Chemobiologic Approach to NAADP Signaling

NAADP 信号传导的化学生物学方法

• 批准号: 8232675
• 负责人: JAMES T SLAMA
• 金额: $ 35.07万
• 依托单位: UNIVERSITY OF TOLEDO HEALTH SCI CAMPUS
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8232675
• 关键词: Affinity; Agonist; Azides; Behavior; Binding; Binding Proteins; Binding Sites; Biological; Calcium Channel; Calcium ion; Cell Extracts; Cells; Chemicals; Chemistry; Complementary DNA; Cultured Cells; Environment; Esters; Fertilization; Fluorescence; Future; Human; Human Cell Line; Image; In Vitro; Invertebrates; Ion Channel; Ions; Label; Laboratories; Lymphocyte; Mammalian Cell; Mass Spectrum Analysis; Measurement; Mediating; Membrane; Methods; Molecular Weight; Muscle Contraction; NAADP; Nucleic Acids; Organelles; Pharmaceutical Preparations; Phosphate-Binding Proteins; Photoaffinity Labels; Population; Preparation; Procedures; Process; Production; Protein Binding; Proteins; Relative (related person); Reporting; Ryanodine Receptor Calcium Release Channel; Sea Urchins; Signal Pathway; Signal Transduction; Smooth Muscle; Solutions; Stream; Strongylocentrotus purpuratus; Structure; System; T-Cell Receptor; Testing; Time; Tissues; Training; Variant; Work; analog; cell type; crosslink; defined contribution; egg; graduate student; human tissue; inorganic phosphate; insulin secretion; new therapeutic target; nicotinic acid adenine dinucleotide; novel; patch clamp; programs; protein protein interaction; receptor; research study; response; tissue/cell culture; tool; undergraduate student

DESCRIPTION (provided by applicant): Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca+2 mobilizing 2nd messenger. This application proposes a collaborative effort to synthesize novel and informative NAADP analogs and to apply these compounds to solve important problems in the field-including the unambiguous identification of the NAADP binding domain, a through elucidation of the structure-activity relation for NAADP recognition, an appreciation for the variation in the SAR between species and between tissues, and the production of potent, pharmacologically well characterized and membrane permanent agonists and antagonists. Evidence strongly suggests that NAADP regulates the two-pore channel (TPC) located on a lysosomal-like acidic organelle, and that NAADP mediated Ca+2 release may serve as a trigger to initiate the release of Ca+2 ion, which is then amplified through other Ca+2 release mechanisms. A variety of tissues and cell types have been shown to respond to NAADP, arguing for the importance and generality of this signaling mechanism. NAADP derivatives will be synthesized using chemo-enzymatic methods as well as through total synthesis. First, we propose to apply a photoaffinity label developed in the PI's laboratory to label NAADP binding proteins in mammalian cell extracts and in sea urchin homogenates, and to determine if the NAADP binding domain is a domain of the TPC, the ryanodine receptor, or if it is a novel protein. Purification and isolation of the photoderivatized binding protein will be facilitated by our synthesis of potent bifunctional photoprobes containing an aromatic azide for photocrosslinking to the receptor as well as an acetylenic substituent that will be used after photocrosslinkage to attach an affinity tag using "click chemistry". This procedure will enable us to purify the small quantities of photoderivatized NAADP binding protein and subsequently to identify the binding protein by obtaining partial sequences using mass spectrometry. NAADP analogs will be characterized using sea urchin egg homogenates and in mammalian cells using patch clamp and single cell fluorescent measurements, enabling us to characterize the behavior of our compounds both in the important invertebrate system and in mammalian cells. Last, we will synthesize and test metabolically stable NAADP derivatives. Compounds demonstrated to be high potency agonists and antagonists will be converted into acytoxymethyl esters and developed as membrane permanent agonists and antagonists. PUBLIC HEALTH RELEVANCE: Changes in of intracellular calcium-ion concentration are a fundamental cell regulatory mechanism. Nicotinic acid adenine dinucleotide 2'-phosphate (NAADP) is one of several interacting intracellular 2nd messengers that mobilize calcium-ion to control biological response. Results of this program will enhance our understanding of NAADP signaling mechanisms, identify new therapeutic targets, and uncover novel drug strategies. It will contribute to understanding fertilization, insulin secretion, smooth muscle contraction, and other important processes.

描述（由申请人提供）：烟酸腺嘌呤二核苷酸磷酸（NAADP）是一种有效的Ca+2动员第二信使。本申请提出了一种合作的努力来合成新的和信息丰富的NAADP类似物，并应用这些化合物来解决该领域的重要问题，包括明确鉴定NAADP结合结构域，通过阐明NAADP识别的结构-活性关系，评价物种之间和组织之间SAR的变化，以及产生有效的，良好表征的膜永久性激动剂和拮抗剂。有证据表明，NAADP调节位于溶酶体样酸性细胞器上的双孔通道（TPC），并且NAADP介导的Ca+2释放可能作为启动Ca+2离子释放的触发器，然后通过其他Ca+2释放机制放大Ca+2离子释放。多种组织和细胞类型已被证明对NAADP有反应，这证明了这种信号传导机制的重要性和普遍性。NAADP衍生物将使用化学-酶促方法以及通过全合成来合成。首先，我们建议应用PI的实验室开发的光亲和标记标记NAADP结合蛋白在哺乳动物细胞提取物和海胆匀浆，并确定是否NAADP结合域是一个域的TPC，ryanodine受体，或者如果它是一种新的蛋白质。光衍生化的结合蛋白的纯化和分离将通过我们合成的有效的双功能光探针来促进，所述双功能光探针含有用于光交联至受体的芳香族叠氮化物以及将在光交联后使用“点击化学”连接亲和标签的炔属取代基。这个程序将使我们能够纯化少量的光衍生NAADP结合蛋白，并随后通过使用质谱法获得部分序列来鉴定结合蛋白。NAADP类似物将使用海胆卵匀浆和哺乳动物细胞中使用膜片钳和单细胞荧光测量进行表征，使我们能够表征我们的化合物在重要的无脊椎动物系统和哺乳动物细胞中的行为。最后，我们将合成和测试代谢稳定的NAADP衍生物。被证明是高效激动剂和拮抗剂的化合物将被转化为乙酰氧基甲酯，并被开发为膜永久激动剂和拮抗剂。 公共卫生相关性：细胞内钙离子浓度的变化是一种基本的细胞调节机制。烟酸腺嘌呤二核苷酸2 '-磷酸（NAADP）是细胞内几种相互作用的第二信使之一，可动员钙离子以控制生物反应。该计划的结果将增强我们对NAADP信号机制的理解，确定新的治疗靶点，并发现新的药物策略。它将有助于理解受精，胰岛素分泌，平滑肌收缩和其他重要过程。

项目成果

Nicotinic Acid Adenine Dinucleotide Phosphate Analogues Substituted on the Nicotinic Acid and Adenine Ribosides. Effects on ReceptorMediated Ca²⁺ Release.

烟酸腺嘌呤二核苷酸磷酸类似物在烟酸和腺嘌呤核苷上取代。对受体介导的Ca²⁺释放的影响。

• DOI: 10.1021/acs.jmedchem.5b00279
• 发表时间: 2015-04-23
• 期刊: JOURNAL OF MEDICINAL CHEMISTRY
• 影响因子: 7.3
• 作者: Trabbic, Christopher J.;Zhang, Fan;Walseth, Timothy F.;Slama, James T.
• 通讯作者: Slama, James T.

A Bioactive Resveratrol Trimer from the Stem Bark of the Sri Lankan Endemic Plant Vateria copallifera.

来自斯里兰卡特有植物 Vateria copallifera 的茎皮的生物活性白藜芦醇三聚体。

• DOI: 10.1021/acs.jnatprod.7b00892
• 发表时间: 2018
• 期刊: Journal of natural products
• 影响因子: 5.1
• 作者: Samaradivakara,SaroopaP;Samarasekera,Radhika;Handunnetti,ShiromaM;Weerasena,OVDSJagathpriya;Al-Hamashi,AyadA;Slama,JamesT;Taylor,WilliamR;Alhadidi,Qasim;Shah,ZahoorA;Perera,Lalith;Tillekeratne,LMViranga
• 通讯作者: Tillekeratne,LMViranga