Supramolecular Approaches to Protein Inhibitors Activated by MicroRNA-21

MicroRNA-21 激活蛋白质抑制剂的超分子方法

• 批准号: 8237569
• 负责人: Janarthanan Jayawickramarajah
• 金额: $ 28.6万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-05 至 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8237569
• 关键词: Address; Adverse effects; Antineoplastic Agents; Attenuated; Basic Science; Binding; Biological Assay; Biological Markers; Carbonic Anhydrase II; Cell Line; Cells; Cellular Structures; Chemistry; Chimera organism; Complex; DNA; DNA Structure; DNA-Binding Proteins; Development; Enzymes; Fluorescence; Fluorescence Spectroscopy; Foundations; Goals; Isoenzymes; Knowledge; Lead; Ligands; Malignant Neoplasms; MicroRNAs; Mission; Modification; Molecular Conformation; Oligonucleotides; Oncogenic; Outcome; Patients; Prodrugs; Protein Dynamics; Proteins; Public Health; Research; Resistance; Seminal; Site; Stimulus; Structure; System; Testing; Tissues; Transfection; United States National Institutes of Health; Vertebral column; Vision; Work; aqueous; base; burden of illness; cancer cell; cell growth; chemotherapy; clinically relevant; design; enzyme substrate; esterase; improved; inhibitor/antagonist; innovation; locked nucleic acid; molecular recognition; novel; nuclease; overexpression; protein activation; research study; response; small molecule; stem

DESCRIPTION (provided by applicant): Prodrugs that are triggered by cancer biomarkers are attractive because of their potential to be activated preferentially in cancer cells thereby reducing the serious adverse effects associated with conventional chemotherapy. While numerous cancer biomarkers have been discovered, prodrugs triggered by enzymes have been the main focus because enzymes can activate prodrugs via covalent transformations. Thus, a barrier exists in using non-enzyme biomarkers for prodrug-activation because of our poor understanding of how to develop molecules that selectively recognize, and are activated by, biomarkers that cannot facilitate covalent transformations. The existence of this knowledge gap hinders the full scope of prodrugs that can be developed. Thus the long-term vision of this project is to generate non-covalently triggered prodrugs. The objective of this basic-science application, which is a seminal step in achieving our long term goal, is to establish fundamental supramolecular mechanisms for the non-covalent activation of protein inhibitors by microRNA-21 (miR-21), a highly deregulated, oncogenic, miR. The central hypothesis of this application, which we will test via two complementary design strategies (Aim 1 and 2, respectively), is that miR-21 inducible protein antagonists can be achieved by developing DNA-small molecule chimeras (DCs), that undergo a structural switch from a de-activated conformation to an activated state upon binding sequence specifically to miR-21. The specific aims for this project are to develop, (Aim 1) hairpin-forming DCs de-activated by host/guest interactions and (Aim 2) bidentate DCs de-activated via duplex formation with a guide strand, as miR-21 responsive inhibitors of clinically relevant carbonic anhydrase-II (CA-II). In both aims, we will synthesize and characterize the respective DC systems and will probe their ability to undergo miR-21 induced structure-switching to an activated state via a number of spectroscopic experiments (including UV-vis and fluorescence quenching). Subsequently, the differential ability of the de-activated and active DC conformations to inhibit CA-II will be evaluated (via a colorimetric esterase assay). After optimization of the structure of the DC systems (including enhancing CA-II binding and nuclease resistance), we will transfect DCs into cell-lines over expressing (and control cell-lines under expressing) miR-21 and probe CA-II inhibition-derived cell growth arrest. This proposal is innovative because it develops the first examples of activating protein inhibitors in response to miRs. Further, this project will provide impetus to shift general research into examining non- covalent mechanisms for prodrug activation. This work is significant because (a) it is a seminal step in a continuum of research aimed, in the long-term, at developing miR-21 triggered prodrugs, and (b) it will exert a sustained influence on the field of supramolecular chemistry by enhancing our understanding of how key principles of molecular recognition (such as host/guest interactions and multivalency) in combination with DNA assembly and structure-switching phenomena can be used to develop dynamic protein-binders. ) PUBLIC HEALTH RELEVANCE: This proposal is relevant to public health because fundamental studies on protein inhibitors that are triggered by oncogenic microRNA-21 has the potential to lead to prodrugs that selectively target cancer cells, thereby attenuating the systemic adverse effects and poor patient outcomes endemic to conventional chemotherapy. Thus, the proposed research is particularly relevant to the mission of the NIH that pertains to reducing the burden of illness. )

说明书(申请人提供)：由癌症生物标志物触发的前体药物很有吸引力，因为它们有可能在癌细胞中优先激活，从而减少与传统化疗相关的严重不良反应。虽然已经发现了许多癌症生物标志物，但由酶引发的前药物一直是主要关注的焦点，因为酶可以通过共价转化激活前药物。因此，在使用非酶生物标记物进行前药物激活方面存在障碍，因为我们对如何开发选择性识别生物标记物并被其激活的分子缺乏了解，而这些生物标记物无法促进共价转化。这种知识鸿沟的存在阻碍了可以开发的前药的全部范围。因此，该项目的长期愿景是产生非共价触发的前药。这一基础科学应用的目标是为microRNA-21(miR-21)非共价激活蛋白质抑制剂建立基本的超分子机制，miR-21是一种高度放松调控的致癌miR。我们将通过两种互补的设计策略(分别为目标1和目标2)验证这一应用的中心假设，即miR-21可诱导的蛋白质拮抗剂可以通过开发DNA-小分子嵌合体(DC)来实现，当与miR-21特异结合序列时，DC经历从失活构象到激活状态的结构转换。该项目的具体目标是开发(目标1)通过宿主/客体相互作用失活的发夹形成的树突状细胞和(目标2)通过具有引导链的双链形成失活的双齿树突状细胞，作为临床相关的碳酸氢酶-II(CA-II)的miR-21反应抑制剂。在这两个目标中，我们将合成和表征各自的DC系统，并通过一些光谱实验(包括UV-Vis和荧光猝灭)来探索它们经历miR-21诱导的结构转换到激活状态的能力。随后，将评估失活和活性DC构象抑制CA-II的不同能力(通过比色酯酶分析)。在优化了DC系统的结构(包括增强CA-II结合和核酸酶抗性)后，我们将DC导入高表达miR-21的细胞系(和表达低表达的细胞系)，并检测CA-II抑制衍生的细胞生长停滞。这一提议是创新的，因为它开发了第一个激活蛋白抑制物以响应miRs的例子。此外，该项目将推动将一般研究转移到检查前药物激活的非共价机制。这项工作具有重要意义，因为(A)从长远来看，这是旨在开发miR-21触发的前药物的连续研究中的开创性步骤，(B)它将通过加强我们对分子识别的关键原理(如主体/客体相互作用和多价性)以及DNA组装和结构切换现象如何被用于开发动态蛋白质结合物的理解，对超分子化学领域产生持续的影响。) 公共卫生相关性：这项建议与公共健康相关，因为对致癌microRNA-21引发的蛋白质抑制物的基础研究有可能导致选择性靶向癌细胞的前药，从而减轻传统化疗特有的全身不良反应和糟糕的患者预后。因此，拟议的研究与美国国立卫生研究院减轻疾病负担的使命特别相关。)