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

描述（由申请人提供）：药物抑制hERG K+通道与心律失常和猝死有关。一些药物由于与hERG相关的心脏毒性而退出市场，需要新的方法来消除hERG与新药的结合。药物开发将从hERG阻滞的结构基础的新实验中获益。Neurion开发了一种独特的方法，hERG MAP(tm)，用于识别药物与hERG通道的关键相互作用。这些数据指导药物化学家合成减少或消除hERG结合。我们在与大型制药公司的成功合作中验证了我们的理性方法。目前我们在爪蟾卵母细胞中表达hERG通道。卵母细胞的卵黄大而疏水，似乎可以吸附药物。重要的是，卵黄结合使得在卵母细胞中测量的药物亲和力估计值比在哺乳动物细胞中测量的估计值要小。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，并符合目前制药公司的做法，我们建议在哺乳动物细胞表达系统中采用hERG MAP。在Aim 1中，我们将阐明六种药物与WT hERG和在哺乳动物细胞中表达的关键结合残基突变的hERG的结合相互作用和通道阻断；在目标2中，我们将应用体内无义抑制方法，使用激光（光学）注射将外源氨基酰化tRNA递送到哺乳动物细胞中，产生非天然hERG突变体。I期工作的结果将广泛适用于基于哺乳动物细胞的hERG检测，可以直接帮助药物化学家消除hERG毒性。在第二阶段，我们将通过在哺乳动物细胞中建立所有相关hERG突变体的表达，并优化技术以实现更高通量的自动化电生理测量，从而使该检测方法产业化。我们还将分析大量分子，并进行所需的计算解释，以创建一个大型药物- herg相互作用数据库。最后，我们还将展示一种已知的heg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.