Targeting anti-apoptotic drugs to failing cardiomyocytes

描述(申请人提供)：心力衰竭是全球发病率和死亡率的主要原因。现已认识到心肌细胞凋亡在心力衰竭中起重要作用。细胞内蛋白水解酶，又称半胱氨酸天冬氨酸氨基转移酶，在细胞凋亡的启动和执行中起着至关重要的作用。实验证据表明，及时给予半胱氨酸天冬氨酸氨基转移酶抑制剂可减少心肌细胞凋亡和心力衰竭。然而，caspase抑制剂的临床开发是困难的，因为作为极性化合物，它们很难穿过细胞膜，最终在肝脏中积累。 我们建议检验一种假说，即早期的心肌细胞凋亡可以通过靶向的caspase抑制剂的细胞内传递来挽救。作为靶向蛋白，我们建议使用膜联蛋白V，这是一种与磷脂酰丝氨酸结合的蛋白，它显示在凋亡细胞的表面，包括凋亡的心肌细胞。最近的证据表明，Annexin V可被凋亡细胞内化，因此可能适合于细胞内给药。 为了增加膜联蛋白V递送的药物分子的数量，我们建议将caspase抑制剂包裹到脂质体中。为了靶向凋亡的心肌细胞，将膜联蛋白V与磷脂偶联，插入到载药脂质体的脂膜中。为了避免随机结合破坏膜联蛋白V，我们将使用N端含有半胱氨酸的标签表达的突变型膜联蛋白V进行位点特异性修饰。我们对该蛋白质的初步数据表明，不同的有效载荷可以在不影响蛋白质活性的情况下与该标签结合。 在这个探索性的项目中，我们将开发膜联蛋白V修饰的脂质体，并测试它们运送足够数量的caspase抑制剂的能力，以从凋亡损伤中拯救心肌细胞。如果我们的构建能够在体外拯救凋亡的心肌细胞，则为在体内检验这一拯救策略提供了实验基础和合理性。

DESCRIPTION (provided by applicant): Heart failure is the major cause of morbidity and mortality worldwide. It is recognized that cardiomyocyte apoptosis plays an important role in heart failure. Intracellular proteases, known as caspases, play crucial role in initiation and execution of apoptosis. Experimental evidence indicates that timely administration of caspase inhibitors diminishes cardiomyocyte apoptosis and heart failure. However, clinical development of caspases inhibitors is difficult because as polar compounds they have difficulties in crossing cell membrane and eventually accumulate in the liver. We propose to test a hypothesis that early apoptotic cardiomyocytes can be rescued by targeted intracellular delivery of caspase inhibitors. As a targeting protein we propose to use annexin V, a protein that binds to phosphatidylserine, which is displayed on the surface of apoptotic cells, including apoptotic cardiomyocytes. Recent evidence indicates that annexin V is internalized by apoptotic cells, and therefore may be suitable for intracellular drug delivery. To increase the number of drug molecules that are delivered by annexin V, we propose to encapsulate caspase inhibitors into liposomes. For targeting to apoptotic cardiomyocytes, annexin V conjugated to phospholipid will be inserted into the lipid membrane of drug-loaded liposomes. To avoid damaging annexin V by random conjugation, we will use a mutant annexin V expressed with N-terminal cysteine-containing tag for site-specific modification. Our preliminary data with this protein indicate that various payloads can be conjugated to this tag without affecting protein activity. In this exploratory project we will develop annexin V decorated liposomes and test their ability to deliver caspase inhibitors in amounts sufficient for rescuing cardiomyocytes from apoptotic injury. If our construct can rescue apoptotic cardiomyocytes in vitro, it provide experimental foundation and rational for testing this rescue strategy in vivo.