Inhibitors of the ENT4 Adenosine Transporter for Cardioprotection

描述(申请人提供)：尽管治疗取得了进步，心脏病仍然是美国头号死因，每年夺去近100万美国人的生命，每年的医疗费用估计高达2865亿美元。缺血-再灌注损伤是心脏手术并发症的主要原因；发病率和死亡率，但目前的心脏保护选择非常有限，在高危患者中不是最理想的。这就迫切需要有效的治疗方法。增强生理性核苷腺苷(ADO)的心脏保护作用的方法仍然很有希望，并仍有待于开发新的心脏保护疗法。该项目将致力于鉴定最近确定的一种新的pH依赖的心脏腺苷转运体ENT4的高亲和力特异性抑制物。相对于其他生理核苷，ENT4选择性地转运腺苷，使其成为研究腺苷在心肌缺血和再灌流中的调节作用的有趣的转运体。目前，还没有已知的有效或特异的ENT4 Ado转运抑制剂。因此，不仅对于心脏研究，而且对于研究ENT4在其他组织中对腺苷的调节，识别有效的特异性抑制物是非常重要的。他们将促进对其在心脏缺血-再灌注中的作用的研究，并表明ENT4是否可以作为心脏保护药物与其他心脏保护剂(如ENT1核苷运输抑制剂)联合使用的靶点。为了寻求腺苷摄取抑制剂作为治疗缺血再灌注损伤的心脏保护剂，我们克隆了人ENT4转运体(HENT4)，并在核苷转运体缺陷型猪PK15(PK15NTD)细胞系中稳定表达。我们随后利用这个表达系统来筛选和鉴定ENT4 Ado转运的小分子抑制剂，其IC50值下降到亚微摩尔水平，并且相对于人质膜平衡的核苷转运体hENT1或hENT2，选择性高达78倍。因此，我们的目标是：1)通过构效关系(SAR)、药效团作图和3D-QSAR研究优化这些先导化合物的效力和选择性；2)探讨ENT4抑制剂在大鼠心肌梗死全心缺血模型中的心脏保护作用。新的ENT4抑制剂将与我们最近发现的心脏保护性ENT1抑制剂(朱等人，AmJ.Physiol.心环。物理。2007、292、H2921-2926)。我们将探索ENT4抑制剂与ENT1抑制剂在心脏保护方面可以相加或协同的可能性。结合并行组合合成、化学信息学/3D-QSAR、生物化学、生理学和细胞生物学方法的多学科方法将被用于实现该项目的目标。这些结果将为深入了解ENT4在心肌缺血中的作用及其抑制剂在心脏保护中的作用，并为开发新的心脏保护药物和/或使用急需的新研究工具来探索ENT4生理学提供潜在的先导化合物。鉴于最近的临床结果表明，他汀类药物通过激活ECTO-5‘-核苷酸酶增加间质ADO水平，可以降低胆固醇水平正常的人的心脏病发作和中风风险，腺苷增强方法对心脏保护再及时不过了(Ridker等，N.Engl。J.Med.以及联合使用低剂量他汀类药物和腺苷摄取抑制剂在心脏保护方面的协同增效作用(Ye等人，2007年)。公共卫生相关性：在这项提案中，我们试图研究一类新的腺苷摄取抑制剂作为心脏保护剂。我们将研究结构-活性关系(SARS)，并在一个孤立的大鼠心脏缺血模型中测试这些化合物的心脏保护特性。

DESCRIPTION (provided by applicant): The advances in treatment notwithstanding, heart disease remains the number one cause of death in the Nation, claiming the lives of nearly one million Americans each year, and running up medical costs estimated at $286.5 billion per year. Ischemia-reperfusion injury is the major cause of complications of cardiac surgery; morbidity and mortality, but current cardioprotective options are very limited and suboptimal in high-risk patients. This leaves a dire need for effective treatments. Approaches to augment the cardioprotective actions of the physiological nucleoside adenosine (Ado) continue to hold promise and remain to be exploited for novel cardioprotective therapies. This project will pursue the identification of high-affinity specific inhibitors of a recently characterized novel pH-dependent cardiac adenosine transporter, ENT4. ENT4 selectively transports adenosine relative to other physiological nucleosides, making it an intriguing transporter to study with respect to adenosine modulation in myocardial ischemia and reperfusion. Currently, there are no known potent or specific inhibitors of ENT4 Ado transport. Thus, the identification of potent specific inhibitors is of high priority, not only for cardiac studies but for studying adenosine regulation by ENT4 in other tissues as well. They will facilitate studies of its role in cardiac ischemia-reperfusion, and show whether or not ENT4 could be a cardioprotection drug target for combination regimens with other cardioprotective agents like ENT1 nucleoside transport inhibitors. In line with our pursuit of adenosine uptake inhibitors as cardioprotective agents for the treatment of ischemia- reperfusion injury, we have cloned and stably expressed the human ENT4 transporter (hENT4) in a nucleoside transporter deficient porcine PK15 (PK15NTD) cell line. We subsequently used this expression system to screen, and identify small molecules inhibitors of ENT4 Ado transport with IC50s values down to submicromolar level, and selectivity up to 78-fold relative to one or the other human plasma membrane equilibrative nucleoside transporters, hENT1 or hENT2. Thus we aim to: 1) optimize the potency and selectivity of these lead compounds through structure- activity relationship (SAR), pharmacophore mapping and 3D-QSAR studies, and to 2) explore the cardioprotective effects of ENT4 inhibitors in an isolated rat heart global ischemia model of myocardial infarction. The new ENT4 inhibitors will be compared with our recently discovered cardioprotective ENT1 inhibitors (Zhu et al., Am. J. Physiol. Heart Circ. Physiol. 2007, 292, H2921-2926). We will explore the possibility that ENT4 inhibitors can be additive or synergistic in combination with ENT1 inhibitors for cardioprotection. A multidisciplinary approach combining parallel combinatorial synthesis, chemoinformatics/3D-QSAR, biochemistry, physiology and cell biology methods will be applied to achieve the aims of the project. The results will provide insights on the role of ENT4 in cardiac ischemia, and its inhibitors in cardioprotection, and provide potential lead compounds for the development of new cardioprotective drugs and/or use as much needed novel research tools to probe ENT4 physiology. The adenosine potentiation approach to cardioprotection could not be more timely, in light of recent clinical results showing that statins, which increase interstitial Ado levels by activating ecto-5'-nucleotidase, can reduce heart attack and stroke risks in people with normal cholesterol levels (Ridker et al., N. Engl. J. Med. 2008; AHA, 2008); and the demonstration of synergism between low dose statin and Ado uptake inhibitor for cardioprotection, when used in combination (Ye et al., 2007). PUBLIC HEALTH RELEVANCE: In this proposal, we seek to study a new class of adenosine uptake inhibitors as cardioprotective agents. We will investigate structure-activity relationships (SARs) and test the cardioprotective properties of the compounds in an isolated rat heart ischemia model.