl)

• NanoBiT‐ and NanoBiT/BRET‐based assays allow the analysis of binding kinetics of Wnt‐3a to endogenous Frizzled 7 in a colorectal cancer model. British Journal of Pharmacology, 181(20), 3819-3835.
  Grätz, Lukas; Sajkowska‐Kozielewicz, Joanna J.; Wesslowski, Janine; Kinsolving, Julia; Bridge, Lloyd J.; Petzold, Katja; Davidson, Gary; Schulte, Gunnar & Kozielewicz, Paweł
  
• Pathway selectivity in Frizzleds is achieved by conserved micro-switches defining pathway-determining, active conformations. Nature Communications, 14(1).
  Grätz, Lukas; Kowalski-Jahn, Maria; Scharf, Magdalena M.; Kozielewicz, Pawel; Jahn, Michael; Bous, Julien; Lambert, Nevin A.; Gloriam, David E. & Schulte, Gunnar
  
• WNT stimulation induces dynamic conformational changes in the Frizzled-Dishevelled interaction. Science Signaling, 16(779).
  Bowin, Carl-Fredrik; Kozielewicz, Pawel; Grätz, Lukas; Kowalski-Jahn, Maria; Schihada, Hannes & Schulte, Gunnar
  
• Frizzleds act as dynamic pharmacological entities. Trends in Pharmacological Sciences, 45(5), 419-429.
  Schulte, Gunnar; Scharf, Magdalena M.; Bous, Julien; Voss, Jan Hendrik; Grätz, Lukas & Kozielewicz, Pawel
  
• Structural and functional insight into the interaction of Clostridioides difficile toxin B and FZD7. Cell Reports, 43(2), 113727.
  Kinsolving, Julia; Bous, Julien; Kozielewicz, Pawel; Košenina, Sara; Shekhani, Rawan; Grätz, Lukas; Masuyer, Geoffrey; Wang, Yuankai; Stenmark, Pål; Dong, Min & Schulte, Gunnar