Development of AMPK Inhibitors for the treatment of leukemia

项目总结 尽管急性髓系白血病(AML)的治疗取得了进展，但只有20%-30%的患者实现了长期 复发AML的无病生存(DFS)和治疗选择极其有限。重现性 急性髓细胞白血病被归因于白血病干细胞(LSCs)，目前的努力集中在针对这种药物的研究上 为了“治愈”急性髓系白血病，需要大量的耐药细胞。我们测量原始人能量代谢的研究 使用活性氧(ROS)作为代谢活性指标的AML样本显示，LSCs 优选地驻留在ROS低状态。此外，高水平激活的5‘AMP激活的蛋白激酶 在LSCs中检测到代谢途径的中央调节因子(AMPK)，并发现AMPK基因被敲除 导致ROS水平升高和随之而来的LSC丢失。基于这些发现，我们建议AMPK 抑制将使LSC脱离ROS低状态，降低其活性，这对LSC来说可能是足够的 消除或可能使他们对常规治疗敏感。有效的、选择性的AMPK抑制剂很少； 然而，多激酶抑制剂Sunitinib已被报道为AMPK活性的有效抑制剂。 因此，我们研究的中心目标是开发有效和选择性的氧化吲哚为基础的AMPK靶向 并在急性髓系白血病模型中检测AMPK抑制或降解的效果。我们已经开发出一种首字母缩写 虽然我们从最初的系列中确定了有效的AMPK抑制剂，但我们相信进一步的 AMPK的选择性和抑制效力是可能的。我们将使用基于计算的建模来指导 开发抑制剂，并用体外激酶法评价它们的AMPK抑制活性。然后，抑制 将通过测量AMPK底物乙酰辅酶A的磷酸化来确定细胞AMPK的含量 用ELISA法测定MOLM13和MOLM14细胞中的羧基酶(ACC)活性，并筛选出适用于KINOME的抑制剂 剖析(目标1)。保留从ATP结合位点延伸出来的氨基烷基侧链的AMPK抑制剂将 与蛋白水解靶向嵌合体(PROTAC)降解剂偶联，其降解细胞AMPK的能力将 被评估。具有末端二甲氨基的氧化吲哚类AMPK抑制剂或降解剂， 与AMPK的DFG基序相互作用将被修改为加入硝基咪唑缺氧激活的前药 旨在引入一层LSC选择性的部分(目标2)。AMPK抑制剂和PROTAC的作用 将在MOLM13和MOLM13中确定作为单一试剂对细胞活力、代谢和ROS水平的降解剂 MOLM14细胞、原代AML细胞、LSCs和正常造血干细胞(HSCs)。然后，它的效果 将在MOLM13和MOLM14中确定AMPK抑制剂或降解剂与万乃馨联合使用的情况 细胞和原代AML细胞中。最后，我们的AMPK抑制剂或降解剂作为单一药物和在 将使用移植到晚期体内的原发AML标本来评估与万乃馨的结合 肿瘤异种移植模型(AIM 3)。这些研究的目标是开发一系列化学工具来 评估AMPK在维持LSC活性中的作用以及靶向AMPK对AML的治疗潜力。

PROJECT SUMMARY Despite advances in the treatment of acute myeloid leukemia (AML), only 20–30% of patients achieve long-term disease-free survival (DFS) and treatment options for relapsed AML are extremely limited. The recurrence of AML has been attributed to leukemic stem cells (LSCs) and efforts are now focused on targeting this drug resistant population of cells in order to “cure” AML. Our studies measuring energy metabolism in primary human AML specimens, using reactive oxygen species (ROS) as an indicator of metabolic activity, revealed that LSCs preferentially reside in a ROS-low state. Furthermore, high levels of activated 5' AMP-activated protein kinase (AMPK), a central regulator of metabolic pathways, were detected in the LSCs and that knockdown of AMPK resulted in increased ROS levels and concomitant loss of LSCs. Based on these findings, we propose that AMPK inhibition will leverage LSCs out of the ROS-low state decreasing their viability which may be sufficient for LSC elimination or may sensitize them to conventional therapy. There are few potent and selective AMPK inhibitors; however, the multi-kinase inhibitor sunitinib has been reported as a potent inhibitor of AMPK kinase activity. Therefore, the central goal of our research is to develop potent and selective oxindole-based AMPK-targeted agents and examine the effect of AMPK inhibition or degradation in AML models. We have developed an initial series of oxindoles and although we identified potent AMPK inhibitors from this initial series, we believe further AMPK selectivity and inhibitory potency is possible. We will use computational-based modeling to guide the development of inhibitors and evaluate their AMPK inhibitory activity using in vitro kinase assays. Then, inhibition of cellular AMPK will be determined by measuring the phosphorylation of the AMPK substrate acetyl-CoA carboxylase (ACC) by ELISA in MOLM13 and MOLM14 cells, and select inhibitors will be submitted for kinome profiling (Aim 1). AMPK inhibitors that retain an aminoalkyl side-chain extending out of the ATP-binding site will be coupled to a proteolysis targeting chimera (PROTAC) degrader and their ability to degrade cellular AMPK will be evaluated. The oxindole-based AMPK inhibitors or degraders that have a terminal dimethylamino group that interacts with the DFG motif of AMPK will be modified to incorporate a nitroimidazole hypoxia-activated prodrug moiety that are designed to introduce a tier of LSC selectivity (Aim 2). The effect of AMPK inhibitors and PROTAC degraders on cell viability, metabolism and ROS levels as single agents will be determined in MOLM13 and MOLM14 cells and in primary AML cells, LSCs, and normal hematopoietic stem cells (HSCs). Then, the effect of AMPK inhibitors or degraders in combination with venetoclax will be determined in MOLM13 and MOLM14 cells and in primary AML cells. Finally, the effect of our AMPK inhibitors or degraders as single agents and in combination with venetoclax will be evaluated using primary AML specimens transplanted into advanced in vivo tumor xenograft models (Aim 3). The objective of these studies are to develop a range of chemical tools to evaluate the role of AMPK in maintaining LSC viability and the therapeutic potential of targeting AMPK in AML.