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.

二酰基甘油激酶(DGKs)是催化二酰甘油磷酸化的多结构域脂蛋白激酶 (DAG)生成磷脂酸(PA)。DAG和PA都是形成细胞的强有力的脂质信使 通过改变基本受体蛋白的亚细胞定位、激活和功能来作出反应(Range 从酶到转录因子)。DAG和PA也是磷脂和 甘油三酯的生物合成以及膜结构和生物能量学的组成部分。我们的重要意义在于 拟议的研究是针对单个DGK的巨大治疗潜力，因为它们 在塑造脂体以支持代谢、结构和信号需求方面的基础作用 病态细胞。尽管它们在近30年前具有临床价值和发现，但关于它们的知识差距 对配体的结合和对生命系统中DGK活性位点的调节扰乱了对 10种具有共同催化结构域的哺乳动物DGK亚型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，即C1和其他非催化结构域，它们 主要区分DGK的异构体、在底物中的功能以及DGK活性部位的抑制剂识别。 拟议的研究计划将测试选择性阻断DGK是否可以恢复缺陷的DAG 克服肿瘤浸润性淋巴细胞活性免疫抑制的信号。遗传学和临床证据 指出DGKs是逆转T细胞免疫抑制的有希望的靶点，尽管分子 DGK代谢受阻与TCR信号增强的耦合机制尚不清楚。我们的机械师 研究将建立一个可测试的模型，以基本了解底物和抑制剂识别 DGK活性部位指导开发新的化学策略以干扰T细胞特异性DGK的活性 用于免疫治疗的活体应用。我们对这项提议的长期目标是在功能上绘制出新的和 T细胞中Dgk和潜在的其他Dgk亚型上的可药物小分子结合位点：1)获得 分子水平对DAG脂肪酰链识别和特异性的洞察，2)确定分子特征 酶活性部位，靶向脂类和蛋白激酶，以及3)开发新的选择性失活抑制剂 在活细胞和动物中的DGK亚型。 我们将测试两个独立但相关的特定目标，旨在：(目标1)识别DAG结合 站点，(目标1)了解单个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抑制剂如何放大T细胞激活，(目标2)了解DGK抑制剂如何 在体内逆转T细胞免疫抑制，以及(目标2)确定DGK抑制剂是否影响膜 易位。我们发现的总体影响将是理解DGK的内在特征是如何相互作用的 与细胞环境的外部特征一起形成脂质信令编码的基础，该编码可以 治疗靶向逆转T细胞的免疫抑制。