Probing the Archazolid Pharmacophore Using Plant-Based V-ATPase Assays

描述（由申请人提供）：古唑烷是最近分离的一类天然产物，基于选择性液泡型atp酶（v - atp酶）抑制，对许多人类癌细胞系显示出强大的生长抑制活性，然而这些化合物实现其活性的机制尚不完全清楚。该研究的长期目标是基于对古唑啉类抑制活性的全面了解，创造一类新的小分子v - atp酶抑制剂治疗药物。本申请的目的是开发一种简化的阿古唑脂衍生的v - atp酶抑制剂的合成和使用方便的基于植物的v - atp酶测定。这是由一个中心假设驱动的，即系统修饰的古唑啉衍生物的v - atp酶抑制活性的比较将提供对古唑啉药效基团的重要见解。提出这项研究的基本原理是，一旦了解了古唑类化合物如何抑制v - atp酶的功能，就有可能合理设计具有增强药理特性的小分子v - atp酶抑制剂，从而为治疗各种疾病提供新的方法。在强有力的初步数据的指导下，这一假设将通过追求两个具体目标来验证：1)开发一种简化的古唑啉类似物的合成；2)开发两种基于植物的v - atp酶检测方法。在第一个目标下，采用立体选择性取代三烯合成的tms -烯丙化/Peterson消除序列的合成策略，已经产生了完整的古唑烷碳框架，将用于制备一系列改性的古唑烷B衍生物。高级合成中间体和完整化合物的v - atp酶抑制活性将使用第二个目标下开发的技术进行测试。具体来说，gfp -拟南芥将用于鉴定活性化合物，然后可以使用拟南芥根生长试验以剂量依赖的方式进行评估。拟议的研究具有创新性，因为它通过提供研究工具来更好地阐明古氮杂生物活性，代表了对v - atp酶研究的传统plecomacrolide方法的背离。这一贡献是重要的，因为它旨在阐明v - atp酶结合和抑制的新模式。除了进一步了解v - atp酶的结构和功能外，该结果还可能为开发v - atp酶抑制剂药物提供新途径，以治疗包括骨质疏松症和癌症在内的各种严重疾病。

DESCRIPTION (provided by applicant): The archazolids are a family of recently isolated natural products that display powerful growth inhibitory activity against a number of human cancer cell lines based on selective vacuolar-type ATPase (V-ATPase) inhibition, however the mechanism by which these compound achieve their activity is not completely understood. A long-term goal of the proposed research is to create a new class of small-molecule V-ATPase inhibitor therapeutics based on a thorough understanding of archazolid inhibitory activity. The objective of this application is to develop a synthesis of simplified archazolid-derived V-ATPase inhibitors and assay using convenient plant-based V-ATPase assays. This is driven by a central hypothesis that the comparison of the V-ATPase inhibitory activity of systematically modified archazolid-derivatives will provide crucial insights into the archazolid pharmacophore. The rationale for the proposed research is that once it is known how the archazolids inhibit V-ATPase function, it will become possible to rationally design small molecule V-ATPase inhibitors with enhanced pharmacological properties resulting in new approaches for the treatment of various diseases. Guided by strong preliminary data, this hypothesis will be tested by pursuing two specific aims: 1) Develop a synthesis of simplified archazolid analogues; and 2) Develop two plant-based V-ATPase assays. Under the first aim, a synthetic strategy featuring a TMS-allylation/Peterson elimination sequence for stereoselective substituted triene synthesis that has already produced the entirety of the archazolid carbon framework will be used to prepare a series of modified archazolid B derivatives. The V-ATPase inhibitory activity of advanced synthetic intermediates and completed compounds will then be tested using the techniques developed under the second aim. Specifically, GFP-Arabidopsis will be used to identify active compounds that can then be evaluated in a dose-dependent manner using an Arabidopsis root-growth assay. The proposed research is innovative because it represents a departure from the traditional plecomacrolide approach to V-ATPase research by providing research tools to better elucidate archazolid activity. This contribution is significant because it aims to elucidate a nove mode of V-ATPase binding and inhibition. In addition to providing further insights into V-ATPase structure and function, the results have the potential to lead to new approaches for the development of V-ATPase inhibitor drugs to treat various severe diseases including osteoporosis and cancer.