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

天然产品在人类医学中的使用已有数千年历史，到目前为止，大多数现代药物都是以天然产品为基础的。这些化合物通常作用于多个蛋白质靶标，但这些分子的大多数蛋白质靶标的身份尚不清楚。鉴定具有生物活性的天然产物的蛋白质靶标可以提供关于这些分子作用方式的有价值的信息，也可以为新的药物靶标和先导结构提供灵感。在这个提案中，我们描述了一个多学科的平台，用于深入描述伊鲁丁·S和芦荟内酯。该平台不仅包括用于靶标识别的化学蛋白质组工具集，还包括用于功能表征的下游方法。伊鲁丁S具有强大而有趣的抗癌特性-特别是对多药耐药癌细胞株。虽然DNA烷基化可能是它的主要作用机制，但它在真核细胞中的蛋白质靶标的身份仍然不清楚。这些蛋白质靶标可能有助于抗肿瘤活性，以及不必要的毒性。在细胞系统中应用伊卢丁探针将使我们能够使用基于质谱学的蛋白质组平台来识别蛋白质靶标。利用这个平台，我们以前已经建立了THID--细菌硫胺素生物合成中的一个关键酶--作为芦古拉内酯的靶标，我们在这里提出了THID的进一步表征。结构分析将揭示芦丁内酯对THID的抑制模式。功能注释将通过基因缺失研究和定量PCR实验进行。这些研究也将应用于已确定的伊鲁丁靶标。此外，抑制细菌代谢途径是寻找新的抗菌靶点的有价值的途径。例如，抑制细菌叶酸的生物合成是抗菌治疗的关键目标，已经使用了60多年。就像叶酸(维生素B9)一样，硫胺素(维生素B1)是所有活着的生物体中必不可少的辅因子，虽然大多数微生物可以从头合成硫胺素，但人类和其他动物完全依靠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关键酶具有极大的潜力，成为亟需的新型抗菌靶点。THID是结核病的病原体--结核分枝杆菌(MT)体内的一种重要酶。MT不包含回收或运输硫胺素的基因，这使得这种有机体完全依赖于这种基本维生素的从头合成。我们认为，抑制MT-THID是发现和开发新型抗结核药物的一个有吸引力的靶点。以芦丁内酯为先导结构，探索MT-thid的抑制作用，并评价其作为新的药物靶点的应用。

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.