Poster Presentation 50th Lorne Proteins Conference 2025

Structure-guided discovery of EphB6 modulators to potentiate tumor suppressor functions (#312)

Jean-Max Mazeri 1 2 , Andrew P Thompson 1 2 , Lurlene Trepout 1 2 , Luca Pozzetti 3 , Toni T Lybeck 3 , David H Drewry 4 , Christopher R M Asquith 3 , Isabelle S Lucet 1 2
  1. Walter and Eliza Hall Institute of Medical Research, Parkville, VICTORIA, Australia
  2. Department of Medical Biology, University of Melbourne, Melbourne, VIC, Australia
  3. School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland
  4. Structural Genomics Consortium, University of North Carolina, Chapel Hill, North Carolina, USA

The Eph family of receptor tyrosine kinases are critical players in cellular communication, relaying signals from the cell surface to the intracellular environment. They contain an extracellular ectodomain that binds to membrane-bound ephrin ligands, enabling cell-cell contact and an intracellular domain containing a kinase domain that initiates signalling cascades. Consequently, Eph:Ephrin interactions play critical roles in regulating cell organisation and tissue patterning and their dysregulation can promote oncogenic processes, such as tumorigenesis or metastasis. Among Eph receptors, the lack phosphorylation activity of EphB6 and EphA10 means they are classified as pseudokinases. However, their role in signalling is non-catalytic and they are thought to act as scaffolds influencing Eph signalling processes via conformational regulation and protein:protein interactions. Dysregulation of EphB6 and EphA10 is associated to multiple cancer types, yet their precise mechanisms remain unknown. The elucidation of these two pseudokinases function is crucial to developing novel Eph receptors-targeted anti-cancer therapies.

In collaboration with the Asquith and Drewry groups, we aim to interrogate the pseudokinase function of EphB6 by developing highly potent and selective small molecules. We hypothesize that conformational modulators that locks EphB6 in a state that regulates its scaffold activity could potentiate its anti-tumor function. Towards this goal, we have produced two functional versions of the EphB6 pseudokinase domain. Using an established differential scanning fluorimetry (DSF), we screened compounds against the EphB6 pseudokinase domain, and identified novel compound series that lock EphB6 in distinct conformational states. We are also currently pursuing crystallography studies and cell functional assays. These parallel efforts will guide the structure-based design of selective compounds that are capable of modulating EphB6 pseudokinase conformation and modify its downstream signalling. This will enable the development of effective tools to probe’s EphB6 structure, function in tumor suppression and the Eph receptor regulation pathway. These will combine to validate EphB6 as a potential therapeutic target for cancer.

  1. Pasquale, E.B. 2024. Eph receptors and ephrins in cancer progression. Nat Rev Cancer 24(1) 5-27.
  2. Liang, L.Y., Roy, M., Horne, C.R. et al. 2021. The intracellular domains of the EphB6 and EphA10 receptor tyrosine pseudokinases function as dynamic signalling hubs. Biochem J 478(17) 3351-3371.
  3. Liang, L.Y., Geoghegan, N.D., Mlodzianoski, M. et al. 2024. Co-clustering of EphB6 and ephrinB1 in trans restrains cancer cell invasion. Commun Biol 7(1) 461.