Inhibitors of the ENT4 Adenosine Transporter for Cardioprotection

描述（由申请人提供）：尽管在治疗方面取得了进步，但心脏病仍然是美国的头号死因，每年夺去近100万美国人的生命，每年耗费的医疗费用估计为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，在高危患者中不理想。这就迫切需要有效的治疗方法。增强生理核苷腺苷（Ado）的心脏保护作用的方法继续保持希望，并继续被用于新的心脏保护疗法。该项目将致力于鉴定一种新发现的ph依赖性心脏腺苷转运蛋白ENT4的高亲和力特异性抑制剂。相对于其他生理核苷，ENT4选择性地转运腺苷，使其成为研究心肌缺血和再灌注中腺苷调节的有趣转运体。目前，还没有已知的有效或特异性的ENT4 Ado转运抑制剂。因此，鉴定有效的特异性抑制剂是重中之重，不仅对心脏研究，而且对研究ENT4在其他组织中的腺苷调节也是如此。它们将有助于研究其在心脏缺血-再灌注中的作用，并表明ENT4是否可以成为与其他心脏保护剂（如ENT1核苷转运抑制剂）联合治疗的心脏保护药物靶点。根据我们对腺苷摄取抑制剂作为治疗缺血-再灌注损伤的心脏保护剂的追求，我们在核苷转运蛋白缺乏的猪PK15 （PK15NTD）细胞系中克隆并稳定表达了人类ENT4转运蛋白（hENT4）。随后，我们使用该表达系统筛选并鉴定了ENT4 Ado运输的小分子抑制剂，其ic50值低至亚微摩尔水平，相对于一种或另一种人质膜平衡核苷转运蛋白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）、药效团定位和3D-QSAR研究来优化这些先导化合物的效价和选择性；2)在离体大鼠心肌梗死全脑缺血模型中探索ENT4抑制剂的心脏保护作用。新的ENT4抑制剂将与我们最近发现的心脏保护ENT1抑制剂进行比较(Zhu et al., Am.；j .杂志。心脏杂志，2007,29(2):2921-2926。我们将探索ENT4抑制剂与ENT1抑制剂联合或协同作用的可能性，以保护心脏。将采用多学科方法结合平行组合合成、化学信息学/3D-QSAR、生物化学、生理学和细胞生物学方法来实现该项目的目标。该结果将为研究ENT4在心脏缺血中的作用及其抑制剂在心脏保护中的作用提供新的见解，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。最近的临床结果显示，他汀类药物通过激活外泌5′-核苷酸酶来增加间质Ado水平，可以降低胆固醇水平正常人群的心脏病发作和中风风险，因此腺苷增强心脏保护的方法再及时不过了（Ridker et al.， N. Engl）。医学杂志；2008；啊哈,2008);以及低剂量他汀类药物和Ado摄取抑制剂联合使用时对心脏保护的协同作用（Ye et al., 2007）。公共卫生相关性：在本提案中，我们寻求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），并在离体大鼠心脏缺血模型中测试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.