Chemobiologic Approach to NAADP Signaling

说明(申请人提供)：烟酸腺嘌呤二核苷酸磷酸(NAADP)是一种强大的钙动员第二信使。这项申请提出了一项合作努力，以合成新颖和信息丰富的NAADP类似物，并将这些化合物应用于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，评价物种和组织之间的SAR差异，以及生产有效的、药理学特征良好的膜永久激动剂和拮抗剂。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)，NAADP介导的Ca+2释放可能是启动Ca+2离子释放的触发因素，然后通过其他Ca+2释放机制放大。多种组织和细胞类型已被证明对NAADP有反应，这证明了这种信号机制的重要性和普遍性。NAADP衍生物将通过化学-酶方法和全合成方法合成。首先，我们建议应用PI实验室开发的光亲和标记来标记哺乳动物细胞提取物和海胆匀浆中的NAADP结合蛋白，并确定NAADP结合结构域是TPC的一个结构域，还是ryanodine受体，或者它是否是一个新的蛋白质。光衍生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，该双功能光探针包含用于与受体进行光交联的芳香族叠氮化合物以及将在光交联后使用的乙炔取代基，以使用“点击化学”来连接亲和性标签。这一过程将使我们能够纯化少量的光衍生物NAADP结合蛋白，并随后通过质谱学获得部分序列来鉴定结合蛋白。NAADP类似物将使用海胆鸡蛋匀浆和哺乳动物细胞中的膜片钳和单细胞荧光测量来表征，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酰氧甲酯，并发展成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基本的细胞调节机制。烟酸腺嘌呤二核苷酸2‘-磷酸(NAADP)是几种相互作用的细胞内第二信使之一，它动员钙离子来控制生物反应。该计划的结果将加深我们对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。

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.