Molecular mechanisms of lithium action on kinases

摘要/摘要 锂是数百万双相情感障碍患者的一线治疗药物， 抑制痴呆症的发展。实验表明，Li+改变生理的主要模式是： 方法是通过降低数量惊人有限的Mg 2+依赖性磷酸转移酶的活性， 酶，包括磷酸单酯酶和蛋白激酶。虽然（Li独立）催化剂 这些酶的机制已经非常清楚，其中很多都是关于其背后的机制细节 锂敏感性仍然未知。毫不奇怪，设计酶变体仍然是一个主要挑战， 是抗锂的，并使用它们来解开与个体的锂耐受性相关的信号通路。 内切酶在这里，我们专注于Li+对激酶的作用，并解决以下问题，以减轻 上面提到的问题。对71种人类激酶的实验显示了广泛的锂敏感性-许多是 其他人受到不同程度的影响。但是没有解释这些变化。 我们通过使用最先进的分子力学来解决我们对Li作用的理解中的这一差距 (MM)，量子力学（QM）和QM/MM模拟，以及诱变实验指导下， 生物信息学和自然选择在实验的支持下，我们探讨了Li+ 通过与其催化位点直接相互作用而影响激酶活性。在目标1中，模拟将研究Li+ 结合激酶，以及Li+结合如何降低激酶活性。此外，模拟将提供洞察力， 调节催化位点活性的潜在变构效应。我们的生化、细胞和体内实验 在Aim 2中，设计用于（i）系统地检查Li-敏感性和Li-敏感性之间的序列差异的影响， 耐药激酶，目标是使Li敏感酶GSK-3对Li+具有抗性;以及（ii）发现关键的 使某些激酶对Li敏感的残基。实验还将验证模拟结果， 与此同时，模拟将为解释突变实验的结果提供分子见解。 对模拟和实验结果的综合分析将产生耐锂GSK-3， 重要的是，它将首次使我们能够解开GSK-3驱动的Li+生理效应 与其他锂敏感酶不同本研究也将为解释观测到的现象提供物理基础 不同的激酶对锂的敏感性不同，这些生物物理发现将为未来的研究奠定基础。 努力使其他Li敏感激酶对Li+具有抗性，并绘制其特异性表型。我们预计 这些努力将改善对Li治疗和剂量的患者反应的理解和预测， 这仍然是一项艰巨的任务。这将加快治疗并避免暴露于副作用。最后 这项研究将探索模拟酶反应的新进展，并产生一个有效的极化力 用于描述Li+/Mg 2+与蛋白质相互作用的字段。这将使未来可靠的研究锂的行动， 蛋白质没有考虑在这个项目和扩大探索的全方位镁结合蛋白。

Summary/Abstract Lithium is a first-line therapy for millions of people suffering from bipolar disorder, and is promising for inhibiting development of dementia. Experiments show that a primary mode by which Li+ alters physiological processes is by reducing activities of a surprisingly limited number of Mg2+-dependent phosphoryl-transferring enzymes, including phosphomonoesterases and protein kinases. While the (Li-independent) catalytic mechanisms of these enzymes are quite well-understood, much about the mechanistic details underlying their Li-susceptibility remain unknown. Not surprisingly, it remains a major challenge to design enzyme variants that are Li-resistant, and use them to disentangle signaling pathways associated with Li-susceptibilities of individual enzymes. Here we focus on Li+'s action on kinases, and address the following problem central to alleviating the issues raised above. Experiments on 71 human kinases show a wide range of Li-susceptibility — many are unaffected and others are affected to varying degrees. But there is no explanation for these variations. We address this gap in our understanding of Li-action by using state-of-the-art molecular mechanics (MM), quantum mechanics (QM) and QM/MM simulations, as well as mutagenesis experiments guided by bioinformatics and natural selection. Supported by experiments, we explore the overarching hypothesis that Li+ affects kinase activity by interacting directly with their catalytic sites. In Aim 1, simulations will examine how Li+ binds kinases, and how Li+ binding reduces kinase activity. Additionally, simulations will provide insights into potential allosteric effects that regulate catalytic site activity. Our biochemical, cellular and in vivo experiments in Aim2 are designed to (i) systematically examine effects of sequence differences between Li-sensitive and Li- resistant kinases, with the goal of making a Li-sensitive enzyme, GSK-3, resistant to Li+; and (ii) discover key residues that make certain kinases Li-sensitive. Experiments will also validate findings from simulations, and at the same time, simulations will provide molecular insights to interpret results from mutational experiments. Combined analysis of results from simulations and experiments will yield a Li-resistant GSK-3, which is significant because it will, for the first time, enable us to disentangle GSK-3-driven physiological effects of Li+ from those of other Li-sensitive enzymes. This study will also provide a physical basis to explain observed variations of Li-sensitivity across kinases, and these biophysical findings will serve as foundations for future efforts to make other Li-sensitive kinases resistant to Li+, and map their specific phenotypes. We expect that such efforts will improve understanding and predictions of patient responses to Li-treatments and dosages, which remains a difficult task. This will both expedite therapy and avoid exposure to side effects. Finally, this study will explore new advancements in modeling enzyme reactions and yield a validated polarizable force field for describing Li+/Mg2+ interactions with proteins. This will enable future reliable studies of Li-action on proteins not considered in this project and broaden exploration of the full range of Mg-binding proteins.