Chemical proteomic investigation of lipid kinase specificity and druggability

脂质激酶特异性和成药性的化学蛋白质组学研究

• 批准号: 10660099
• 负责人: Ku-Lung Hsu
• 金额: $ 39.13万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-13 至 2023-08-24
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10660099
• 关键词: Active Sites; Address; Affect; Anabolism; Animals; Architecture; Attenuated; Azoles; Binding; Binding Sites; Biochemical; Bioenergetics; Biological; Biological Assay; Biology; CD8B1 gene; Catalytic Domain; Cell membrane; Cell physiology; Cells; Cellular biology; Chemicals; Clinical; Code; Coupling; DAG/PE-Binding Domain; Development; Diacylglycerol Kinase; Diglycerides; Disease; Drug Targeting; Environment; Enzymes; Family; Genetic; Goals; Human; Immunosuppression; Immunotherapy; Impairment; Individual; Investigation; Knock-out; Knowledge; Ligand Binding; Ligands; Link; Lipids; Location; Lysine; MAP Kinase Gene; Mammals; Maps; Mediating; Membrane; Metabolic; Metabolism; Methods; Modeling; Modification; Molecular; Mutation; Organism; Outcome Study; Phosphatidic Acid; Phospholipids; Phosphorylation; Phosphotransferases; Protein Engineering; Protein Isoforms; Protein Kinase; Proteins; Proteome; Proteomics; Receptor Signaling; Recombinants; Regulation; Reporting; Research; Role; Series; Shapes; Signal Transduction; Site; Specificity; Substrate Specificity; System; T cell regulation; T cell response; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; Testing; Therapeutic; Translating; Triglycerides; Tumor Antigens; Tumor-Infiltrating Lymphocytes; Tyrosine; VAV1 gene; candidate identification; chemotherapy; drug discovery; experimental study; extracellular; in vivo; inhibitor; insight; interest; kinase inhibitor; lipid metabolism; lipidome; lipidomics; metabolomics; mimetics; mouse model; novel; overexpression; pharmacologic; programs; receptor; response; small molecule; therapeutic target; transcription factor; tumor

Diacylglycerol kinases (DGKs) are multi-domain lipid kinases that catalyze phosphorylation of diacylglycerol (DAG) to generate phosphatidic acid (PA). Both DAG and PA serve as potent lipid messengers to shape cellular responses by altering subcellular localization, activation, and function of essential receptor proteins (ranging from enzymes to transcription factors). DAG and PA also serve as building blocks for phospholipid and triglyceride biosynthesis and integral to membrane architecture and bioenergetics. The significance of our proposed studies is the enormous therapeutic potential of targeting individual DGKs because of their fundamental role in sculpting the lipidome to support metabolic, structural, and signaling demands of healthy and diseased cells. Despite their clinical value and discovery nearly 30 years ago, gaps in knowledge with regards to ligand binding and regulation of DGK active-sites in living systems have confounded basic understanding of how 10 mammalian DGK isoforms, which share a common catalytic domain, are capable of regulating distinct metabolic and signaling functions. We will test our hypothesis that C1 and other non-catalytic domains, which largely differentiate DGK isoforms, function in substrate and inhibitor recognition of DGK active sites. The proposed research program will test whether selective blockade of DGK can restore deficient DAG signaling to overcome immunosuppression of tumor infiltrating lymphocyte activity. Genetic and clinical evidence point to DGKs as promising targets for reversing immunosuppression of T cells although the molecular mechanisms coupling disrupted DGK metabolism to enhanced TCR signaling are not clear. Our mechanistic studies will establish a testable model for fundamental understanding of substrate and inhibitor recognition in DGK active sites to guide development of new chemical strategies to perturb activity of T cell specific DGKs in vivo for immunotherapy applications. Our long-term goals for this proposal are to functionally map novel and druggable small molecule binding sites on DGK and potentially other DGK isoforms in T cells to: 1) gain molecular level insights into DAG fatty acyl chain recognition and specificity, 2) identify molecular features of enzyme active sites to target lipid versus protein kinases, and 3) develop new inhibitors for selective inactivation of DGK isoforms in live cells and animals. We will test 2 independent yet related specific aims directed at: (Aim 1) identification of the DAG binding site, (Aim 1) understanding how individual DGK domains couple extracellular signals to shape T cell responses, (Aim 2) determining how DGK inhibitors amplify T cell activation, (Aim 2) understanding how DGK inhibitors reverse T cell immunosuppression in vivo, and (Aim 2) determining if DGK inhibitors affect membrane translocation. The overall impact of our findings will be to understand how intrinsic features of DGKs cross-talk with extrinsic features of cellular environments to form the basis of a lipid signaling code that can be therapeutically targeted for reversing immunosuppression of T cells.

二酰甘油激酶（DGK）是催化二酰甘油磷酸化的多结构域脂质激酶 (DAG)以产生磷脂酸（PA）。DAG和PA都是有效的脂质信使， 通过改变必需受体蛋白的亚细胞定位、激活和功能（范围 从酶到转录因子）。DAG和PA还可以作为磷脂的构建模块， 甘油三酯生物合成和膜结构和生物能量学的组成部分。我们的重要性 提出的研究是靶向个体DGK的巨大治疗潜力，因为它们 在塑造脂质体以支持健康和肥胖的代谢，结构和信号需求方面发挥着重要作用。 病变细胞尽管它们的临床价值和近30年前的发现， 配体结合和调节DGK活性位点在生活系统中混淆了基本的理解， 10种哺乳动物DGK同工型共享一个共同的催化结构域，它们如何能够调节不同的 代谢和信号功能。我们将测试我们的假设，C1和其他非催化结构域， 在很大程度上区分DGK亚型，在底物中起作用，并抑制DGK活性位点的识别。 拟议的研究计划将测试选择性阻断DGK β是否可以恢复缺陷的DAG 信号传导以克服肿瘤浸润淋巴细胞活性的免疫抑制。遗传和临床证据 指出DGK是逆转T细胞免疫抑制的有希望的靶点，尽管分子水平 将破坏的DGK代谢与增强的TCR信号传导偶联的机制尚不清楚。我们的机械论 研究将建立一个可测试的模型，用于基本了解底物和抑制剂的识别， DGK活性位点，以指导开发新的化学策略来干扰T细胞特异性DGK的活性， 用于免疫治疗应用。我们这项提案的长期目标是在功能上映射新的和 在T细胞中DGK β和潜在的其他DGK同种型上的可药物化的小分子结合位点，以：1）获得 分子水平的见解DAG脂肪酰基链的识别和特异性，2）确定分子特征， 靶向脂质与蛋白激酶的酶活性位点，以及3）开发用于选择性失活的新抑制剂 活细胞和动物中的DGK亚型。 我们将测试2个独立但相关的具体目标，目的是：（目标1）鉴定DAG结合 位点，（目的1）了解单个DGK结构域如何偶联细胞外信号以形成T细胞应答， (Aim 2）确定DGK抑制剂如何放大T细胞活化，（目的2）了解DGK抑制剂如何 逆转体内T细胞免疫抑制，以及（目的2）确定DGK抑制剂是否影响膜 易位我们的研究结果的总体影响将是了解DGK串扰的内在特征 与细胞环境的外在特征形成脂质信号传导密码的基础， 治疗靶向逆转T细胞的免疫抑制。