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

点击翻译按钮获取中文摘要

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.