Small molecule probes for elucidating necrotic cell death mechanisms

描述（由申请人提供）：具有坏死形态学特征的受调控的细胞死亡途径最近成为重要的基本生物学过程。人们开始认识到这些途径在人类疾病（例如急性神经损伤）中的相关性。鉴定可以用作探针来询问关键生物靶点的作用的小有机分子对于实现对受调节的坏死细胞死亡途径的机制理解和制定治疗人类疾病的治疗策略至关重要。在过去的几年里，我们描述了一个受调节的坏死细胞死亡途径（称为坏死上睑垂），确定了该途径中的一个关键分子靶点（RIP1激酶），并报道了四种不同的靶向RIP1激酶的小分子抑制剂系列。其中三个系列直接抑制RIP1激酶活性，而其中一个系列间接抑制该途径。最近我们发现了一种新的间接抑制剂（Nec-10）。初步数据表明，这种间接抑制剂的作用机制可能是通过抑制RIP1泛素化，这已被证明对RIP1信号传导至关重要。本研究的主要目标是优化Nec-10的效价，设计和合成基于Nec-10类似物的机制探针，并利用这些化合物阐明RIP1泛素化在受调节的坏死细胞死亡中的作用。

DESCRIPTION (provided by applicant): Regulated cell death pathways with morphological characteristics of necrosis have recently emerged as important fundamental biological processes. The relevance of these pathways in human diseases, e.g. acute neurological injuries, is beginning to be appreciated. Identification of small organic molecules that can be used as probes to interrogate the roles of key biological targets are crucial for achieving a mechanistic understanding of regulated necrotic cell death pathways and for formulating therapeutic strategies for treating human diseases. Over the past several years we have described a regulated necrotic cell death pathway (termed necroptosis), identified a key molecular target (RIP1 kinase) in the pathway and reported on four distinct series of small molecule inhibitors that target RIP1 kinase. Three of these series inhibit RIP1 kinase activity directly, while one of these series inhibits the pathway indirectly. Recently we have identified a new indirect inhibitor (Nec-10). Preliminary data suggests that the mechanism of action for this indirect inhibitor may be through inhibiting RIP1 ubiquitination, which has been shown to be critical for RIP1 signaling. The primary goal of this proposal is to optimize Nec-10 for potency, to design and synthesize mechanistic probes based on Nec-10 analogs and to utilize these compounds for elucidating the role of RIP1 ubiquitination in regulated necrotic cell death. PUBLIC HEALTH RELEVANCE: Cells are generally thought to die through two processes, called apoptosis (a regulated process) and necrosis (a non-regulated process). Although the necrosis mechanism is prevalent in many neurological diseases, such as stroke and traumatic brain injury, it has not been targeted by therapies due to the belief that it can not be modulated. However, we and others have found that in some cases this is not true and that therapeutics could potentially be developed for the necrosis form of cell death if its molecular mechanism was better understood. We propose to develop small organic compounds that can be used as molecular probes to investigate necrosis so that therapeutic strategies can be devised for the treatment of neurological diseases where this form of cell death is present.