Degrader probes for necroptosis pathway

摘要 受体相互作用蛋白激酶1和3(RIPK1和RIPK3)以及混合血统激酶结构域- 与(MLKL)一样，伪激酶在受调控的坏死性细胞死亡过程中也是成熟的参与者，称为 坏死性下垂。这些蛋白的活性与许多疾病状态有关，包括免疫学和 炎症状态。然而，RIPK1和RIPK3支架的功能不同于它们的催化 人们对活动的探索要少得多。此外，到目前为止所开发的“激活域”结合的MLKL配体缺乏。 统一的职能活动，表明需要针对这一关键的执行者制定新的战略 坏死性下垂。因此，能够选择性降解这些蛋白质的分子探针将使 通过阐明这些蛋白的支架功能来了解坏死性下垂的进一步进展。这部作品 也将为新的治疗范例提供指导。为了评估这一假设，蛋白水解靶向 将利用先前确定的配体产生RIPK3和MLKL(AIM 1)的嵌合体(PROTAC)。 将评估这些探针诱导RIPK3和MLKL的细胞降解的能力， 因此，提供对病理性坏死性下垂的保护。独立地，诱导共同- 将开发RIPK1和凋亡抑制蛋白(IAPs)的降解(AIM 2)，基于 在我们的初步数据中确定了这些分子的新起点。这些共降解物分子 将为诱导耐坏死性肿瘤和活化的髓系细胞的细胞死亡提供新的选择。 总体而言，该项目将提供关键数据，揭示靶向RIPK1、RIPK3和RIPK3激酶的可行性 假性激酶MLKL的降解--一种理解其非依赖性功能的新途径 这也可以被用于创新的治疗方向。

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.