E ubiquitin ligases are key players
E3 ubiquitin ligases are key players in the ubiquitin-proteasome pathway because they catalyse ubiquitination of substrate proteins.39, 40, 41 As important regulators of cellular ubiquitination, E3 ligases are emerging as attractive drug targets, particularly in cancer.42, 43, 44 However, E3 ligases have proven difficult to target using small molecule inhibitors. So far only few high-quality inhibitors have been developed, mainly against the ligases MDM2, VHL, and IAPs. E3 ligases lack deep mole calculator to accommodate endogenous small-molecule cofactors or substrates, as is the case for ATP in protein kinases. Targeting E3 ligases therefore requires disruption (or modulation) of protein-protein interactions. E3 ligase inhibitors face particular challenges: first, the difficulty to compete with high-affinity endogenous substrates, which increase in level as a result of E3 blockade; and second, the observation that small molecules that bind to E3 ligases may modulate the surface of the targeted E3 in such a way that new substrate proteins are recruited for degradation, as shown for the E3 ligases CRBN,37, 51, 52 and DCAF15.53, 54
We hypothesized that the E3 ligases themselves might be hijacked against one another using a PROTAC approach, thus inducing E3 ligase degradation as opposed to E3 blockade. In 2017, we disclosed the first report of a small molecule dimerizer of an E3 ligase as a means to induce its own degradation, an approach that we called “homo-PROTAC”. We designed bifunctional molecules made up of the same ligand for the ubiquitously expressed VHL protein, connected via a linker, that would induce VHL dimerization as the key step to trigger VHL ubiquitination and subsequent degradation. The best degrader, the symmetric homo-PROTAC CM11 (Figure 1), dimerized VHL in vitro with high avidity (cooperativity) of ∼20-fold, leading to potent, complete and prolonged degradation of VHL in different cell lines. With CM11, we confirmed the hypothesized mechanism and qualified a novel chemical probe degrader for VHL. Subsequently, the same idea was applied by Krönke, Gütschow and co-workers, who reported homo-PROTACs for the CRBN ligase, and showed compound 15a (CC15a in Figure 1) to be the most active compound. As an extension of our homo-PROTAC approach, we envisaged that two different E3 ligases could be brought together using hetero-bifunctional PROTACs made of a ligand handle for one ligase and another handle for a different ligase. We hypothesized that with such compounds the two E3 ligases might be hijacked against one another, leading to two potential scenarios: 1) both ligases being degraded in cell; 2) one of the two being preferentially degraded – resulting in one ligase ‘winning’ over the other one. In the present study, we describe the design, synthesis and cellular activity of VHL-CRBN heterodimerizing PROTACs, and interrogate the outcome of hijacking these two E3 ligases against each other.
Results and discussion
Discussion We described dually targeting CRBN-VHL PROTACs, developed with the aim of investigating the relative ability of CRBN and VHL E3 ligase to induce degradation of one other. Among the three series of compounds developed, we observed preferential degradation of one ligase i.e. CRBN over the other one (VHL) with some of the compounds from two of the series. The most potent PROTAC, compound 14a, induced CRBN degradation with high potency (DC50 of 200 nM) and to profound levels (Dmax of up to 98%) and rapidly (within 1 h of treatment). Further structure-activity relationships could help to better understand and improve the already high potency and efficiency of CRBN degradation achieved with 14a. Our data thus suggests that VHL can ‘win the battle’ with CRBN when the two ligases are brought together by a PROTAC. Future mechanistic studies are warranted to attempt to elucidate the contributors for this preferential unilateral outcome of our ‘double-hijacking’ approach. We also cannot exclude that different combinations of conjugation patterns (via different attachment points for example) and linker lengths and structures of CRBN-VHL PROTACs might be able to discriminate different relative orientation of the ternary complex in such a way that the outcome might become reverse, i.e. VHL being preferentially degraded over CRBN – a hypothesis that will be tested in future work. In this regard, it is interesting to note that minor concentration-dependent depletion of pVHL30 was observed at the lower end of the concentration range (5–50 nM) in HeLa (Figure 5) as well as in the screen at lower compound concentration (10 nM, Figure S2). pVHL30 is the VHL isoform that is preferentially degraded by the homo-PROTAC CM11. No observable PROTAC-induced degradation of pVHL19 was instead observed with any of our compounds, consistent with the cellular outcome observed with CM11. These observations together suggest an enticing possibility that differential ligase degradation might apply at distinct ranges of concentration of CRBN-VHL dimerizers. Differential absolute concentration between the two E3 ligases, and/or differential binding affinities of each end of the bivalent molecule for its respective ligase, are likely to be amongst the contributing factors that could effectively skew the hook effect towards one ligase versus the other one depending on the PROTAC concentration, ultimately imparting differential protein degradation outcomes. Such an effect could be of relevance in a broader context for other E3 ligase pairs. It is noteworthy that a recent study reported MDM2 PROTAC degraders, designed by linking an MDM2 inhibitor via either a thalidomide-based CRBN ligand or a VHL ligand. Potent and selective PROTAC-induced degradation of MDM2 was observed for the CRBN-MDM2 heterodimers. However notably, protein level of the hijacked CRBN or VHL ligases were not monitored. Hetero-bifunctional VHL-CRBN PROTACs were also disclosed in a study recently published by Steinebach et al. Preferential degradation of CRBN over VHL was also observed by Steinebach et al., with their most potent compound (CRBN-6-5-5-VHL) being a conjugate of pomalidomide and VHL ligand via the terminal acetyl group, as with 14a, albeit with a different linker structure.