Degrader probes for necroptosis pathway

摘要 受体相互作用蛋白激酶1和3（RIPK 1和RIPK 3）沿着混合谱系激酶结构域- 类（MLKL）假激酶是公认的参与者，在调节坏死细胞死亡过程中，被称为 坏死性凋亡这些蛋白质的活性与许多疾病状态有关，包括免疫学和免疫学疾病。 炎性条件。然而，RIPK 1和RIPK 3支架功能与其催化活性不同， 活动探索得更少。此外，迄今为止开发的“激酶结构域”结合MLKL配体缺乏 统一的职能活动，这表明需要一个新的战略，针对这一关键的刽子手， 坏死性凋亡因此，能够选择性降解这些蛋白质的分子探针将使 通过阐明这些蛋白质的支架功能，进一步了解坏死性凋亡。这项工作 也将为新的治疗模式提供指导。为了评估这一假设，蛋白水解靶向 将利用先前鉴定的配体产生RIPK 3和MLKL（Aim 1）的嵌合体（PROTAC）。 将评估这些探针诱导RIPK 3和MLKL细胞降解的能力， 从而提供对病理性坏死性凋亡的保护。独立地，诱导共- RIPK 1和凋亡蛋白抑制剂（IAP）的降解（目标2）将基于 在我们的初步数据中识别这些分子的新起点。这些协同降解分子 将为诱导坏死性凋亡抗性癌症和活化的骨髓细胞中的细胞死亡提供新的选择。 总的来说，该项目将提供关键数据，揭示靶向RIPK 1、RIPK 3激酶和 用于降解的假激酶MLKL作为了解其激酶非依赖性功能的新方法 这也可以在创新的治疗方向上发挥作用。

ABSTRACT Receptor-interacting Protein Kinases 1 and 3 (RIPK1 and RIPK3) along with Mixed Lineage Kinase Domain- like (MLKL) pseudokinase are well-established players in a regulated necrotic cell death process, known as necroptosis. Activity of these proteins has been implicated in many disease states, including immunologic and inflammatory conditions. However, RIPK1 and RIPK3 scaffold functions that are distinct from their catalytic activities are much less explored. In addition, “kinase-domain” binding MLKL ligands developed thus far lacked uniform functional activities, suggesting need for a new strategy targeting this critical executioner of necroptosis. Consequently, molecular probes capable of selective degradation of these proteins will enable further progress in understanding necroptosis by elucidation of scaffold functions for these proteins. This work will also provide guidance for new therapeutic paradigms. To assess this hypothesis, proteolysis targeting chimeras (PROTACs) of RIPK3 and MLKL (Aim 1) will be generated leveraging previously identified ligands. These probes will be assessed for their ability to induce cellular degradation of RIPK3 and MLKL and, consequentially, to provide protection against pathologic necroptosis. Independently, probes that induce co- degradation of RIPK1 and Inhibitor of Apoptosis Proteins (IAPs) (Aim 2) will be developed, based on identification of new starting points for such molecules in our preliminary data. These co-degrader molecules will provide new options for inducing cell death in necroptosis-resistant cancer and activated myeloid cells. Overall, this project will provide critical data revealing the feasibility of targeting RIPK1, RIPK3 kinases and the pseudokinase MLKL for degradation as a new approach to understanding their kinase-independent functions that can also be leveraged in innovative therapeutic directions.