Electrophilic PROTACs that degrade nuclear proteins by engaging DCAF16

Abstract

Ligand-dependent protein degradation has emerged as a compelling strategy to pharmacologically control the protein content of cells. So far, however, only a limited number of E3 ligases have been found to support this process. Here, we use a chemical proteomic strategy that leverages broadly reactive, cysteine-directed electrophilic fragments coupled to selective ligands for intracellular proteins (e.g., SLF for FKBP12, JQ1 for BRD4) to screen for heterobifunctional degrader compounds (or PROTACs) that operate by covalent adduction of E3 ligases. This approach identified DCAF16 – a poorly characterized substrate recognition component of CUL4-DDB1 E3 ubiquitin ligases – as a target of electrophilic PROTACs that promote the nuclear-restricted degradation of proteins. We find that only a modest fraction (~10-40%) of DCAF16 needs to be modified to support protein degradation, pointing to the potential for electrophilic PROTACs to induce neo-substrate degradation without substantially perturbing the function of the participating E3 ligase.

Covalent Small Molecules as Enabling Platforms for Drug Discovery

“…Finally, a different class of covalent PROTACs was concurrently reported by the Cravatt and Nomura laboratories. These bifunctional molecules covalently engage with the E3 ligase, while only reversibly binding to the POI (Figure C).…”

“…Despite the large number of different E3 ligases known in the human genome (≈600), only VHL, Cereblon, IAP and MDM2 had yet been successfully exploited. To this aim, Cravatt et al . first attached reactive fragments to the SLF ligand that binds selectively and with high affinity to FKBP12.…”

Bifunctional chemical probes inducing protein–protein interactions

“…Complementary approaches involve de-orphanizing E3 ligases with new non-covalent ligands and degron peptides [31,[84][85][86], and modifying covalently nucleophilic residues e.g. cysteines on the E3 protein using electrophilic warheads [87][88][89] (Figure 5b). Beyond PROTACs and the targeting to E3 ligases, it will be interesting to watch the range of combinations being exploited to recruit proteins on-demand for purposefully modulating cellular signaling.…”

Drug Discovery Researches on Modulators of Lysine-Modifying Enzymes Based on Strategic Chemistry Approaches

“…60,61) Since this methodology was successful, it has been widely used and applied to target several proteins. [62][63][64][65][66][67] In addition, not only IAP ligands but also other E3 ligands have been used for protein knockdown, [68][69][70][71] where some of them are especially adaptable to in vivo studies or to the clinical trial stages. 44,63,[72][73][74] Thus, our drug discovery studies on ubiquitination inducers have driven the establishment of protein degradation approach using small molecules.…”