In the course of this project, a series of novel PROTACs targeting the von Hippel-Lindau (VHL) E3 ligase has been designed and synthesized. The performance of these novel PROTAC molecules has been studied in cellular and biophysical assays. In an initial degradation assay, four of the novel VHL-targeting PROTACs induced significant reduction of VHL protein levels. These compounds were further evaluated in dose- and time-dependent treatments in comparison to the previously reported state-of the-art VHL degrader, CM11. Two of these degraders showed improved efficacy with regard to both half-degradation times (t½) and half-maximal degradation concentration (DC50) values. Target engagement assays in live cells compared to permeabilized cells indicated improved cell permeability of these PROTACs compared to CM11, which can be attributed to the newly introduced functional motif in the PROTACs’ linkers. Furthermore, the first PROTAC degrading both isoforms of VHL has been identified within this series of novel VHL-targeting PROTACs.
Biophysical assays confirmed that degrader efficiency correlates with their ability to induce a ternary complex. To gain structural insight into the structural arrangement of the ternary complex, co-crystallization of protein with VHL-targeting PROTACs was attempted, yet unsuccessfully. As an alternative approach, cryo-EM was trialled on a VHL:PROTAC:VHL protein assembly, and a low-resolution 3D reconstruction of this protein assembly was generated, which serves as promising starting point for further sample optimization.
Finally, using this series of PROTACs featuring a novel functional motif within their linkers, a hit-identification assay has been designed and optimized, that is anticipated to identify PROTACs that efficiently form ternary complexes at an early stage in PROTAC development.
The results of this project have been presented at two international conferences and will be published in due course.
