Mechanistic Dissection of Cancer Cell Susceptibility to Smac Mimetics

对于前一个资助期，我们建议开发一种促凋亡蛋白的小分子模拟物， Smac蛋白这个想法是促进或敏感癌细胞凋亡的情况下，他们抵制 执行路径。我们已经成功地实现了这一目标。Smac通常是一种线粒体蛋白， 仅在凋亡期间释放到胞质溶胶中。一旦在细胞质中，Smac结合到几个抑制剂- 细胞凋亡蛋白（IAP），并中和它们抑制半胱天冬酶（一组细胞内蛋白酶）的能力 执行细胞凋亡。Smac的功能基序位于其N端，只有四个残基 （AVPI）是体外Smac样活性的充分和必要条件。根据这些信息，我们设计了 并表征了一种小分子，其在以下方面的表现与天然蛋白质一样好，如果不是更好的话， 解除半胱天冬酶的IAP抑制。更重要的是，我们的化合物是细胞渗透性的， 浓度与死亡诱导细胞因子如肿瘤坏死因子α（TNF-α）和 肿瘤坏死因子相关凋亡诱导配体（TRAIL）促进不应答的癌细胞系中的细胞死亡 这些细胞因子的作用该化合物对非癌细胞无毒。我们现在已经评估了 超过80个培养的人类癌细胞系，发现反应从高度敏感到 抵抗这给了我们一个独特的机会，以进一步了解细胞凋亡中的信号转导。特别是 了解为什么某些癌细胞在单独的Smac模拟物存在下经历程序性细胞死亡， 而另一些人则只在增加刺激的情况下才这样做，或者根本不这样做。这一提议概述了详细的生物化学， 分子和细胞生物学实验来探索这个问题。 这项研究与公共卫生有关，因为它可以帮助开发一种新的癌症疗法，并指导其治疗。 在诊所使用。

For the previous funding period we proposed to develop a small molecule mimetic of the pro-apoptotic protein Smac. The idea was to promote or sensitize cancer cells to apoptosis in situations where they resist executing the pathway.We have succeeded in this goal. Smac is normally a mitochondrial protein and is released into the cytosol only during apoptosis. Once in the cytoplasm, Smac binds to several Inhibitor-of- Apoptosis Proteins (lAPs) and neutralizes their ability to inhibit caspases, a group of intracellular proteases that execute apoptosis. The functional motif in Smac is found at its very N-terminus, with just four residues (AVPI) being sufficient and necessary for Smac like activity in vitro. Based on this information, we designed and characterized a small molecule that performs just as well, if not better, than the native protein in terms of relieving IAP inhibition of caspases. More importantly, our compound is cell permeable and at subnanomolar concentrations synergizes with death inducing cytokines such as tumor necrosis factor alpha (TNF-a) and TNF-related apoptosis-inducing ligand (TRAIL) to facilitate cell death in cancer cell lines that do not respond to these cytokines alone. The compound is not toxic towards non-cancerous cells. We have now evaluated more than 80 cultured human cancer cell lines and found that responses vary from highly sensitive to resistant. This gives us a unique opportunity to further understand signaling in apoptosis. In particular, by learning why certain cancer cells undergo programmed cell death in the presence of a Smac mimetic alone, while others do so only with an added stimulus, or not at all.This proposal outlines detailed biochemical, molecular and cell biological experiments to probe this issue. This research is relevant to public health because it could help develop a new cancer therapy and guide its use in the clinic.