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Eukaryotic initiation factor 4A (eIF4A) is a member of the DEAD-box family of putative RNA helicases whose members are involved in many aspects of RNA metabolism. eIF4A is thought to facilitate binding of 43S preinitiation complexes to mRNAs by unwinding secondary structures present in the 5' untranslated region. Pateamine A, a small-molecule inhibitor of translation initiation, acts in an unusual manner by stimulating eIF4A activity. Herein, we report the elucidation of pateamine's mode of action. We demonstrate that Pateamine A is a chemical inducer of dimerization that forces an engagement between eIF4A and RNA and prevents eIF4A from participating in the ribosome-recruitment step of translation initiation.

RNA helicases are the largest group of enzymes in eukaryotic RNA metabolism. The DEXD/H-box putative RNA helicases form the helicase superfamily II, whose members are defined by seven highly conserved amino acid motifs, making specific targeting of selected members a challenging pharmacological problem. The translation initiation factor eIF4A is the prototypical DEAD-box RNA helicase that works in conjunction with eIF4B and eIF4H and as a subunit of eIF4F to prepare the mRNA template for ribosome binding, possibly by unwinding the secondary structure proximal to the 5' m7GpppN cap structure. We report the identification and characterization of a small molecule inhibitor of eukaryotic translation initiation that acts in an unusual manner by stimulating eIF4A-associated activities. Our results suggest that proper control of eIF4A helicase activity is necessary for efficient ribosome binding and demonstrate the feasibility of selectively targeting DEAD-box RNA helicases with small molecules.

Intraspecific variation in the composition of three cytotoxic secondary metabolites from the New Zealand marine sponge Mycale hentscheli collected at two sites in central New Zealand was quantified by 1H NMR techniques. A total of 275 sponges were analyzed bimonthly over 15 mo to compare intersite (approximately 100 km) and intrasite (approximately 100 m) spatial and temporal variations in the metabolites. Biological and physical characteristics of sponge size, morphology, depth, and temperature were recorded at each site. Metabolite concentrations were found to vary in space and time. Metabolite composition was site-specific; mycalamide A, pateamine, and peloruside A were present at Pelorus Sound, whereas pateamine was absent from sponges at Kapiti Island. Pateamine and peloruside A concentrations in sponges at Pelorus Sound varied seasonally; no such patterns were observed at Kapiti Island. Relationships of compound concentration with volume and depth were complex. High levels of peloruside A in Pelorus Sound sponges from between 8 and 10 m depth coincided with a density boundary layer and chlorophyll a maximum.

Peloruside A (peloruside), a compound isolated from the marine sponge Mycale hentscheli , inhibits growth of human (HL-60) and mouse (32D-ras) myeloid leukemic cells, as well as non-transformed 32D cells. Using the MTT cell proliferation assay and trypan blue dye exclusion tests, little difference was seen in growth inhibition between 32D and 32D- ras cells; however, peloruside was more cytotoxic to the oncogene-transformed cells. Peloruside also blocked 32D- ras cells more readily in G2/M of the cell cycle, leading to apoptosis. Annexin-V/propidium iodide staining of 32D and 32D- ras cells showed that 1.6 microM peloruside induced significant cell death by 36 hours in 32D cells (16% survival), but to comparable levels as early as 14 hours in 32D- ras cells (11% survival). There was no evidence for activation of either of the initiator caspases-8 or -9 by 0.1 microM peloruside following 12 hours of exposure. Peloruside inhibited T cell proliferation and IL-2 and IFN gamma production in both the mixed lymphocyte reaction and following CD3 cross-linking, and this effect was shown to be a non-specific cytotoxic effect. It is concluded that peloruside preferentially targets oncogene-transformed cells over non-transformed cells by inducing transformed cells to undergo apoptosis.

A neuroblastoma assay for determination of brevetoxins in shellfish was developed together with a method for sample cleanup that allows separation of brevetoxins from most of the components that cause matrix interference in the assay. This improved assay method was applied to a range of shellfish samples with different characteristics. Extracts of naturally contaminated and nontoxic shellfish together with extracts spiked with known amounts of toxin were tested. The results demonstrated that brevetoxins could be reliably detected in shellfish extracts at concentrations below the regulatory limit. Brevetoxin activity was detected in 15 of 23 samples from 5 separate toxicity incidents in which shellfish tested positive in the neurotoxic shellfish poisoning (NSP) mouse bioassay. Twelve of these positive NSP results came from 2 toxicity incidents. Yessotoxin was the major contributor to toxicity in 2 other incidents, although some samples contained both yessotoxin and brevetoxin. The sample from the remaining incident contained an unidentified toxin bioactivity, together with gymnodimine. In contrast to earlier versions of the neuroblastoma assay, gymnodimine was not detected by this modified method.

The marine sponge metabolites mycalamide A (mycalamide) and pateamine are extremely cytotoxic. While mycalamide has been shown to inhibit protein synthesis, the mechanism by which these compounds induce cell death is unknown. Using DNA laddering, Annexin-V staining, and morphological analysis, we demonstrate that both metabolites induce apoptosis in several different cell lines. Furthermore, both mycalamide and pateamine were more potent inducers of apoptosis in the 32D myeloid cell line after transformation with either the ras or bcr-abl oncogenes. This increased sensitivity was also observed in response to the protein synthesis inhibitors cycloheximide and puromycin, and cytosine-beta-D-arabinofuranoside (Ara-C), an inducer of DNA damage. We propose, therefore, that in 32D cells where Ras signalling has been altered either by constitutive expression of oncogenic ras or by Bcr/abl-mediated perturbation of upstream signalling events, increased susceptibility to apoptosis by a range of stimuli is conferred.

A novel secondary sponge metabolite, peloruside A (peloruside), isolated from the marine sponge Mycale sp. (New Zealand), was tested for its cytotoxic effects on mammalian cells in culture. The macrolide structure of peloruside is similar to that of the protein kinase C (PKC) activator, bryostatin-1 (bryostatin), both containing a pyranose ring adjacent to a gemdimethyl moiety. Peloruside is a potent inhibitor of cell proliferation. Treatment of different mammalian cell lines with peloruside for 48-96 h gave IC50 values ranging from 4 to 15 nM, using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium (MTT) cell proliferation assay. Peloruside was shown to be both cytostatic and cytotoxic by trypan blue dye exclusion tests. Peloruside induced apoptosis in a dose-dependent manner in murine (32D) and human (HL-60) myeloid cell lines, revealed by DNA laddering in agarose gels and flow cytometric analysis of annexin-V- and propidium iodide-stained cells. Treatment of HL-60 cells caused vacuolisation, partial substrate adherence, and the appearance of multi-lobed nuclei, suggesting the induction of a differentiation pathway. Vacuolisation was also observed in a human lung cancer cell line (H441). Opening of the pyranose ring of peloruside by sodium borohydride reduction increased the 48 h IC50 value by 26-fold in 32D cells, suggesting a similar active site to that proposed for bryostatin. However, unlike bryostatin, peloruside failed to bind to PKC in HL-60 cells and was unable to synergize with the calcium ionophore, ionomycin, or with interleukin-2, to activate T-lymphocytes in culture. In summary, although structurally similar to bryostatin, peloruside is a potent inhibitor of cell proliferation, has apoptosis-inducing properties and has a unique mode of action independent of PKC.

Pyrroindomycins A and B were isolated from fermentations of culture LL-42D005, a strain of Streptomyces rugosporus. Pyrroindomycins possess potent antimicrobial activities against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci. Their structures have been determined by using 1- and 2-D NMR, mass spectroscopy and chemical degradations. Pyrroindomycins are the first natural products that contain the highly unsaturated pyrroloindole moiety.