Protein targets of rugulactone and illudin S: An analysis of their function and mechanism of action

天然产物在人类医学中的应用已有数千年的历史，迄今为止，大多数现代药物都是基于天然产物。 这些化合物通常作用于多个蛋白质靶点，但这些分子的大多数蛋白质靶点的身份仍然未知。生物活性天然产物的蛋白质靶点的鉴定可以提供关于这些分子的作用模式的有价值的信息，以及为新的药物靶点和先导结构提供灵感。在这个建议中，我们描述了一个多学科的平台，深入表征伊鲁丁S和rugulactone。 该平台不仅包括用于靶标鉴定的化学蛋白质组学工具集，还包括用于功能表征的下游方法。Illudin S具有有效和有趣的抗癌特性-特别是针对多药耐药癌细胞系。 虽然DNA烷基化可能是其主要作用机制，但其在真核细胞中的蛋白质靶点的身份仍然不清楚。 这些蛋白质靶标可能有助于抗肿瘤活性以及不需要的毒性。在细胞系统中的应用illudin探针将允许我们使用基于质谱的蛋白质组学平台来识别蛋白质靶点。使用这个平台，我们以前已经建立了ThiD -一种在细菌硫胺素生物合成的必需酶-作为rugulactone的目标，我们在这里提出进一步表征ThiD。结构分析将揭示rugulactone抑制ThiD的模式。通过基因缺失研究和定量PCR实验进行功能注释。这些研究也将应用于确定的目标的伊鲁定。此外，抑制细菌代谢途径是一种有价值的方法，以新的抗菌靶标。 例如，细菌叶酸生物合成的抑制是抗菌治疗的关键靶标，并且已经使用了60多年。与叶酸（维生素B 9）类似，硫胺素（维生素B1）是所有生物体中必不可少的辅助因子，虽然大多数微生物可以从头合成硫胺素，但人类和其他动物仅依赖于饮食摄入来获得硫胺素。因此，抑制硫胺素生物合成途径中的必需酶对于急需的新型抗菌靶标具有巨大的潜力。 ThiD已被鉴定为结核分枝杆菌（MT）（结核病（TB）的病原体）中的体内必需酶。MT不包含硫胺素补救或运输的基因，使这种生物体完全依赖于这种必需维生素的从头合成。我们认为MT ThiD的抑制是发现和开发新型抗结核药物的一个有吸引力的靶点。使用rugulactone作为进一步SAR努力的先导结构，我们建议探索MT ThiD的抑制作用，并评估其作为新型药物靶标的用途。

Natural products have found use in human medicine for millennia, and to date, most modern medicines are based on natural products. These compounds typically act on multiple protein targets, yet the identity of most of the protein targets for these molecules remains unknown. Identification of the protein targets of bioactive natural products can provide valuable information concerning the mode of action of these molecules, as well as provide inspiration for novel drug targets and lead structures. In this proposal, we describe a multidisciplinary platform for the in depth characterization of illudin S and rugulactone. This platform includes not only the chemoproteomic toolset for target identification but also incorporates downstream methodologies for functional characterization. Illudin S possesses potent and interesting anticancer properties - particularly against multidrug resistant cancer cell lines. While DNA alkylation is likely its primary mechanism of action, the identity of its protein targets in eukaryotic cells remains obscure. These protein targets may contribute to antitumor activity, as well as unwanted toxicity. Application of illudin probes in cellular systems will allow us to identify the protein targets using a mass spectrometry-based proteomic platform. Using this platform, we have previously established ThiD - an essential enzyme in bacterial thiamin biosynthesis- as a target of rugulactone, and we here propose further characterization of ThiD. Structural analysis will reveal the mode of inhibition of ThiD by rugulactone. Functional annotation will be carried out by gene deletion studies and quantitative PCR experiments. These studies will also be applied to the identified targets of the illudins. Moreover, the inhibition of bacterial metabolic pathways is a valuable approach to novel antibacterial targets. For example, the inhibition of bacterial folate biosynthesis is a key target for antibacterial therapy and has been in use for over 60 years. Much like folate (vitamin B9), thiamin (vitamin B1) is an essential cofactor in all living organisms, and while most microorganisms can synthesize thiamine de novo, humans and other animals rely solely on dietary intake to obtain thiamine. Therefore, inhibition of essential enzymes in the thiamine biosynthetic pathway has great potential for much-needed novel antibacterial targets. ThiD has been identified as an in vivo essential enzyme in Mycobacterium tuberculosis (MT), the causative agent for tuberculosis (TB). MT does not contain the genes for thiamine salvage or transport, making this organism entirely dependent on de novo synthesis for this essential vitamin. We propose that the inhibition of MT ThiD represents an attractive target for the discovery and development of novel antitubercular drugs. Using rugulactone as a lead structure for further SAR efforts, we propose to explore the inhibition of MT ThiD and evaluate its use as a novel drug target.