Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research (LACDR), Leiden University, 2333 CC Leiden, The Netherlands.
Safety Innovation and PROTAC Safety, Clinical Pharmacology & Safety Sciences, R&D, AstraZeneca, Cambridge CB2 0AA, UK.
Int J Mol Sci. 2024 Aug 18;25(16):8984. doi: 10.3390/ijms25168984.
CC chemokine receptor 2 (CCR2) has been linked to many inflammatory and immune diseases, making it a relevant drug target. Yet, all CCR2 antagonists developed so far have failed in clinical trials; thus, novel strategies are needed to target this receptor. Targeted protein degradation represents a novel approach to inhibit protein function by hijacking the cellular degradation machinery, such as the proteasome, to degrade the protein of interest. Here, we aimed to determine the amenability of CCR2 to chemically induced degradation by using a CCR2 fusion protein containing a HaloTag7 and HiBiT tag (CCR2-HaloTag-HiBiT). After characterization of the CCR2 construct, we used luminescence-based assays and immunofluorescence to quantify CCR2 levels, as well as a label-free, phenotypic assay to investigate the functional effect of CCR2 degradation. Treatment with HaloPROTAC3, which selectively degrades HaloTag fusion proteins, led to concentration- and time-dependent degradation of CCR2-HaloTag-HiBiT. HaloPROTAC3 induced degradation via the proteasome, as degradation was fully blocked with proteasomal inhibitors. Finally, functional assays showed that degradation of CCR2-HaloTag-HiBiT leads to a reduced functional response after agonist stimulation. Overall, our results indicate that CCR2 is amenable to targeted degradation, paving the way for the future development of CCR2 chemical degraders.
CC 趋化因子受体 2（CCR2）与许多炎症和免疫性疾病有关，使其成为一个相关的药物靶点。然而，迄今为止开发的所有 CCR2 拮抗剂都在临床试验中失败了；因此，需要新的策略来靶向该受体。靶向蛋白降解代表了一种通过劫持细胞降解机制（如蛋白酶体）来降解靶蛋白来抑制蛋白功能的新方法。在这里，我们旨在通过使用含有 HaloTag7 和 HiBiT 标签的 CCR2 融合蛋白（CCR2-HaloTag-HiBiT）来确定 CCR2 是否适合化学诱导降解。在对 CCR2 构建体进行表征后，我们使用基于发光的测定法和免疫荧光法来定量 CCR2 水平，以及使用无标记、表型测定法来研究 CCR2 降解的功能影响。用 HaloPROTAC3 处理，该化合物选择性降解 HaloTag 融合蛋白，导致 CCR2-HaloTag-HiBiT 的浓度和时间依赖性降解。HaloPROTAC3 通过蛋白酶体诱导降解，因为蛋白酶体抑制剂完全阻断了降解。最后，功能测定表明 CCR2-HaloTag-HiBiT 的降解导致激动剂刺激后的功能反应降低。总体而言，我们的结果表明 CCR2 适合靶向降解，为开发 CCR2 化学降解剂铺平了道路。
