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.

项目总结/摘要 这项研究的目的是阐明这种精致的生物化学机制。 两种磷脂酰肌醇磷酸5-激酶（PIP 5 K，PIKfyve）的底物结合和催化特异性。 PIPK家族的脂质激酶包括PIP 5 K（1型）、PIP 4K（2型）和PIKfyve（3型），并且主要是 负责将磷脂酰肌醇单磷酸脂质转化为PI（4，5）P2和PI（3，5）P2。尽管 序列同源性，这些激酶在底物结合中具有高度选择性[PIP 5 K结合PI（4）P，PIP 4K结合PI（4）P]。 PI（5）P，PIKfyve结合PI（3）P]和在催化活性中[PIK 5 K和PIKfyve磷酸化PI（5）P的C5羟基]。 脂质的肌醇头基，而PIP 4K磷酸化C4羟基]。我们和其他人已经 以前确定了激酶结构域中的两个结构元件，特异性环和保守的 PIP结合基序，这有助于底物的选择性，但这两个元素如何合作，赋予 激酶特异性在结构水平上仍不确定。在目标1中，我们计划交联策略以稳定 特异性环以促进与脂质底物的共结晶。我们还计划产生和具体化一个 PIKfyve的最小催化核心结构域。在目标2中，我们提出遗传和化学生物学 研究PIKfyve在SARS-CoV-2生命周期中的作用。几种大型药物 再利用项目已经确定了PIKfyve抑制剂阿吡莫德作为抑制SARS-CoV-2的首要药物 在细胞培养中复制（阿吡莫德治疗COVID-19的II期临床试验正在耶鲁中心进行 临床研究）。这一发现，加上早期的观察，阿吡莫德也减少感染 埃博拉病毒和马尔堡病毒引起了人们对靶向PIKfyve的病毒的极大兴趣。绘图 关于脂质激酶家族的结构和生物化学知识，以及以前的化学工具， 针对PIP 4K开发，我们发现了一类新的有效PIKfyve抑制剂，并计划使用它们 与阿吡莫德一起研究PIKfyve抑制如何破坏SARS-CoV-2感染。所有先前 已知的PIKfyve抑制剂在结构上与阿吡莫德相关，并且它们与脂质激酶的结合模式是 未知新的抑制剂类别是重要的，因为它不仅增加了建议的信心， PIKfyve参与SARS-CoV-2感染，但也具有已知的与PIPK的结合模式，这应该 便于将来通过药物化学进行优化。