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.

靶向蛋白质降解（Targeted protein degradation）已成为药物研发领域的强大策略，可靶向原本不可成药蛋白质（undruggable proteins）进行蛋白酶体降解（proteasomal degradation）。该策略通常采用异双功能降解剂（heterobifunctional degrader），由靶向蛋白质的配体与E3泛素连接酶（E3 ligase）招募剂相连，通过泛素化标记目标蛋白以进行蛋白酶体降解。然而该方法存在一项挑战：尽管人类基因组中已发现数百种E3泛素连接酶，但目前可用于靶向蛋白质降解的E3连接酶招募剂却寥寥无几。本研究利用基于活性的蛋白质谱分析（activity-based protein profiling, ABPP）的共价配体筛选方法，鉴定出可与E3泛素连接酶RNF4结合的半胱氨酸反应性小分子，为设计基于RNF4的降解剂提供了化学起始骨架。本次筛选得到的阳性共价配体可与RING结构域中两个锌配位半胱氨酸残基C132和C135结合，且对RNF4的活性无影响。我们进一步优化了该阳性配体的活性，并将这种潜在的RNF4招募剂整合至与JQ1相连的双功能降解剂中，JQ1是BET家族溴结构域蛋白的抑制剂。实验证明，所得化合物CCW 28-3可通过蛋白酶体依赖且RNF4依赖的方式降解BRD4蛋白。本研究证实，借助基于化学蛋白质组学的共价配体筛选平台，可拓展可用于靶向蛋白质降解的E3连接酶招募剂的范围，证明了该策略的可行性。