Isolation and Identification of Eukaryotic Initiation Factor 4A as a Molecular Target for the Marine Natural Product Pateamine A
Natural products continue to demonstrate their utility both as therapeutics and as molecular probes for the discovery and mechanistic deconvolution of various cellular processes. However, this utility is dampened by the inherent difficulties involved in isolating and characterizing new bioactive natural products, in obtaining sufficient quantities of purified compound for further biological studies, and in developing bioactive probes. Key to characterizing the biological activity of natural products is the identification of the molecular target(s) within the cell. The marine sponge-derived natural product Pateamine A (PatA) has been found to be an inhibitor of eukaryotic translation initiation. Herein, we describe the methods utilized for identification of the eukaryotic translation initiation factor 4A (eIF4A) as one of the primary protein targets of PatA. We begin by describing the synthesis of an active biotin conjugate of PatA (B-PatA), made possible by total synthesis, followed by its use for affinity purification of PatA binding proteins from cellular lysates. We have attempted to present the methodology as a general technique for the identification of protein targets for small molecules including natural products.
[Show abstract] [Hide abstract] ABSTRACT: Marine sponges are an excellent source of bioactive secondary metabolites with potential therapeutic value in the treatment of diseases. One group of compounds of particular interest is the microtubule-stabilizing agents, the most well-known compound of this group being paclitaxel (Taxol), an anti-cancer compound isolated from the bark and leaves of the Pacific yew tree. This review focuses on two of the more recent additions to this important class of drugs, peloruside A and zampanolide, both isolated from marine sponges. Peloruside A was isolated from Mycale hentscheli collected in New Zealand coastal waters, and it already shows promising anti-cancer activity. Two other potent bioactive compounds with different modes of action but isolated from the same sponge, mycalamide A and pateamine, will also be discussed. The fourth compound, zampanolide, most recently isolated from the Tongan sponge Cacospongia mycofijiensis, has only recently been added to the microtubule-stabilizing group of compounds, and further work is in progress to determine its activity profile relative to peloruside A and other drugs of this class.0Comments 41Citations
- "To our knowledge, however, no further work using this approach has been published. The cellular target of pateamine was subsequently identified by affinity chromatography to be the protein initiation factor eIF4A727374. By stimulating eIF4A, pateamine inhibits protein synthesis. Interestingly, it was also shown to inhibit nonsense-mediated mRNA decay . "
[Show abstract] [Hide abstract] ABSTRACT: Natural products, and their derivatives and mimics, have contributed to the development of important therapeutics to combat diseases such as infections and cancers over the past decades. The value of natural products to modern drug discovery is still considerable. However, its development is hampered by a lack of a mechanistic understanding of their molecular action, as opposed to the emerging molecule-targeted therapeutics that are tailored to a specific protein target(s). Recent advances in the mass spectrometry-based proteomic approaches have the potential to offer unprecedented insights into the molecular action of natural products. Chemical proteomics is established as an invaluable tool for the identification of protein targets of natural products. Small-molecule affinity selection combined with mass spectrometry is a successful strategy to "fish" cellular targets from the entire proteome. Mass spectrometry-based profiling of protein expression is also routinely employed to elucidate molecular pathways involved in the therapeutic and possible toxicological responses upon treatment with natural products. In addition, mass spectrometry is increasingly utilized to probe structural aspects of natural products-protein interactions. Limited proteolysis, photoaffinity labeling, and hydrogen/deuterium exchange in conjunction with mass spectrometry are sensitive and high-throughput strategies that provide low-resolution structural information of non-covalent natural product-protein complexes. In this review, we provide an overview on the applications of mass spectrometry-based techniques in the identification and characterization of natural product-protein interactions, and we describe how these applications might revolutionize natural product-based drug discovery.0Comments 38Citations
- "Biotin–streptavidin affinity assay was also used to search for protein targets of Pateamine A (PatA), a potent anti-proliferative agent first isolated from marine sponges (Northcote, Blunt, & Munro, 1991). Structure–activity studies have indicated its C-3 amino group as a suitable site for derivatization (Low et al., 2007). Biotin was thus incorporated into PatA via this position, and the probe was screened with RKO cell extracts. "
- [Show abstract] [Hide abstract] ABSTRACT: The survival and proliferation of cancer cells depend largely on the elevation of oncogene expression. Oncogenic expression can be affected by alterations in translation, or protein synthesis. Translation initiation, the rate limiting step of translation, closely regulates protein synthesis which both healthy and cancerous cells require for division and growth. Inhibitors of the eukaryotic translation initiation factor 4F (eIF4F) have been shown to selectively repress the expression of oncogenes in multiple in vitro studies. Although chemotherapeutic application of translation initiation inhibition is in its early stages, promising in vitro results demonstrate the potential of eIF4F inhibitors for use in clinical settings. This review offers a brief overview of the mechanisms that underlie eIF4F inhibitor activity in combating cancer.0Comments 1Citation