Development of AMPK Inhibitors for the treatment of leukemia

用于治疗白血病的 AMPK 抑制剂的开发

• 批准号: 10299304
• 负责人: Philip Reigan
• 金额: $ 33.29万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 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/10299304
• 关键词: 4-nitroimidazole; 5' -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/antagonist; 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

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 尽管急性髓细胞白血病（AML）的治疗取得了进展，但只有20-30%的患者实现了长期生存。 复发性AML的无病生存期（DFS）和治疗选择极其有限。再次发生 AML被归因于白血病干细胞（LSC），现在的努力集中在靶向这种药物 耐药细胞群，以“治愈”AML。我们的研究测量能量代谢在初级人类 AML标本，使用活性氧（ROS）作为代谢活性的指标，显示LSC 优先处于ROS低状态。此外，高水平的活化的5'AMP活化蛋白激酶 在LSC中检测到AMPK（一种代谢途径的中枢调节因子），AMPK的敲低 导致ROS水平增加和伴随的LSC损失。基于这些发现，我们建议AMPK 抑制将使LSC脱离ROS-低状态，降低其活力，这对于LSC可能是足够的。 消除或可能使其对常规治疗敏感。几乎没有有效的和选择性的AMPK抑制剂; 然而，据报道，多激酶抑制剂舒尼替尼是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的和选择性的基于羟吲哚的AMPK靶向 药物的作用，并检查AMPK抑制或降解在AML模型中的作用。我们开发了一个初始的 虽然我们从最初的系列中鉴定出了有效的AMPK抑制剂，但我们相信进一步的研究表明， 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 开发抑制剂并使用体外激酶测定法评估其AMPK抑制活性。然后是抑制 通过测量AMPK底物乙酰辅酶A的磷酸化， 在MOLM 13和MOLM 14细胞中通过ELISA测定羧化酶（ACC），并将提交选择的抑制剂用于激酶组 剖析（目标1）。保留延伸出ATP结合位点的氨基烷基侧链的AMPK抑制剂将 与蛋白水解靶向嵌合体（PROTAC）降解剂偶联，并且它们降解细胞AMPK的能力将 被评价。基于羟吲哚的AMPK抑制剂或降解剂具有末端二甲基氨基， 与AMPK的DFG基序相互作用将被修饰以掺入硝基咪唑低氧激活的前药 设计用于引入一系列LSC选择性的基团（目的2）。AMPK抑制剂和PROTAC的作用 将在MOLM 13中测定降解剂作为单一试剂对细胞活力、代谢和ROS水平的影响， M0 LM 14细胞和原代AML细胞、LSC和正常造血干细胞（HSC）中。那么， 将在MOLM 13和MOLM 14中测定AMPK抑制剂或降解剂与维奈托克的联合给药 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性AML标本评价与维奈托克的联合治疗。 肿瘤异种移植模型（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活力中的作用以及靶向AMPK在AML中的治疗潜力。