Targeting anti-apoptotic drugs to failing cardiomyocytes

描述（由申请人提供）：心力衰竭是全球发病率和死亡率的主要原因。心肌细胞凋亡在心力衰竭中起重要作用。细胞内蛋白酶，称为半胱天冬酶，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，及时给予半胱天冬酶抑制剂减少心肌细胞凋亡和心力衰竭。然而，半胱天冬酶抑制剂的临床开发是困难的，因为作为极性化合物，它们难以穿过细胞膜并最终在肝脏中积累。 我们建议测试一个假设，即早期凋亡的心肌细胞可以通过靶向细胞内递送半胱天冬酶抑制剂来拯救。作为靶向蛋白，我们建议使用膜联蛋白V，一种与磷脂酰丝氨酸结合的蛋白质，其显示在凋亡细胞（包括凋亡的心肌细胞）的表面上。最近的证据表明，膜联蛋白V被凋亡细胞内化，因此可能适合于细胞内药物递送。 为了增加由膜联蛋白V递送的药物分子的数量，我们建议将半胱天冬酶抑制剂封装到脂质体中。为了靶向凋亡的心肌细胞，将与磷脂缀合的膜联蛋白V插入到载药脂质体的脂质膜中。为了避免通过随机缀合破坏膜联蛋白V，我们将使用用N-末端含半胱氨酸的标签表达的突变膜联蛋白V进行位点特异性修饰。我们对这种蛋白质的初步数据表明，各种有效载荷可以与这种标签缀合而不影响蛋白质活性。 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，并测试它们提供足够数量的半胱天冬酶抑制剂以拯救心肌细胞免于凋亡损伤的能力。如果我们的构建体能够在体外挽救凋亡的心肌细胞，这将为在体内验证这种挽救策略提供实验基础和理论依据。

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.