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

描述(申请人提供)：肾素-血管紧张素系统(RAS)含有逆调节成分的发现彻底改变了人们对其功能的思考。血管紧张素II和血管紧张素III(Ang II和Ang III)作用于AT1 Ang II受体亚型 引起升压、纤维化、有丝分裂和炎症反应，损害心血管系统以及易感靶器官，如肾脏、心脏和大脑。然而，当Ang II和Ang III作用于AT2受体亚型时，它们具有降压和抗纤维化作用。最近，RAS的另一个更强大的反调控臂被证明存在：ACE-2/Ang 1-7/ma轴。血管紧张素转换酶-2(ACE-2)是2000年发现的一种单羧基肽酶，作用于血管紧张素转换酶-2，作用于血管紧张素Ⅱ，形成血管紧张素转换酶1-7，是癌基因编码的去变形G蛋白偶联受体的激动剂配体。Ang 1-7作用于Mas受体，具有降压、抗纤维化和抗炎作用，可对抗Ang II和Ang III在AT1血管紧张素受体上的病理生理效应。由于ACE-2在RAS的这个反向调节臂中具有双重功能：它使Ang II失活，并在AT1受体上形成Ang II和Ang III的生理拮抗剂，因此它在治疗与Ang II/Ang III/AT1受体轴过度活动相关的疾病方面具有重要的治疗意义。这项提议将开发和应用一种可用于测量功能性ACE-2活性的放射性配基，以补充目前的检测方法，并弥补这些方法的缺点。这项建议的概念来自于使用351a，它是赖诺普利(广泛使用的抗高血压ACE抑制剂)的类似物，可以被放射性碘化(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是一种特异的、高亲和力(IC50~440 PM)的ACE-2拮抗剂，但对放射性碘不敏感。该方案的假设是，在该伪二肽的羧基末端用酪氨酸侧链取代亮氨基侧链的MLN-4760类似物，将保留MLN-4760对ACE-2的高亲和力和特异性，无论是否有碘-125分子连接到酪氨酸侧链上。建议的研究包括1)合成符合选择性、高亲和力ACE-2抑制剂的MLN-4760类似物(JFS101/102)；2)放射性碘化和改进 3)该放射性配基在多种心血管疾病和中风动物模型中的应用，用于测量功能性ACE-2蛋白及其调节。最初的研究将集中于在被认为影响ACE-2表达的条件下测量大脑中的ACE-2，例如，在转基因小鼠或大鼠中过表达或缺失ACE-2，这些转基因小鼠或大鼠通过病毒转导ACE-2 mRNA或使用抑制ACE-2合成的小干扰RNA。研究还将包括使用放射自显影和125I-MLN-4760类似物结合的定量密度分析来确定ACE-2在啮齿类动物大脑中的分布。这些研究的未来计划结果将包括在其他组织中检测ACE-2，例如在疾病动物模型中的心、肾、肺、胰腺、生殖器官。预计通过这项研究，我们对ACE-2作为传统RAS的反向调节因子的表达和功能意义的理解将大大增强。而且，针对ACE-2的新的治疗药物可能被开发来促进其拮抗RAS的病理生理作用的有益方面。

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.