Project 4: VDAC Opening Small Molecules to Revert Warburg Metabolism and Induce Oxidative Stress

总结 糖酵解的增强和线粒体代谢的抑制表征了脑内的瓦尔堡现象， 癌细胞代谢酶通过外膜上的一个通道进入和离开线粒体：电压 依赖性阴离子通道（VDAC）。这一提议的中心假设是高水平的游离微管蛋白 在癌细胞中阻断VDAC并抑制瓦尔堡代谢中的氧化磷酸化， 微管蛋白对VDAC抑制的逆转具有抗Warburg效应，其增强了氧化应激， 磷酸化，促进氧化应激和减少糖酵解。我们进一步假设， VDAC对微管蛋白高表达线粒体的抑制作用的分子拮抗剂， 增加活性氧（ROS）产生，导致线粒体功能障碍和细胞 死亡因此，在具体目标1中，我们将描述erastin和其他VDAC-微管蛋白的作用。 拮抗剂对细胞生物能量学（ATP，ADP，AMP，Pi，NADH氧化还原状态，AMP激酶， 呼吸和糖酵解）。我们还将 在Huh 7小鼠异种移植模型中评估erastin/VDAC-微管蛋白拮抗剂对线粒体膜电位的影响。 膜电位（Δ E）和糖酵解表型。高通量鉴定的先导化合物 筛选将通过电生理学确认为VDAC-微管蛋白拮抗剂，评价对细胞的影响， 生物能量学和用于创建药效团。在具体目标2中，我们将评估蛋白质的影响 激酶A（PKA）依赖性VDAC磷酸化对HCC细胞生物能量的影响。我们将使用激动剂， PKA以及PKA过表达和siRNA沉默的抑制剂， erastin/VDAC-微管蛋白拮抗剂和在VDAC同种型双敲低后。此外，蛋白质组学分析 将决定VDAC同种型磷酸化的特定位点。在具体目标3中，我们将确定 VDAC-微管蛋白拮抗剂的细胞毒性机制。我们预计erastin和铅化合物将增加 Δ ε和ROS的形成，导致线粒体通透性转换，生物能量衰竭， 死亡我们还将确定细胞死亡是否通过细胞凋亡、坏死或坏死性凋亡发生，以及线粒体 功能可以通过抗氧化剂来保持。总的来说，该项目将产生基本的新知识， 在HCC中引起线粒体代谢抑制的机制，并将确定新的药物 阻断VDAC-微管蛋白相互作用以逆转促增殖瓦尔堡代谢表型， 选择性促进细胞毒性氧化应激。

SUMMARY Enhanced glycolysis and suppression of mitochondrial metabolism characterize the Warburg phenomenon in cancer cells. Metabolites enter and exit mitochondria through one channel in the outer membrane: the voltage dependent anion channel (VDAC). The central hypothesis of this proposal is that high free tubulin levels in cancer cells blocks VDAC and suppresses oxidative phosphorylation in Warburg metabolism and that reversal of tubulin inhibition of VDAC has an anti-Warburg effect that enhances oxidative phosphorylation, promotes oxidative stress and decreases glycolysis. We further hypothesize that small molecules antagonists of the inhibitory effect of VDAC on tubulin hyperpolarize mitochondria and increase generation of reactive oxygen species (ROS), leading to mitochondrial dysfunction and cell death. Accordingly in Specific Aim 1, we will characterize the effects of erastin and other VDAC-tubulin antagonists on cellular bioenergetics (ATP, ADP, AMP, Pi, NADH redox state, AMP kinase, and rates of respiration and glycolysis) in HepG2, Huh7 and FOCUS human hepatocarcinoma (HCC) cells. We will also assess in a Huh7 mouse xenograft model the effect of erastin/VDAC-tubulin antagonists on mitochondrial membrane potential (ΔΨ) and the glycolytic phenotype. Lead compounds identified in a high throughput screening will be confirmed by electrophysiology as VDAC-tubulin antagonists, evaluated for effects on cellular bioenergetics and used to create a pharmacophore. In Specific Aim 2, we will assess the effects of protein kinase A (PKA)-dependent VDAC phosphorylation on the bioenergetic of HCC cells. We will use agonists and inhibitors of PKA as well as PKA overexpression and siRNA silencing in the presence and absence of erastin/VDAC-tubulin antagonists and after VDAC isoform double knockdown. Additionally, proteomic analysis will determine specific sites of VDAC isoform phosphorylation. In Specific Aim 3, we will determine the cytotoxic mechanisms of VDAC-tubulin antagonists. We expect that erastin and lead compounds will increase ΔΨ and ROS formation, leading to the mitochondrial permeability transition, bioenergetic failure, and cell death. We will also determine if cell death occurs by apoptosis, necrosis or necroptosis and if mitochondrial function can be preserved by antioxidants. Overall, the project will generate fundamental new knowledge on mechanisms causing suppression of mitochondrial metabolism in HCC and will identify new agents that block VDAC-tubulin interaction to revert the pro-proliferative Warburg metabolic phenotype and selectively promote cytotoxic oxidative stress.