Development of AMPK Inhibitors for the treatment of leukemia

用于治疗白血病的 AMPK 抑制剂的开发

基本信息

批准号：
10661503
负责人：
Philip Reigan
金额：
$ 34.04万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-01 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10661503
关键词：
5&apos -AMP-activated protein kinase AMP-activated protein kinase kinase Acetyl-CoA Carboxylase Acute Myelocytic Leukemia BCL2 gene Binding Sites Biological Assay Catalytic Domain Cell Culture Techniques Cell Hypoxia Cell Survival Cells Chemicals Clinic Consumption Coupled Development Disease Disease-Free Survival Drug Targeting Drug resistance Energy Metabolism Enzyme-Linked Immunosorbent Assay Fluorescence Resonance Energy Transfer Glucose Goals Hematopoietic stem cells Human Hydrophobicity Hypoxia In Vitro Indazoles Lead Maintenance Measures Mediating Metabolic Metabolic Pathway Metabolism Modeling Molecular Monitor Nitroimidazoles Oxidative Phosphorylation Oxidoreductase Pathway interactions Patients Pattern Peripheral Phosphorylation Phosphotransferases Population Prodrugs Production Property Protac Protein Isoforms Pyrroles Reactive Oxygen Species Recurrence Regimen Relapse Reporting Research Resistance Role Series Specimen System Therapeutic Translating Transplantation Treatment Efficacy Western Blotting Xenograft Model acute myeloid leukemia cell chemotherapy conventional therapy design effective therapy improved in vivo inhibitor interest kinase inhibitor knock-down leukemia leukemia treatment leukemic stem cell novel oxindole patient derived xenograft model protein kinase inhibitor pyridine small molecule inhibitor stem cell survival targeted agent therapeutic target tool tumor xenograft

项目摘要

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.

项目摘要尽管治疗急性髓样白血病（AML），但只有20-30％的患者长期实现无病生存期（DFS）和复发AML的治疗选择极为有限。复发 AML归因于白血病干细胞（LSC），现在努力旨在针对该药物抗性细胞种群以“治愈” AML。我们的研究测量原代人的能量代谢使用活性氧（ROS）作为代谢活性的指标，AML标本显示LSCS 优先驻留在ROS-低状态。此外，高水平的活化5'AMP激活的蛋白激酶（AMPK）是代谢途径的中央调节剂，在LSC中检测到AMPK的敲低导致ROS水平升高和LSC的伴随损失。基于这些发现，我们建议AMPK 抑制作用将利用LSC从ROS-LOW状态中降低其生存能力，这对于LSC可能就足够消除或可能会感觉到他们接受常规疗法。潜力和选择性AMPK抑制剂几乎没有；但是，多激酶抑制剂舒尼尼已被报道为AMPK激酶活性的潜在抑制剂。因此，我们研究的核心目的是开发以氧丝的为基础的潜力和选择性的AMPK目标代理并检查AML模型中AMPK抑制或降解的效果。我们已经开发了一个初始一系列的Oxindole，尽管我们从这个初始系列中确定了潜在的AMPK抑制剂，但我们相信进一步 AMPK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使用体外激酶测定法的开发抑制剂并评估其AMPK抑制活性。然后，抑制细胞AMPK的含量将通过测量AMPK底物乙酰辅酶A的磷酸化来确定 ELISA在MOLM13和MOLM14细胞中的羧化酶（ACC），将对Kinome提交选择抑制剂分析（目标1）。保留氨基烷基侧链的AMPK抑制剂将从ATP结合位点延伸与靶向嵌合体（protac）降解器的蛋白水解耦合及其降解细胞AMPK的能力将进行评估。基于氧吲哚的AMPK抑制剂或具有末端二甲基氨基的降解器，与AMPK的DFG基序相互作用将被修改以结合硝基咪唑低氧激活前药旨在引入LSC选择性层的部分（AIM 2）。 AMPK抑制剂和Protac的作用在MOLM13和 MOLM14细胞以及原代AML细胞，LSC和正常的造血干细胞（HSC）。然后，效果将在MOLM13和MOLM14中确定AMPK抑制剂或降解器与Venetoclax结合使用细胞和原代AML细胞。最后，我们的AMPK抑制剂或降解器作为单一药物的效果以及将使用移植到晚期体内的主要AML标本来评估与Venetoclax的结合肿瘤特征模型（AIM 3）。这些研究的目的是开发一系列化学工具评估AMPK在维持LSC生存能力和靶向AMPK靶向AMPK的治疗潜力方面的作用。