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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要拟议研究的目的是阐明精致的生物化学机制两种磷脂酰肌醇磷酸 5-激酶（PIP5K、PIKfyve）的底物结合和催化特异性。 PIPK 脂质激酶家族包括 PIP5K（1 型）、PIP4K（2 型）和 PIKfyve（3 型），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。尽管由于序列同源性，这些激酶在底物结合方面具有高度选择性 [PIP5K 结合 PI(4)P，PIP4K 结合 PI(5)P 和 PIKfyve 结合 PI(3)P] 并具有催化活性 [PIP5K 和 PIKfyve 磷酸化 C5 羟基脂质的肌醇头基，而 PIP4K 磷酸化 C4 羟基]。我们和其他人有先前确定了激酶结构域内的两个结构元件，特异性环和保守的 PIP 结合基序，有助于底物选择性，但这两个元件如何配合赋予激酶特异性在结构水平上仍然未定义。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。我们还计划生成并结晶 PIKfyve 的简约催化核心域。在目标 2 中，我们提出遗传和化学生物研究 PIKfyve 在 SARS-CoV-2 生命周期中的作用的实验。多个大型药品重新利用计划已确定 PIKfyve 抑制剂阿匹莫德 (apilimod) 是抑制 SARS-CoV-2 的首要药物细胞培养中的复制（耶鲁大学中心正在进行阿匹莫德治疗 COVID-19 的 II 期临床试验用于临床研究）。这一发现与早期观察到的阿匹莫德也能减少感染埃博拉病毒和马尔堡病毒引起的药物靶标 PIKfyve 引起了人们的极大兴趣。绘画有关脂质激酶家族的结构和生化知识以及以前的化学工具针对 PIP4K 开发的，我们发现了一类新的强效 PIKfyve 抑制剂并计划使用它们与阿匹莫德一起研究 PIKfyve 抑制如何破坏 SARS-CoV-2 感染。之前全部已知的 PIKfyve 抑制剂在结构上与阿匹莫德相关，它们与脂质激酶的结合模式为未知。新的抑制剂类别意义重大，因为它不仅增加了拟议的信心 PIKfyve 参与 SARS-CoV-2 感染，而且还具有已知的与 PIPK 的结合模式，这应该促进未来药物化学的优化。