pyroside (a natural pyrethrum extract) showed synergism with the mixture giving higher than the additive kill rate. Similarly, against Colorado potato beetles the paw paw extract synergized well with a standardized neem (azadirachtin) extract. Such experiments clearly demonstrated that the acetogenins need not be purified, beyond a crude level of concentration, to produce effective pesticidal products. Furthermore, the application levels can be reduced, using synergistic mixtures, saving money and reducing the environmental loads of individual components.

Evaluation of various parts of the paw paw tree, using the brine shrimp test, identified the small twigs as the optimum plant part for commercial harvest of biomass for extraction;72 thus, by pollarding the trees, the collection of biomass is renewable through regrowth, and the trees are not killed. A subsequent study of paw paw twigs collected from the same tree every month for a year used the brine shrimp test and HPLC/MS/MS to determine that the bioactivity is highest in the month of May, and the concentrations of the major bioactive acetogenins, asimicin (1), bullatacin (6), and trilobacin (7), peak concurrently in May/June;73 thus, seasonal variations can affect the concentrations of phytochemicals, and paw paw biomass is collected in May for commercial purposes.

Neal Peterson has established a beautiful plantation of over 600 paw paw trees at the Western Maryland Regional Education Center (Keedysville, MD); to study the infraspecific variations in biological activity from tree to tree, we used the brine shrimp test to evaluate twig samples collected at Keedysville on the same day from 135 individual trees. Table 1 lists the highest and lowest acetogenin producers and illustrates that the trees can vary up to 1000 times in twig potency.[74] These results are important to keep in mind when making plant collections for phytochemical work: one should collect materials from as many individual plants as possible to avoid the collection of those genotypes that are low producers. The highest producing genotypes of paw paw are, thus, available for grafting and/or clonal reproduction through plant tissue cultures.

Londerhausen et al.75 initially observed that the toxicities caused by the annonaceous acetogenins in insects resulted in lethargy and decreased mobility prior to death; treated insects had substantially lower levels of adenosine triphosphate (ATP), similar to the effects of antimycin A, a known inhibitor of the mitochondrial electron transport system (ETS). Mitochondrial enzymes were tested, and the acetogenins were 2.5 to 5 times as potent as rotenone in inhibiting complex I (NADH:ubiquinone oxidoreductase). Concurrently, at Thor Arnason's laboratory at the University of Ottawa, Lewis et al.76 observed a lower level of oxygen consumption in treated European corn borer larvae and located the site of action of asimicin (1) and paw paw extract at mitochondrial complex I. Meanwhile, at Bob Hollingworth's laboratory at Michigan State University, bullatacin (6) was tested in SF9 insect cells, with mitochondria from rat liver and Manduca sexta, and with complex I isolated from beef heart and arrived at the same conclusion.77 This group later determined that 6 is among the most potent of the known inhibitors of complex I.78 Other workers have found that the acetogenins bind competitively with respect to the ubiquinone site at complex I.79,80 Hiroko Shimada, in my laboratory, prepared deuterated liposomes and used intermolecular nuclear Overhauser effects with 1H NMR spectroscopy to investigate and possibly predict the orientation and positioning of the acetogenins in biological membranes.81,82 Jennifer Landolt, Trina Colman, and Dorothe Alfonso isolated rat liver mitochondria to determine the structure—activity relationships (SAR) of the acetogenins at the subcellular level.83,84

1315

Other workers have found that the acetogenins bind competitively with respect to the ubiquinone site at complex I.79,80 Hiroko Shimada, in my laboratory, prepared deuterated liposomes and used intermolecular nuclear Overhauser effects with 1H NMR spectroscopy to investigate and possibly predict the orientation and positioning of the acetogenins in biological membranes.81,82 Jennifer Landolt, Trina Colman, and Dorothe Alfonso isolated rat liver mitochondria to determine the structure–activity relationships (SAR) of the acetogenins at the subcellular level.83,84

Kan He et al.85 determined the SARs among 44 of the acetogenins in yellow fever mosquito larvae and in the brine shrimp test. In general, the optimum activities, in most of the SAR bioassay test systems, reside in the C37 acetogenins bearing bis-THF rings with flanking hydroxyls, usually at the C-15 to C-24 positions, and a third hydroxyl somewhere along the hydrocarbon chain; the stereochemistry of the ring systems was not as important as one might think. We supplied a set of 22 acetogenins to Hideto Miyoshi at Kyoto University for SAR determinations in submitochondrial preparations of complex I; to our surprise, most of the compounds were similarly potent, suggesting that the intact mitochondrial membrane plays a crucial role in determining the SARs.86 Molecular pharmacologists who attempt to design drug screens using isolated enzymes that are naturally embedded in complex lipid membranes should, perhaps, take note. Useful SARs are determined only when the membranes are intact.

Using bullatacin (6) supplied by us, Schuler et al.87 at the University of California (Berkeley) localized the acetogenin binding site in complex I to the 23-kDa PSST subunit. Miyoshi et al.88 synthesized a photoreactive acetogenin analogue that binds to a 30 kDa protein that is in the ND1 subunit of complex I; other complex I inhibitors, such as piericidin A and rotenone, efficiently suppressed the binding of the acetogenin analogue, indicating that they all share a common binding domain.

Jim Morré at Purdue University has shown that bullatacin (6) potently inhibits the NADH oxidase that is found in the plasma membranes of tumor cells;89 this enzyme permits the tumor cell to produce ATP under anaerobic conditions by restoring NAD levels and permitting glycolysis and substrate level phosphorylation to continue. Thus, inhibition of ATP production is the result of both of the proposed actions of the acetogenins, and both aerobic (oxidative) and anaerobic (substrate level) phosphorylations are being inhibited. ATP depletion, as seen in the early studies with insects,75 occurs, and apoptosis (programmed cell death) would be expected to be the result.90 Indeed, Bob Geahlen at Purdue University has demonstrated DNA laddering (apoptosis) in cancerous human B-lymphocytes that had been treated with bullatacin (6). and 6 has now been reported to induce apoptosis.91

As mentioned above, uvaricin (4), the first acetogenin to be reported, was isolated by monitoring the fractionation with the 3PS in vivo murine leukemia assay (157% T/C at 1.4 mg/kg).27 Asimicin (1), rolinone, and rolliniastatin, other early isolated acetogenins, were also determined to be 3PS actives (124% T/C at 25 μg/kg, 147% T/C at 1.4 mg/kg, and 128% T/C at 250 μg/kg).29 However, with the demise of in vivo testing at the NCI in the 1980s, we decided to seek help at the pharmaceutical companies to secure additional in vivo results. Pat McGovren at the Upjohn Corporation (Kalamazoo, MI) determined that asimicin (1), bullatacin (6), and the bullatacinones (5) were all active in conventional mice bearing ip implanted L-1210 leukemia (131% T/C at 200 μg/kg, 138% T/C at 50 μg/kg, and 144% T/C at 400 μg/kg).77 Taxol (paclitaxel) gives 139% T/C at 15 000 μg/kg against L-1210, so bullatacin (6) was 300 times as potent as taxol in this in vivo test system. Also, the mice treated with taxol lost 10% of their body weight during the 10-day test period, while the bullatacinone-treated mice gained 5%, suggesting, potentially, less toxicity than taxol.