Abstract
The von Hippel-Lindau (VHL) and cereblon (CRBN) proteins are substrate recognition subunits of two ubiquitously expressed and biologically important Cullin RING E3 ubiquitin ligase complexes. VHL and CRBN are also the two most popular E3 ligases being recruited by bifunctional Proteolysis-targeting chimeras (PROTACs) to induce ubiquitination and subsequent proteasomal degradation of a target protein. Using homo-PROTACs, VHL and CRBN have been independently dimerized to induce their own degradation. Here we report the design, synthesis and cellular activity of VHL-CRBN hetero-dimerizing PROTACs featuring diverse conjugation patterns. We found that the most active compound 14a induced potent, rapid and profound preferential degradation of CRBN over VHL in cancer cell lines. At lower concentrations, weaker degradation of VHL was instead observed. This work demonstrates proof of concept of designing PROTACs to hijack different E3 ligases against each other, and highlights a powerful and generalizable proximity-induced strategy to achieve E3 ligase knockdown.
Highlights
Targeting proteins for degradation by hijacking the ubiquitin-proteasome system with small molecules is a powerful modality of intervention into biology, and an emerging therapeutic strategy.[1,2,3,4] A primary approach to targeted protein degradation involves the design of Proteolysis-targeting chimeras (PROTACs) (PROteolysis-Targeting Chimeras)
PROTACs are bifunctional compounds that form a ternary complex with a target protein of interest and an E3 ubiquitin ligase, such that the target protein is ubiquitinated by the hijacked E3 ligase and subsequently degraded by the proteasome.[5,6]
Within the past four years, potent and selective PROTACs have been designed to hijack either the von Hippel-Lindau (VHL) or cereblon (CRBN) E3 ligase against a target protein of interest.[18,19]
Summary
Targeting proteins for degradation by hijacking the ubiquitin-proteasome system with small molecules is a powerful modality of intervention into biology, and an emerging therapeutic strategy.[1,2,3,4] A primary approach to targeted protein degradation involves the design of PROTACs (PROteolysis-Targeting Chimeras). With CM11, we confirmed the hypothesized mechanism and qualified a novel chemical probe degrader for VHL.[11] Subsequently, the same idea was applied by Krönke, Gütschow and co-workers, who reported homoPROTACs for the CRBN ligase, and showed compound 15a (CC15a in Figure 1) to be the most active compound.[55] 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.[56] 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. 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
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