The biochemical mechanism and pharmacological inhibition of phosphatidylinositol phosphate kinases

磷脂酰肌醇磷酸激酶的生化机制及药理抑制作用

• 批准号: 10711064
• 负责人: YA HA
• 金额: $ 32.2万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-15 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10711064
• 关键词: 2019-nCoV; Binding; Biochemical; Biological; Biology; CCR; COVID-19 treatment; Catalytic Domain; Cell Culture Techniques; Cell Line; Cells; Chemicals; Complex; Crystallization; Crystallography; Development; Disease; Ebola virus; Elements; Endosomes; Epithelial Cells; Family; Future; Genetic; Goals; Head; Human; Hydroxyl Radical; In Vitro; Infection; Inositol; Integration Host Factors; Knock-in; Knock-out; Knowledge; Lead; Life Cycle Stages; Lipids; Lung; Marburgvirus; Membrane; Molecular Conformation; Pattern; Pharmaceutical Chemistry; Pharmacology Study; Phase II Clinical Trials; Phosphatidylinositol Phosphates; Phosphatidylinositols; Phosphorylation; Phosphotransferases; Positioning Attribute; Refractory; Research; Resolution; Role; SARS-CoV-2 infection; SARS-CoV-2 inhibitor; Sequence Homology; Specificity; Structure; Substrate Specificity; Testing; Viral; Viral Physiology; Work; analog; clinical center; clinical investigation; crosslink; drug repurposing; experimental study; inhibitor; interest; member; mutant; pharmacologic; phosphatidylinositol 5-phosphate; programs; small molecule inhibitor; tool; trafficking

Project Summary / Abstract The objective of the proposed research is to elucidate the biochemical mechanisms underlying the exquisite substrate binding and catalytic specificity of two phosphatidylinositol phosphate 5-kinases (PIP5K, PIKfyve). The PIPK family of lipid kinases include PIP5K (type 1), PIP4K (type 2) and PIKfyve (type 3), and is primarily responsible for converting phosphatidylinositol monophosphate lipids into PI(4,5)P2 and PI(3,5)P2. Despite sequence homology, these kinases are highly selective in substrate binding [PIP5K binds PI(4)P, PIP4K binds PI(5)P, and PIKfyve binds PI(3)P] and in catalytic activity [PIP5K and PIKfyve phosphorylate the C5 hydroxyl of the lipid's inositol head group, whereas PIP4K phosphorylates the C4 hydroxyl]. We and others have previously identified two structural elements within the kinase domain, the specificity loop and a conserved PIP-binding motif, that contribute to substrate selectivity, but how these two elements cooperate to confer kinase specificity remains undefined at the structural level. In aim 1, we plan crosslinking strategies to stabilize the specificity loop to facilitate co-crystallization with lipid substrates. We also plan to generate and crystallize a minimalistic catalytic core domain of PIKfyve. In aim 2, we propose genetic and chemical biological experiments to examine the role of PIKfyve in the life cycle of SARS-CoV-2. Several large-scale drug repurposing programs have identified apilimod, a PIKfyve inhibitor, as a top lead in suppressing SARS-CoV-2 replication in cell culture (a phase II clinical trial of apilimod in treating COVID-19 is ongoing at the Yale Center for Clinical Investigation). This discovery, together with earlier observations that apilimod also reduces infection by Ebola and Marburg viruses, has generated great interest in pharmacologically targeting PIKfyve. Drawing on structural and biochemical knowledge about the lipid kinase family, as well as chemical tools previously developed to target PIP4K, we have discovered a new class of potent PIKfyve inhibitors and plan to use them together with apilimod to interrogate how PIKfyve inhibition disrupts SARS-CoV-2 infection. All previously known PIKfyve inhibitors are structurally related to apilimod, and their binding mode to the lipid kinase is unknown. The new inhibitor class is significant because it not only adds confidence to the proposed involvement of PIKfyve in SARS-CoV-2 infection, but also has a known binding mode to PIPK, which should facilitate future optimization by medicinal chemistry.

项目摘要/摘要 这项拟议的研究的目的是阐明精致的 两种磷脂酰肌醇磷酸5-激酶(PIP5K，PIKfyve)的底物结合和催化专一性。 PIPK家族包括PIP5K(类型1)、PIP4K(类型2)和PIKfyve(类型3)，主要是 负责将磷脂酰肌醇单磷酸脂转化为PI(4，5)P2和PI(3，5)P2。尽管 序列同源性，这些酶在底物结合上具有很高的选择性[PIP5K结合PI(4)P，PIP4K结合 PI(5)P和PIKfyve结合PI(3)P]，并在催化活性上[PIP5K和PIKfyve磷酸化 脂质的肌醇头部基团，而PIP4K磷酸化C4羟基]。我们和其他人有 先前在该蛋白的结构域中发现了两个结构元件，即特异环和一个保守的 PIP结合基序，有助于底物选择性，但这两个元件是如何协同作用来授予 在结构水平上，激酶的特异性仍未确定。在目标1中，我们计划使用交联策略来稳定 专一性环，以促进与脂质底物的共结晶。我们还计划生成并结晶一种 PIKfyve的极简主义催化核心区。在目标2中，我们提出了遗传和化学生物学 检测PIKfyve在SARS-CoV-2生命周期中的作用的实验。几种大规模毒品 重新调整用途的计划已经确定阿匹莫德是一种PIKfyve抑制剂，是抑制SARS-CoV-2的主要药物 细胞培养中的复制(阿皮莫特治疗新冠肺炎的II期临床试验正在耶鲁中心进行 用于临床研究)。这一发现，加上早期的观察结果，阿比利莫特也可以减少感染 埃博拉和马尔堡病毒引起了人们对以PIKfyve为靶标的药理学研究的极大兴趣。绘图 关于脂激酶家族的结构和生化知识，以及以前的化学工具 针对PIP4K的开发，我们发现了一类新的有效的PIKfyve抑制剂并计划使用它们 与apilimod一起询问PIKfyve抑制如何破坏SARS-CoV-2感染。都是以前的事 已知的PIKfyve抑制剂在结构上与apilimod相关，它们与脂激酶的结合模式是 未知。新的抑制器类别意义重大，因为它不仅为提议的 PIKfyve参与SARS-CoV-2感染，但也与PIPK有已知的结合模式，这应该是 通过药物化学促进未来的优化。