Article

Isolation and identification of eukaryotic initiation factor 4A as a molecular target for the marine natural product Pateamine A

Department of Pharmacology, Johns Hopkins School of Medicine, Baltimore, Maryland, USA.
Methods in Enzymology (Impact Factor: 2.19). 02/2007; 431:303-24. DOI: 10.1016/S0076-6879(07)31014-8
Source: PubMed

ABSTRACT 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.

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    • "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 eIF4A [72–74]. By stimulating eIF4A, pateamine inhibits protein synthesis. "
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    • "ction mechanism of EKA . 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 . MALDI - MS analysis revealed eIF4A and the serine / threonine kinase receptor - associated protein as the targets of PatA ( Low et al . , 2005 ) . eIF4A participates in eukaryotic translation initiation ( Gingras , Raught ,"
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