MKP5 allostery in MAPK regulation and signaling in the heart

MKP5 变构在心脏 MAPK 调节和信号传导中的作用

• 批准号: 10375784
• 负责人: Anton M Bennett
• 金额: $ 62.03万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-18 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10375784
• 关键词: Adopted; Allosteric Site; Binding; Binding Sites; Biology; CRISPR/Cas technology; Cardiac development; Catalysis; Catalytic Domain; Cells; Cellular biology; Chemicals; Clinical; Complex; Coupled; Crystallization; Data; Development; Disease; Effectiveness; Fingerprint; Generations; Genetic; Goals; Heart; Heart failure; Intervention; Kinetics; Knock-in; Knock-in Mouse; MAPK phosphatase; MAPK8 gene; Mediating; Mitogen-Activated Protein Kinases; Modeling; Molecular; Morbidity - disease rate; Mus; Outcome; Pathway interactions; Pharmaceutical Preparations; Pharmacology; Phosphoric Monoester Hydrolases; Physiological; Play; Protein Kinase Interaction; Proteomics; Regulation; Resolution; Role; Signal Transduction; Site; Specificity; Structure; Therapeutic; Tissues; Transforming Growth Factors; United States; Work; biophysical techniques; coronary fibrosis; druggable target; experimental study; fibrogenesis; genetic approach; in vivo; inhibitor; insight; mortality; mouse genetics; mutant; novel; operation; p38 Mitogen Activated Protein Kinase; phosphatase inhibitor; phosphoproteomics; pressure; small molecule; structural biology; therapeutic target; transcriptomics

ABSTRACT The catalytic activity and specificity of the mitogen-activated protein kinase (MAPK) phosphatases (MKPs) is governed through their ability to interact with the MAPKs. The MKPs set the fidelity and kinetics of MAPK signaling by regulating, in a spatially distinct manner, both the magnitude and duration of MAPK activity. However, these regulatory mechanisms alone are insufficient to explain the complexity of MKP/MAPK- mediated signaling. This is exemplified by the observation that inactivation of MKPs deliver unique, and in some cases, non-obvious physiological and pathophysiological outcomes that has led to the realization that MKPs represent valuable therapeutic targets for the treatment of certain diseases. We have found that MKP5, which directly dephosphorylates p38 MAPK and JNK, is involved in the development of cardiac fibrosis, a serious sequalae that precedes heart failure. Mice lacking MKP5 are protected from the development of cardiac fibrosis and heart failure in a model of pressure overload. A high throughput small molecule screen identified a first-in-class inhibitor of MKP5. Resolution of the co-crystal structure of the inhibitor in complex with MKP5 revealed that the inhibitor bound to an allosteric site on MKP5. We hypothesize that the MKP5 allosteric site represents an additional mode of MKP5 regulation that governs signaling specificity and that this allosteric site represents the “Achilles heel” of MKP5 for small molecule targeting. The broad goal of this work is two-fold. First, we will define the molecular basis for how the allosteric site regulates MKP5 catalysis and MAPK signaling. Second, whether disruption of the allosteric site produces outcomes in a disease context that provide proof-of-concept that if successfully “drugged” this site represents a platform for development of first-in-class MKP inhibitors. We will execute the specifics of these two broad goals through three specific aims. Aim 1, will employ structural biology and biophysical approaches to elucidate the structural determinants and binding mechanisms conferred by the MKP5 allosteric site to determine how this site regulates MKP5 catalysis and MAPK binding. A co-complex between MKP5 and p38 MAPK will be obtained to define how p38 MAPK modulates MKP5 through the allosteric site. In Aim 2, the effects of mutants in the MKP5 allosteric site on MAPK signaling will be determined by generating knockin cells harboring MKP5 allosteric site mutants using CRISPR-Cas approaches. The specificity of the MKP5 allosteric site to “fine-tune” MAPK-mediated signaling will be determined using non-biased phosphoproteomic and transcriptomic approaches. In Aim 3, the effects of targeting the MKP5 allosteric site in cardiac fibrosis and heart failure will be determined through the generation of a novel MKP5 allosteric site mutant knock-in mouse. These studies will define a new mode of operation for MKP5 that will provide information on the actions of the allosteric site at the atomic level. How the MKP5 allosteric site impacts physiological signaling and the validity of the allosteric site as a “druggable” target in disease therapeutics will be defined.

摘要 丝裂原活化蛋白激酶(MAPK)磷酸酶(MKPs)的催化活性和特异性是 通过他们与MAPK互动的能力来管理。MKP决定了MAPK的保真度和动力学 通过以空间上不同的方式调节MAPK活性的大小和持续时间来传递信号。 然而，仅靠这些调控机制不足以解释MKP/MAPK- 中介信号。MKP的失活提供了独特的，并且在 在某些情况下，不明显的生理和病理生理结果导致了意识到 MKP是治疗某些疾病的有价值的治疗靶点。我们发现MKP5， 它直接去磷酸化p38MAPK和JNK，参与了心脏纤维化的发展，a 心力衰竭前的严重后遗症。缺乏MKP5的小鼠受到保护，不会发生 压力超负荷模型中的心脏纤维化和心力衰竭。一种高通量的小分子筛 确定了一种MKP5的一流抑制剂。缓蚀剂在络合物中共晶结构的解析 MKP5显示该抑制剂结合在MKP5上的变构位点上。我们假设MKP5变构 SITE代表了MKP5调节的另一种模式，该模式管理信号特异性，并且这种变构 SITE代表了小分子靶向MKP5的“阿喀琉斯之踵”。这项工作的总体目标有两个。 首先，我们将定义变构位置如何调节MKP5催化和MAPK的分子基础 发信号。第二，变构位点的破坏是否在疾病背景下产生结果，从而提供 概念证明，如果成功地给这个网站下了药，它代表着一个一流的开发平台 MKP抑制剂。我们将通过三个具体目标来执行这两个广泛目标的具体内容。目标1，意志 使用结构生物学和生物物理方法来阐明结构决定因素和结合 MKP5变构位点决定该位点如何调节MKP5催化和 MAPK结合。将获得MKP5和p38MAPK之间的共同复合体，以定义p38MAPK如何 通过变构位点调节MKP5。在目标2中，MKP5变构位点上的突变对 MAPK信号将通过产生携带MKP5变构位点突变体的敲门细胞来确定 CRISPR-CAS接近。MKP5变构位点“微调”MAPK介导信号的特异性 将使用无偏倚的磷酸蛋白质组学和转录组学方法来确定。在目标3中， 靶向心脏纤维化和心力衰竭中的MKP5变构位点将通过生成 一种新的MKP5变构位点突变敲入小鼠。这些研究将确定一种新的运营模式 MKP5将提供关于变构中心在原子水平上的作用的信息。MKP5如何 变构部位影响生理信号和变构部位作为“药物”靶点的有效性 疾病治疗学将被定义。