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节细胞死亡途径最近已成为重要的基本生物学过程。这些途径在人类疾病中的相关性，例如急性神经损伤，开始得到重视。鉴定可用作探针以询问关键生物靶标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用于治疗人类疾病的治疗策略至关重要。在过去的几年中，我们已经描述了一种受调节的坏死细胞死亡途径（称为坏死性凋亡），确定了该途径中的关键分子靶点（RIP 1激酶），并报道了四种不同系列的靶向RIP 1激酶的小分子抑制剂。其中三个系列直接抑制RIP 1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RIP 1泛素化，这已被证明是RIP 1信号转导的关键。该提案的主要目标是优化Nec-10的效力，设计和合成基于Nec-10类似物的机制探针，并利用这些化合物来阐明RIP 1泛素化在调节性坏死细胞死亡中的作用。 公共卫生相关性：细胞通常被认为通过两个过程死亡，称为凋亡（一种调节过程）和坏死（一种非调节过程）。虽然坏死机制在许多神经系统疾病中普遍存在，例如中风和创伤性脑损伤，但由于认为它不能被调节，因此尚未被治疗靶向。然而，我们和其他人发现，在某些情况下，这是不正确的，如果更好地理解其分子机制，可能会开发出治疗坏死形式的细胞死亡的疗法。我们建议开发小的有机化合物，可以用作分子探针来研究坏死，以便可以设计治疗策略来治疗存在这种形式的细胞死亡的神经系统疾病。

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.