Natural Products Cellular Receptor Isolation
Natural products have not only offered important compounds for the discovery and development of new therapeutic agents, particularly antibiotic and anticancer drugs, but have also served as increasingly useful probes to manipulate and elucidate the mechanisms of various biological processes, from signal transduction to cell cycle progression. A rate-limiting step in the use of natural products as probes in biology is the chemical synthesis of useful bioactive probes, such as affinity reagents or fluorescent probes, that can be readily applied by biochemists and cell biologists. Pateamine A Was initially discovered as a potent antiproliferative agent against the murine P388 tumor cell line. Subsequently, it was found to possess potent immunosuppressive activity in animal models and later at the cellular level. Although PatA undoubtedly inhibits T cell receptor-mediated signaling leading to interleukin (IL)-2 production, the overall immunosuppressive effect of PatA is likely to result from its effect on eukaryotic translation initiation and, hence, T lymphocyte proliferation. Nevertheless, the IL-2 reporter assay proved useful in our initial structure/activity studies, which enabled the synthesis of an active biotin-PatA (B-PatA) conjugate, as the potencies of PatA analogs used in the studies were similar to their potency in blocking cell proliferation and translation. In collaboration with Prof. Jun Liu (Johns Hopkins University), We trace the route by combining synthetic organic chemistry and protein biochemistry that led to the detection, isolation, and identification of two PatA-interacting proteins, one of which is the eukaryotic translation initiation factor (eIF)-4A.
1) "Inhibition of Eukaryotic Translation initiation by the Marine Natural Product Pateamine A" Low, W.-K.; Dang, Y.; Schneider-Poetsch, T.; Shi, Z; Choi, N. S.; Merrick, W. C. ; Romo, D. ; Liu, J. O. Mol. Cell, 2005, 20, 709-722. [Highlighted in C&EN, December 19, 2005 and Featured in Chem. Biol. (Viewpoint by J. Clardy), 2006, 1, 17
2) "Isolation and laentification of Eukaryotic Initiation Factor 4A as a Molecular Target for the Marine Natural Product Pateamine A" Low, W.-K.; Dang, Y.; Schneider-Poetsch, T.; Shi, Z.; Choi, N. S.; Rzasa, R. M.; Shea, H. A.; Li, S.; Park, K.; Ma, G. ; Romo, D. ; Liu, J. O. Methods in Enzymology, 2007, 431, 303324.
Translation initiation in eukaryotes is accomplished through the coordinated and orderly action of a large number of proteins, including the eIF4 initiation factors. Herein, We report that pateamine A (PatA), a potent antiproliferative and proapoptotic marine natural product, inhibits cap-dependent eukaryotic translation initiation. PatA bound to and enhanced the intrinsic enzymatic activities of eIF4A, yet it inhibited eIF4A- eIF4G association and promoted the formation of a stable ternary complex between eIF4A and eIF4B. These changes in eIF4A affinity for its partner proteins upon binding to PatA caused the stalling of initiation complexes on mRNA in vitro and induced stress granule formation in vivo. These results suggest that PatA will be a valuable molecular probe for future studies of eukaryotic translation initiation and may serve as a lead compound for the development of anticancer agents.
3) "Substrate-Dependent Targeting of Eukaryotic Translation Initiation Factor 4A by Pateamine A. Negation of Domain-Linker Regulation of Activity" Low, W.-K.; Dang, Y.; Bhat, S.; Romo, D.; Liu, J. O. Chem. & Biol., 2007, 14, 1-13.
Central to cap-dependent eukaryotic translation initiation is the eIF4F complex, which is composed of the three eukaryotic initiation factors eIF4E, eIF4G, and eIF4A. eIF4A is an RNA-dependent ATPase and an ATP-dependent helicase that unwinds local secondary structure in mRNA to allow binding of the 43S ribosomal complex. The marine natural product pateamine A (PatA) has been demonstrated to inhibit cap-dependent initiation by targeting eIF4A and disrupting its protein-protein interactions while increasing its enzymatic activities. Here We demonstrate that the increased activity is caused by the induction of global conformational changes within eIF4A. Furthermore, binding of PatA is dependent on substrate (RNA and ATP) binding, and the increased activity upon PatA binding is caused by relief of a negative regulatory function of the eIF4A unique domain linker.
4) “Inhibition of Nonsense-mediated mRNA Decay by the Natural Product Pateamine A Through Eukaryotic Initiation Factor 4AIII" Dang, Y.; Low, W-K., Xu, J.; Gehring, N. H. ; Dietz, H. C. ; Romo, D.; Liu, J.O. J. Biol. Chem., 2009, 35, 23613-23621.
Nonsense-mediated mRNA decay (NMD) in mammalian cells is a key mechanism for the removal of mRNA containing premature stop codons and is mediated by the coordinated function of numerous proteins that dynamically associate with the exon junction complex. The information communicated by these interactions and the functional consequences from a mechanistic perspective, however, are not completely documented. Herein, we report that the natural product pateamine A (PatA) is capable of inhibiting NMD through direct interaction with eIF4All, which is independent of its inhibition of translation initiation. Furthermore, we have characterized the mechanisms by which PatA and cycloheximide modulate NMD. Unlike CHX, PatA was found to inhibit NMD by a novel mechanism that is independent of the phosphorylation of Up-frameshift protein 1.