Mechanistic Dissection of Cancer Cell Susceptibility to Smac Mimetics

在之前的资助期间，我们建议开发一种模拟促细胞凋亡的小分子。 蛋白质Smac。这个想法是为了在癌细胞抵抗的情况下促进或使其对凋亡敏感。 执行这条道路。我们已经成功地实现了这个目标。Smac通常是一种线粒体蛋白， 只有在细胞凋亡时才释放到细胞质中。一旦进入细胞质，Smac就会与几种抑制因子结合。 细胞凋亡蛋白(LAPS)，并中和其抑制caspase的能力，caspase是一组细胞内的蛋白酶 执行细胞凋亡的细胞。Smac的功能基序位于其N端，仅有四个残基 (AVPI)是体外Smac样活性的充分和必要条件。基于这些信息，我们设计了 并对一种小分子进行了表征，在以下方面，这种小分子的表现与天然蛋白质一样好，甚至更好 解除IAP对半胱氨酸酶的抑制。更重要的是，我们的化合物是细胞渗透性的，是亚纳摩尔的。 浓度与致死细胞因子协同作用，如肿瘤坏死因子-α和 肿瘤坏死因子相关的凋亡诱导配体(TRAIL)促进无反应癌细胞系的死亡 只对这些细胞因子起作用。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株，发现反应从高度敏感到 抵抗力强。这给了我们一个独特的机会来进一步了解细胞凋亡中的信号转导。特别是，通过 了解为什么某些癌细胞在Smac模拟物单独存在的情况下经历程序性细胞死亡， 而其他人只有在增加刺激的情况下才能做到这一点，或者根本不刺激。这项提议概述了详细的生化， 分子和细胞生物学实验来探索这个问题。 这项研究与公共健康相关，因为它可以帮助开发一种新的癌症疗法，并指导其 在临床上使用。

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.