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

• 批准号: 8433896
• 负责人: Robert Charles Speth
• 金额: $ 39.37万
• 依托单位: NOVA SOUTHEASTERN UNIVERSITY
• 依托单位国家: 美国
• 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/8433896
• 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; public health relevance; radioligand; radiotracer; receptor; reproductive; research study; small hairpin RNA; tool

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）：肾素-血管紧张素系统（RAS）含有反调节成分的发现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）作用于 AT1 Ang II 受体亚型， 引起升压、纤维化、促有丝分裂和炎症反应，损害心血管系统以及易受影响的靶器官，例如肾、心和脑。然而，当 Ang II 和 Ang III 作用于 AT2 受体亚型时，它们具有抑制和抗纤维化作用。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 年发现的单羧肽酶血管紧张素转换酶 2 (ACE-2) 作用于 Ang II 形成 Ang 1-7，它是由 mas 癌基因编码的去孤儿 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、抗纤维化和抗炎作用，抵消 Ang II 和 Ang III 对 AT1 血管紧张素受体的病理生理作用。由于 ACE-2 在 RAS 的反调节臂中具有双重功能：它使 Ang II 失活并形成 Ang II 和 Ang III 在 AT1 受体上的作用的生理拮抗剂，因此它在治疗与 Ang II/Ang III/AT1 受体轴过度活跃相关的疾病方面具有治疗意义。 该提案将开发和应用一种可用于测量功能性 ACE-2 活性的放射性配体，以补充当前的检测方法并弥补这些方法的缺点。该提案的概念来自于使用 351A，它是赖诺普利（广泛使用的抗高血压 ACE 抑制剂）的类似物，可以通过放射性碘标记 (125I-351A) 来测量功能性 ACE 蛋白并确定其在体内组织中的分布。 MLN-4760 是一种特异性、高亲和力 (IC50 ~ 440 pM) 的 ACE-2 拮抗剂，但不易进行放射性碘标记。该提案的假设是，MLN-4760 的类似物（其中亮氨酸侧链在该假二肽的羧基末端侧被酪氨酸侧链取代）将保留 MLN-4760 对 ACE-2 的高亲和力和特异性，无论酪氨酸侧链是否连接有碘 125 分子。 拟议的研究涉及 1) MLN-4760 类似物 (JFS101/102) 的合成，该类似物符合选择性、高亲和力 ACE-2 抑制剂的规格，2) 放射性碘化和改进 Mono125I（和 127I）类似物的 HPLC 纯化，以及 3) 应用这种放射性配体测量功能性 ACE-2 蛋白及其在多种心血管疾病和中风动物模型中的调节。初步研究将侧重于在被认为影响 ACE-2 表达的条件下测量大脑中的 ACE-2，例如，用 ACE-2 mRNA 或抑制 ACE-2 合成的小干扰 RNA 病毒转染的转基因小鼠或大鼠中 ACE-2 的过度表达或缺失。研究还将包括使用放射自显影术和 125I-MLN-4760 类似物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成果将包括在其他组织中检测 ACE-2，例如组织中的 ACE-2。心脏、肾脏、肺、胰腺、生殖器官疾病动物模型。预计这项研究将大大增强我们对 ACE-2 作为传统 RAS 反调节因子的表达和功能意义的理解。并且，可能会开发出针对 ACE-2 的新型治疗剂，以促进其拮抗 RAS 病理生理作用的有益方面。