Stopping Trouble before It Starts Jon Clardy* Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115
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he translation of mRNA into proteins is a fundamental, tightly controlled, and complex process involving an elaborate molecular machine with both ribonucleic acids and proteins (Figure 1). Our understanding of translation has benefited from several small molecules that stall the process, many of which have become drugs. Many common antibiotics, such as tetracycline and erythromycin, stall prokaryotic translation in the processive stages where new amino acids are added to a growing chain, while cycloheximide, a fungicide, and anisomycin, an antiprotozoal, stall eukaryotic translation in the processive stages. A very recent publication (1 ) reports the first selective inhibition of prokaryotic initiation by the curious naturally occurring peptide GE81112 (Figure 2, 2) from Streptomyces sp. Eukaryotic initiation (Figure 1) involves several initiation factors such as the eIF4 complex in which eIF4A, eIF4E, and eIF4G play critical roles. The precise role of eIF4A, which is both an RNA-dependent ATPase and an ATP-dependent RNA helicase, is not known. Now, Liu et al. (2 ) show that pateamine A (Figure 2, 1) interferes with the interaction between eIF4A and eIF4G (Figure 1). These findings open the door for mechanistic studies of eIF4A and once again show the utility of natural products in understanding complex processes. A brief review of the discovery and development of Taxol (Figure 2, 3), a natural product that became an important anticancer drug, provides a historical perspective for this latest revelation. Taxol (Figure 2, 3) illustrates both the enormous potential of natural products, www.acschemicalbiolog y.o rg
that is, identifying new targets and providing the basis for new therapies, along with their frustrating liabilities, that is, identifying their mechanism of action, assuring an adequate supply, and dealing with a molecular template ill-suited to traditional medicinal chemistry (3 ). Work on the molecule that was to become Taxol (Figure 2, 3) began in 1965 when a large-scale screening effort by the National Cancer Institute (NCI) found that a crude extract of the Pacific yew (Taxus brevifolia) had in vivo activity in a mouse leukemia model (L1210 and P388; 4 ). The active compound, Taxol, was isolated and identified in 1969, although it was not published until 1971 (4 ). At that time, Taxol was just one of many possible leads that had come out of the NCI program, but with the 1975 discovery of its pronounced activity against B16 melanoma, it became a development candidate. When the Horwitz laboratory defined Taxol’s mechanism of action as promoting tubulin polymerization and stabilizing microtubules against depolymerization in 1979, drug development began in earnest. Although Taxol entered phase I clinical trials in 1983, it did not receive approval from the Food and Drug Administration (FDA) until 1992, a delay attributable both to supply and formulation issues (4 ). Taxol was first marketed in 1993; in the next 10 years, it had sales of $9 billion, and in 2005, its sales will be a little over $1 billion. Pateamine A has now taken a significant step along this path. In 1991, the Munro and Blunt laboratory reported its isolation from a sponge (Mycale sp.) collected off
A B S T R A C T A recent publication revealing that the cytotoxic marine natural product pateamine A targets eukaryotic initiation factor eIF4A continues a story with lessons for both chemists and biologists, that is, the significance of natural products, the importance of synthetic organic chemistry, the small molecule regulation of eukaryotic translation machinery, and possibly a new approach to cancer chemotherapy.
*To whom correspondence should be addressed. E-mail: jon_clardy@ hms.harvard.edu
Published online February 17, 2006 10.1021/cb0600029 CCC: $33.50 © 2006 American Chemical Society
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Figure 1. The role of the eIF4 complex in eukaryotic translation. The 5´-end of eukaryotic mRNAs contains a 7-methyl guanosine (m7G) cap structure that serves as a scaffold for the formation of a large protein complex containing various eukaryotic initiation factors (eIFs) including eIF4A, eIF4E, eIF4G, and PolyA binding protein (PABP). This complex brings the 5´- and 3´‑ends of the mRNA in proximity and marks the mRNA for translation. The role of eIF4A in this complex is not clear, but it is known to bind to eIF4G. The 43S pre-initiation complex containing the 40S ribosomal subunit, initiator tRNA (stick model), eIF1, eIF1A, eIF2, eIF3, and eIF5 joins the cap complex, and scanning for the start codon (AUG) begins. Upon encountering the start codon, the complex is remodeled, the 60S large ribosomal subunit joins the process, and translation of the mRNA into protein begins.
the coast of New Zealand (5 ). The isolation scheme followed pateamine A’s activity against P388 leukemia (using cell lines, not mice) along with additional studies that highlighted its selective cytotoxicity for rapidly growing cells (5 ). At that time, pateamine A was just one of many sponge metabolites with selective cytotoxicity against cancer cell lines, an unknown target, and an uncertain source of supply. In 1995 the Romo laboratory set about exploring the chemistry of pateamine A 18
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(Figure 2, 1) with two related goals: (1 ) find simpler active analogues and (2 ) identify pateamine A’s cellular target. Key discoveries in this decade-long effort were an active compound lacking both the C-3 amino and C-5 methyl groups and close analogues with no activity, and the preparation of these analogues with deep-seated structural changes depended on efficient total syntheses. The ability to acylate the C-3 amino group without loss of activity led to the synthesis of a biotin-linked affinity
reagent (Figure 2, 4). In the Liu laboratory, this affinity reagent (Figure 2, 4) was used to identify two potential targets: STRAP, a serine-threonine kinase receptorassociated protein, and eukaryotic initiation factor eIF4A (2 ). The inability of the inactive pateamine A analogues to displace pateamine in the STRAP and eIF4A complexes confirmed the specificity of the binding. A study using HeLa-derived cell lines overexpressing each protein showed increased resistance to pateamine A for the eIF4A line, and no increased resistance for the STRAP line indicated that the physiologically relevant target was eIF4A. An indepen dent study (6 ) using a high-throughput screen for protein synthesis inhibitors and secondary assays by the Pelletier laboratory reached the same conclusion a few months earlier. While agreeing on the target, the two laboratories differ on the detailed mechanism by which pateamine A disrupts protein synthesis. These studies suggest the following two things: (1 ) our ability to identify cellular targets for small molecules identified in simple cytotoxicity screens has not improved, as identifying the targets for Taxol and pateamine A both took roughly a decade and (2 ) including natural products in high-content screens more efficiently links natural products, biological activities, and targets. Pateamine A is the first published (7 ) ligand to specifically modify eIF4A-based activity, and its discovery will undoubtedly lead to an improved understanding of eIF4A’s role in protein translation. Somewhat paradoxically, pateamine A enhances both catalytic activities of eIF4A but inhibits protein translation. Low et al. resolved the paradox by using standard molecular biology techniques to show that pateamine A disrupts the formation of the eIF4 complex by decreasing the interaction between eIF4A and eIF4G (2 ). Further studies, which will undoubtedly employ pateamine A and its analogues, will be needed to precisely define eIF4A’s role in w w w. a c s c h e m i ca l biology.org
initiation and its regulation by small molecules. After the initial discovery of Taxol as a tubulin-interacting agent, many other tubulin-interacting molecules with very different molecular structures were discovered, and it is likely that many small molecules that target eIF4A will now emerge. Pateamine A and its analogues could conceivably be drug development candidates, but development will have to overcome the current move towards targets specific to cancer cells (think Gleevec) and away from targets with widespread and essential activities such as protein synthesis. One could argue the case for pateamine A by noting that Taxol targets the widespread and essential cellular micro-
tubules, but trends are changing as more specific drugs show encouraging clinical efficacy. If development of an anticancer drug based on pateamine A, or possibly pateamine A itself, proceeds, supply will be a major concern. Taxol supply was a major focus of the organic chemistry community with total synthesis, semisynthesis, and extraction from Pacific yew all contributing. Eventually, both extraction and semisynthesis starting with more readily available plant metabolites provided the most economical solution (4 ). Pateamine A represents a much bigger challenge. Marine sponges have proved to be notoriously difficult sources to recollect, and even the original paper noted the sporadic occurrence of pateamine A in Mycale sponges. However, recent work suggests that many so-called sponge metabolites are biosynthesized by bacterial symbionts, and either these bacterial producers can be cultured in the laboratory or their biosynthetic genes can be placed into alternative hosts for heterologous expression. Two papers last year on the spongederived anticancer agent patellamide Figure 2. Pateamine A (1), which disrupts translation at the initiation (Figure 2, 5) illusstage by binding to eIF4A, was isolated from a marine sponge Mycale sp. GE81112 (2), which disrupts prokaryotic translations, was trate the power of isolated from a Streptomyces sp. Taxol (3), which binds to tubulin, was the latter approach isolated from the Pacific yew (Taxus brevifolia). The affinity reagent (8, 9 ). The one sure (4) was used to identify the cellular target of pateamine A. Patellamide prediction is that (5), which has shown some promise as an anticancer agent, was originally isolated from a marine sponge, but later work showed that it the pateamine A was produced by a cyanobacterial symbiont. story will continue.
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REFERENCES 1. Brandi, L., Fabbretti, A., La Teana, A., Abbondi, M., Losi, D., Donadio, S., and Gualerzi, C. O. (2006) Specific, efficient, and selective inhibition of prokaryotic translation initiation by a novel peptide antibiotic, Proc. Natl. Acad. Sci. U.S.A. 103, 39–44. 2. Low, W. K., Dang, Y., Schneider-Poetsch, T., Shi, Z., Choi, N. S., Merrick, W. C., Romo, D., and Liu, J. O. (2005) Inhibition of eukaryotic translation initiation by the marine natural product pateamine A, Mol. Cell 20, 709–722. 3. Clardy, J., and Walsh, C. (2004) Lessons from natural molecules, Nature 432, 829–837. 4. Cragg, G. M. (1998) Paclitaxel (Taxol): a success story with valuable lessons for natural product drug discovery and development, Med. Res. Rev. 18, 315–331. 5. Northcote, P. T., Blunt, J. W., and Munro, M. H. G. (1991) Pateamine: a potent cytotoxin from the New Zealand marine sponge, Mycale sp., Tetrahedron Lett. 32, 6411–6414. 6. Bordeleau, M. E., Matthews, J., Wojnar, J. M., Lindqvist, L., Novac, O., Jankowsky, E., Sonenberg, N., Northcote, P., Teesdale-Spittle, P., and Pelletier, J. (2005) Stimulation of mammalian translation initiation factor eIF4A activity by a small molecule inhibitor of eukaryotic translation, Proc. Natl. Acad. Sci. U.S.A. 102, 10460–10465. 7. Justman, C. J. (1999) Torreyanic acid: affinity chromatographic identification of receptors and biochemical analysis of the torreyanic acid‑eIF4A complex, Ph.D. Thesis, Harvard University, Cambridge, MA. 8. Schmidt, E. W., Nelson, J. T., Rasko, D. A., Sudek, S., Eisen, J. A., Haygood, M. G., and Ravel, J. (2005) Patellamide A and C biosynthesis by a microcin-like pathway in Prochloron didemni, the cyanobacterial symbiont of Lissoclinum patella, Proc. Natl. Acad. Sci. U.S.A. 102, 7315–7320. 9. Long, P. F., Dunlap, W. C., Battershill, C. N., and Jaspars, M. (2005) Shotgun cloning and heterologous expression of the patellamide gene cluster as a strategy to achieving sustained metabolite production, ChemBioChem 6, 1760–1765.
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