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：
10438847
负责人：
Philip Reigan
金额：
$ 33.11万
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/10438847
关键词：
4-nitroimidazole 5&apos -AMP-activated protein 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 Cyclic AMP-Dependent Protein Kinases 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 Protein Kinase Pyrroles Reactive Oxygen Species Recurrence Regimen Relapse Reporting Research Resistance Role Series Side Specimen System Therapeutic Translating Transplantation Treatment Efficacy Western Blotting Xenograft Model acute myeloid leukemia cell base chemotherapy conventional therapy design effective therapy improved in vivo inhibitor interest 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.

项目概要尽管急性髓系白血病 (AML) 的治疗取得了进展，但只有 20-30% 的患者实现了长期治疗复发性 AML 的无病生存期 (DFS) 和治疗选择极其有限。 AML 已被归因于白血病干细胞 (LSC)，现在的工作重点是针对这种药物我们的研究测量了原代人类的能量代谢。使用活性氧 (ROS) 作为代谢活动指标的 AML 样本显示，LSC 优先处于 ROS 低状态，此外，激活的 5' AMP 激活蛋白激酶水平较高。 (AMPK) 是代谢途径的中央调节因子，在 LSC 中检测到，并且 AMPK 的敲低导致 ROS 水平增加并伴随 LSC 损失。根据这些发现，我们建议 AMPK。抑制作用将使 LSC 脱离 ROS 低状态，降低其生存能力，这对于 LSC 来说可能就足够了有效且选择性的 AMPK 抑制剂很少；然而，多激酶抑制剂舒尼替尼已被报道为 AMPK 激酶活性的有效抑制剂。因此，我们研究的中心目标是开发有效且选择性的基于羟吲哚的 AMPK 靶向药物药物并检查 AMPK 抑制或降解在 AML 模型中的影响。系列的羟吲哚，虽然我们从这个最初的系列中鉴定出了有效的 AMPK 抑制剂，但我们相信进一步 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。抑制剂的开发并使用体外激酶测定评估其 AMPK 抑制活性。细胞 AMPK 的变化将通过测量 AMPK 底物乙酰辅酶 A 的磷酸化来确定通过 ELISA 在 MOLM13 和 MOLM14 细胞中检测羧化酶 (ACC)，并将选择抑制剂用于激酶组保留延伸出 ATP 结合位点的氨基烷基侧链的 AMPK 抑制剂将进行分析（目标 1）。与蛋白水解靶向嵌合体 (PROTAC) 降解剂偶联，它们降解细胞 AMPK 的能力将具有末端二甲氨基的基于羟吲哚的 AMPK 抑制剂或降解剂。与 AMPK 的 DFG 基序相互作用将被修饰以掺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。作为单一药物对细胞活力、代谢和 ROS 水平的降解剂将在 MOLM13 和 MOLM14 细胞以及原代 AML 细胞、LSC 和正常造血干细胞 (HSC) 中的效果。 AMPK 抑制剂或降解剂与 Venetoclax 联合使用的效果将在 MOLM13 和 MOLM14 中确定最后，我们的 AMPK 抑制剂或降解剂作为单一药物和在原发性 AML 细胞中的作用。与 Venetoclax 的组合将使用移植到高级体内的原发性 AML 标本进行评估肿瘤异种移植模型（目标 3）。评估 AMPK 在维持 LSC 活力中的作用以及针对 AML 中 AMPK 的治疗潜力。