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

描述（由申请人提供）：发现肾素-血管紧张素系统（RAS）含有反调节成分，彻底改变了对其功能的认识。血管紧张素II和III（Ang II和Ang III）作用于AT 1 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敏感靶器官，例如肾脏、心脏和大脑。然而，当Ang II和Ang III作用于AT 2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以形成Ang 1-7，其是由mas癌基因编码的去磷酸化G蛋白偶联受体的激动剂配体。作用于mas受体，Ang 1-7具有抑制、抗纤维化和抗炎作用，其抵消Ang II和Ang III对AT 1血管紧张素受体的病理生理作用。由于ACE- 2在RAS的这种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对AT 1受体的作用的生理拮抗剂，因此它在治疗与Ang II/Ang III/AT 1受体轴过度活性相关的疾病中具有治疗意义。 该提案将开发和应用可用于测量功能性ACE-2活性的放射性配体，以补充当前的测定方法并弥补这些方法的缺点。该建议的概念来自于使用351 A，其是赖诺普利（广泛使用的抗高血压ACE抑制剂）的类似物，其可以被放射性碘标记（125 I-351 A）以测量功能性ACE蛋白并确定其在体内组织中的分布。 MLN-4760是ACE-2的特异性、高亲和力（IC 50 ~ 440 pM）拮抗剂，但不容易进行放射性碘标记。该建议的假设是，在该假二肽的羧基末端侧用酪氨酸侧链替换亮氨酰侧链的MLN-4760类似物将保留MLN-4760对ACE-2的高亲和力和特异性，无论酪氨酸侧链是否连接有碘-125分子。 拟定的研究涉及1）合成MLN-4760类似物（JFS 101/102），其符合作为选择性、高亲和力ACE-2抑制剂的质量标准，2）放射性碘化和改进的 单125 I（和127 I）类似物的HPLC纯化，和3）该放射性配体用于测量功能性ACE-2蛋白及其在多种心血管疾病和中风的动物模型中的调节的应用。最初的研究将集中于在被认为影响ACE-2表达的条件下测量脑中的ACE-2，例如，在用ACE-2 mRNA或用抑制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的过表达或缺失。研究还将包括使用放射自显影和125 I-MLN-4760类似物结合的定量光密度分析测定ACE-2在啮齿动物脑中的分布。 这些研究的未来计划结果将包括在疾病动物模型中测定其他组织中的ACE-2，例如心脏、肾脏、肺、胰腺、生殖器官。本研究将有助于我们进一步了解ACE-2作为传统RAS的反调节因子的表达和功能意义。并且，可以开发靶向ACE-2的新型治疗剂以促进其拮抗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.