hERG MAP-A rational approach to eliminate ion channel related cardiotoxicity

描述(申请人提供)：药物抑制HERG K+通道与心律失常和猝死有关。由于HERG相关的心脏毒性，几种药物被从市场上撤下。需要新的方法来消除HERG与新药的结合。药物开发将大大受益于对HERG区块结构基础的新的实验见解。Neurion公司开发了一种独特的方法--HERG MAP(Tm)，它可以识别药物与HERG通道之间的关键相互作用。这些数据指导药物化学家综合减少或消除HERG结合。我们在与主要制药公司的成功合作中验证了我们的Rational方法。目前，我们在非洲爪哇卵母细胞中表达HERG通道。卵母细胞的卵黄很大，疏水性，似乎能吸附药物。重要的是，卵黄结合使得在卵母细胞中测得的药物亲和力比在哺乳动物细胞中测得的要小。这限制了我们直接估计亲和力和测量弱结合药物的能力。为了使我们目前的方法更广泛地适用于并符合当前制药公司的实践，我们建议将HERG MAP应用于哺乳动物细胞表达系统。在目标1中，我们将阐明六种药物与在哺乳动物细胞中表达的关键结合残基突变的WT、HERG和HERG的结合作用和通道阻断；在目标2中，我们将应用体内无义抑制方法，通过激光(光学)注射将外源氨基酰化tRNA转移到哺乳动物细胞中，产生非自然的HERG突变体。第一阶段工作的结果将是广泛适用的、基于哺乳动物细胞的HERG分析，可以直接帮助药物化学家消除HERG毒性。在第二阶段，我们将通过建立所有相关HERG突变体在哺乳动物细胞中的表达并优化高通量、自动化电生理测量的技术来实现检测的工业化。我们还将分析大量的分子，并进行创建药物-HERG相互作用的大型数据库所需的计算解释。最后，我们还将演示一种已知的HERG阻断分子的合成“拯救”。

DESCRIPTION (provided by applicant): hERG K+ channel inhibition by drugs is implicated in cardiac arrhythmias and sudden death. Several drugs were removed from the market due to hERG-related cardiotoxicity New methods are needed for eliminating hERG binding to new drugs. Drug development will benefit substantially from new experimentally-derived insights into the structural basis for hERG block. Neurion has developed a unique approach, the hERG MAP(tm) that identifies the key interactions drugs make with the hERG channel. The data guide medicinal chemists in synthetically reducing or eliminating hERG binding. We validated our rational approach in successful collaborations with major pharmaceutical companies. Currently we express hERG channels in Xenopus oocytes. The yolk of the oocyte is large and hydrophobic, and appears to adsorb drugs. Importantly, yolk binding makes drug affinities estimates measured in oocytes appear less than those measured in mammalian cells. This limits our ability to directly estimate affinity and to measure weakly binding drugs. In order make our current approach more widely applicable and in line with current pharmaceutical company practice, we propose to adopt hERG MAP for mammalian cell expression systems. In Aim 1 we will elucidate the binding interactions and channel block of six drugs with WT hERG and hERG mutated at critical binding residues expressed in mammalian cells; in Aim 2 we will apply the in vivo nonsense suppression methodology to generate unnatural hERG mutants using laser based (optical) injection to deliver exogenous amino acylated tRNA into mammalian cells. The result of the Phase I work will be wildly applicable, mammalian cell-based hERG assays that can directly aid medicinal chemists in eliminating hERG toxicity. In Phase II we will industrialize the assay by establishing the expression of all relevant hERG mutants in mammalian cells and optimizing the technology for higher- throughput, automated electrophysiology measurements. We will also assay a substantial number of molecules and conduct the computational interpretation needed to create a large database of drug-hERG interactions. Finally, we will also demonstrate the synthetic "rescue" of a known hERG-blocking molecule.