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)生成磷脂酸(PA)。 DAG 和 PA 均作为有效的脂质信使来塑造细胞 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 还可以作为磷脂和 甘油三酯生物合成以及膜结构和生物能学的组成部分。我们的意义 拟议的研究表明，针对个体 DGK 具有巨大的治疗潜力，因为它们具有 在塑造脂质组以支持健康和健康的代谢、结构和信号需求方面发挥着重要作用 患病细胞。尽管它们在近 30 年前就已具有临床价值和发现，但在这方面的知识仍存在差距 生命系统中 DGK 活性位点的配体结合和调节混淆了对 共享共同催化结构域的 10 种哺乳动物 DGK 亚型如何能够调节不同的 代谢和信号传导功能。我们将检验我们的假设，即 C1 和其他非催化结构域， 很大程度上区分了 DGK 亚型、底物功能和 DGK 活性位点的抑制剂识别。 拟议的研究计划将测试选择性阻断 DGKα 是否可以恢复有缺陷的 DAG 信号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 DGKs 作为逆转 T 细胞免疫抑制的有希望的靶标，尽管分子 DGK 代谢紊乱与 TCR 信号传导增强的耦合机制尚不清楚。我们的机械 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 活性位点指导新化学策略的开发，以干扰 T 细胞特异性 DGK 的活性 用于免疫治疗应用的体内。我们对该提案的长期目标是在功能上映射新颖且 DGKα 上的可药物小分子结合位点以及 T 细胞中潜在的其他 DGK 亚型可以： 1) 获得 对 DAG 脂肪酰基链识别和特异性的分子水平洞察，2) 识别 DAG 脂肪酰基链的分子特征 靶向脂质与蛋白激酶的酶活性位点，以及 3) 开发新的选择性失活抑制剂 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，旨在：（目标 1）DAG 结合的识别 站点，（目标 1）了解各个 DGK 结构域如何耦合细胞外信号来塑造 T 细胞反应， （目标 2）确定 DGK 抑制剂如何增强 T 细胞活化，（目标 2）了解 DGK 抑制剂如何 逆转体内 T 细胞免疫抑制，并（目标 2）确定 DGK 抑制剂是否影响细胞膜 易位。我们研究结果的总体影响将是了解 DGK 串扰的内在特征 具有细胞环境的外在特征，形成脂质信号传导代码的基础，可以 治疗靶向逆转 T 细胞的免疫抑制。