Development of a radioligand for assay of angiotensin-converting enzyme-2(ACE-2)

开发用于检测血管紧张素转换酶-2（ACE-2）的放射性配体

基本信息

批准号：
8665088
负责人：
Robert Charles Speth
金额：
$ 1.09万
依托单位：
NOVA SOUTHEASTERN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-01-10 至 2015-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8665088
关键词：
AGTR2 gene Affect Affinity Agonist American Angiopoietin-2 Angiotensin II Angiotensin Receptor Angiotensin-Converting Enzyme Inhibitors Animal Disease Models Animal Model Anti-Inflammatory Agents Anti-inflammatory Antihypertensive Agents Autoradiography Binding Biological Assay Brain Cardiovascular Diseases Cardiovascular system Cell Nucleus Communities Complement Development Disease Enzymes Future G-Protein-Coupled Receptors Genes Goals Health Heart High Pressure Liquid Chromatography Hypertension Hypotension I125 isotope In Vitro Inflammatory Response Inhibitory Concentration 50 Ischemic Stroke Kidney Knockout Mice Leucine Ligands Lisinopril Lung MLN4760 Measurement Measures Messenger RNA Methodology Methods Mono-S Mus Neurons Oncogenes Organ Pancreas Peptidyl-Dipeptidase A Phase Phenols Physiological Procedures Proteins Quantitative Autoradiography Radiolabeled Radioligand Assay Rattus Receptor, Angiotensin, Type 1 Renin-Angiotensin System Research Research Project Grants Rodent Series Side Small Interfering RNA Specificity Stroke Technology Testing Therapeutic Therapeutic Agents Thinking Tissue Sample Tissues Transgenic Mice Translations Tyrosine Viral Vector analog arm chloramine-T compound 351 A improved inhibitor/antagonist interest meetings novel therapeutics overexpression radioligand radiotracer receptor reproductive research study small hairpin RNA tool

项目摘要

DESCRIPTION (provided by applicant): The discovery that the renin-angiotensin system (RAS) contains counter-regulatory components has revolutionized thinking about its functionality. Angiotensins II and III (Ang II & Ang III) act at the AT1 Ang II receptor subtype to cause pressor, fibrotic, mitogenic, and inflammatory responses that damage the cardiovascular system as well as susceptible target organs, e.g. kidney, heart and brain. However, when Ang II and Ang III act at the AT2 receptor subtype they have depressor and antifibrotic effects. Recently, another, more powerful counter-regulatory arm of the RAS has been shown to exist: the ACE-2/Ang 1- 7/mas axis. The monocarboxypeptidase angiotensin-converting enzyme-2 (ACE-2), discovered in 2000, acts upon Ang II to form Ang 1-7, which is an agonist ligand for the deorphanized G protein coupled receptor encoded by the mas oncogene. Acting upon the mas receptor, Ang 1-7 has depressor, antifibrotic and anti-inflammatory actions that counteract the pathophysiological effects of Ang II and Ang III at the AT1 angiotensin receptor. Since ACE- 2 has a dual function in this counter-regulatory arm of the RAS: it inactivates Ang II and forms a physiological antagonist of the actions of Ang II and Ang III at the AT1 receptor, it is of therapeutic interest in treating diseases associated with overactivity of the Ang II/Ang III/AT1 receptor axis. This proposal will develop and apply a radioligand that can be used to measure functional ACE-2 activity to complement current assay methodologies and compensate for the shortcomings of these methodologies. The concept for this proposal comes from the use of 351A, an analog of lisinopril (the widely used antihypertensive ACE inhibitor) which can be radioiodinated (125I-351A) to measure functional ACE protein and determine its distribution in tissues in the body. MLN-4760 is a specific, high affinity (IC50 ~ 440 pM) antagonist of ACE-2, but it is not readily amenable to radioiodination. The hypothesis of this proposal is that an analog of MLN-4760 in which a leucinyl side chain is replaced with a tyrosinyl side chain at the carboxy terminal side of this pseudodipeptide will retain the high affinity and specificity of MLN-4760 for ACE-2, with and without an iodine-125 molecule attached to the tyrosinyl side chain. The proposed studies involve 1) synthesis of MLN-4760 analogs (JFS101/102) that meet the specifications of being a selective, high affinity ACE-2 inhibitor, 2) radioiodination and improved HPLC purification of the mono125I (and 127I) analog, and 3) application of this radioligand for measurement of functional ACE-2 protein and its modulation in a variety of animal models of cardiovascular disease and stroke. Initial studies will focus on measurement of ACE-2 in the brain under conditions thought to affect ACE-2 expression, e.g., overexpression or deletion of ACE-2 in transgenic mice or rats virally transfected with ACE-2 mRNA or with a small interfering RNA that inhibits ACE-2 synthesis. Studies will also include determination of the distribution of ACE-2 in the rodent brain using autoradiography and quantitative densitometric analysis of 125I-MLN-4760 analog binding. A future planned outgrowth of these studies will include assay of ACE-2 in other tissues, e.g. heart, kidney, lung, pancreas, reproductive organs in animal models of disease. It is anticipated that our understanding of the expression and functional significance of ACE-2 as a counter-regulator of the traditional RAS will be considerably enhanced by this research. And, that novel therapeutic agents targeting ACE-2 might be developed to promote the beneficial aspects of its antagonism of the pathophysiological actions of the RAS.

描述（由申请人提供）：发现肾素 - 血管紧张素系统（RAS）包含反调节组件的发现已彻底改变了对其功能的思考。血管紧张素II和III（ANG II和ANG III）在AT1 ANG II受体亚型上作用导致压力，纤维化，有丝分裂和炎症反应，损害心血管系统以及易感目标器官，例如肾脏，心脏和大脑。但是，当Ang II和Ang III在AT2受体亚型上作用时，它们会具有抑制剂和抗纤维化作用。最近，RAS的另一个更强大的反调节臂已被证明存在：ACE-2/ANG 1-7/MAS轴。 2000年发现的单羧酸肽酶转化酶-2（ACE-2）作用于ANG II形成ANG 1-7，这是Mas Oncogene编码的去甲化G蛋白偶联受体的激动剂配体。 ANG 1-7作用于MAS受体，具有抑制剂，抗纤维化和抗炎作用，可抵消AT1血管紧张素受体ANG II和ANG III的病理生理作用。由于Ace-2在RAS的此反调节部门中具有双重功能：它使ANG II失活并形成ANG II和ANG III在AT1受体中的作用的生理拮抗剂，因此它在治疗与Ang Ang Ang II/Ang II/Ang1受体轴超过相关的疾病方面具有治疗兴趣。该建议将开发和应用一种放射线，可用于测量功能ACE-2活性以补充当前测定方法并补偿这些方法的缺点。该提案的概念来自使用351a，这是一种赖诺普利的类似物（广泛使用的降压ACE抑制剂），可以被放射性化（125-351A）测量功能ACE蛋白质并确定其在体内组织中的分布。 MLN-4760是ACE-2的特定，高亲和力（IC50〜440 PM）的拮抗剂，但不容易被放射性化。该提议的假设是MLN-4760的类似物，其中该假二肽的羧基末端侧的酪氨酸侧链被替代了ACE-2的MLN-4760的高亲和力和特异性，用于ACE-2的高亲和力和特异性。拟议的研究涉及1）MLN-4760类似物的合成（JFS101/102），符合选择性，高亲和力ACE-2抑制剂的规格，2）放射性施加和改善 Mono125i（和127i）类似物的HPLC纯化，以及3）应用这种放射性物体用于测量功能ACE-2蛋白的测量及其在多种心血管疾病和中风的动物模型中的调节。初步研究将集中于在被认为会影响ACE-2表达的条件下的ACE-2测量大脑中的ACE-2，例如，在转基因小鼠或大鼠ACE-2 mRNA或用小的干扰RNA或抑制ACE-2合成的小型干扰RNA中，ACE-2的过表达或缺失。研究还将包括使用自显影术和125i-MLN-4760模拟结合的定量光密度分析来确定ACE-2在啮齿动物大脑中的分布。这些研究的未来计划生长将包括在其他组织中的ACE-2分析，例如心脏，肾脏，肺，胰腺，疾病动物模型中的生殖器官。可以预见，这项研究将大大增强ACE-2作为传统RA的反调节剂的表达和功能意义的理解。并且，可以开发针对ACE-2的新型治疗剂，以促进其对RAS病理生理作用的拮抗作用的有益方面。