Covalent Ligand Screening Uncovers a RNF4 E3 Ligase Recruiter for Targeted Protein Degradation Applications

Targeted
protein degradation has arisen as a powerful strategy
for drug discovery allowing the targeting of undruggable proteins
for proteasomal degradation. This approach most often employs heterobifunctional
degraders consisting of a protein-targeting ligand linked to an E3
ligase recruiter to ubiquitinate and mark proteins of interest for
proteasomal degradation. One challenge with this approach, however,
is that only a few E3 ligase recruiters currently exist for targeted
protein degradation applications, despite the hundreds of known E3
ligases in the human genome. Here, we utilized activity-based protein
profiling (ABPP)-based covalent ligand screening approaches to identify
cysteine-reactive small-molecules that react with the E3 ubiquitin
ligase RNF4 and provide chemical starting points for the design of
RNF4-based degraders. The hit covalent ligand from this screen reacted
with either of two zinc-coordinating cysteines in the RING domain,
C132 and C135, with no effect on RNF4 activity. We further optimized
the potency of this hit and incorporated this potential RNF4 recruiter
into a bifunctional degrader linked to JQ1, an inhibitor of the BET
family of bromodomain proteins. We demonstrate that the resulting
compound CCW 28-3 is capable of degrading BRD4 in a proteasome- and
RNF4-dependent manner. In this study, we have shown the feasibility
of using chemoproteomics-enabled covalent ligand screening platforms
to expand the scope of E3 ligase recruiters that can be exploited
for targeted protein degradation applications.