COMMENTARY

Tracking the cryptic pumiliotoxins Stanton Q. Smith and Tappey H. Jones* Department of Chemistry, Virginia Military Institute, Lexington, VA 24450

lthough the concept of chemical prospecting, cataloging the chemical expression of an ecosystem, is gaining credence, exceptional biological activity observed in particular groups of plants, animals, or microorganisms has always attracted the attention of biologists and chemists. A fascinating example is the account of the alkaloids found in the skins of the ‘‘poison dart’’ frogs. First documented in the early 19th century, the chemistry and biological activity of the toxic skin secretions of these tropical frogs have been the primary interest of John W. Daly for nearly 40 years (1–3). This work has uncovered ⬎20 classes of alkaloids, the biological properties of which vary from highly toxic, cardiotonic, or anesthetic to nearly inactive. For the most part, the presence of these compounds depends on where the frogs are collected, and a dietary hypothesis has been put forth for their sequestration (1). The source of one of the most active of these classes, the pumiliotoxins, has remained a mystery. The work of Saporito et al. (4) in this issue of PNAS demonstrates the presence of the pumiliotoxins in two genera of formicine ants, noteworthy because of their ubiquity in poison dart frogs and their physiological activity. Amphibian alkaloids with the largest diversity are found in the order Anura (frogs and toads), and the skin extracts from frogs of the Dendrobatidae family are an especially fruitful source of alkaloids. The frogs are notoriously toxic, and the natives of Western Columbia still use skin secretions from three Columbian species of dendrobatid frogs (genus Phyllobates) to poison the grooved tips of blow darts used in hunting small birds and game (1). In the mid-1960s the first group of these alkaloids, the highly toxic steroidal batrachotoxins, were identified (5). Batrachotoxin prevents the closing of sodium-ion channels in the surface membrane of muscle and nerve cells, inhibiting their function (6). Other physiologically active alkaloids elucidated since then include epibatidine, a potent analgesic (7, 8), and the pumiliotoxins, having marked myotonic/cardiac activity (3). Although not extremely toxic, the remaining alkaloids in the other classes almost certainly serve as a repellant to predation. Possibly the most widespread class of alkaloids from toads and frogs are the

A

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Fig. 1.

Pumiliotoxins and sources.

⬎180 pumiliotoxins found in virtually all anurans that contain defensive skin compounds (3). The primary impetus for the study of pumiliotoxins is their remarkable bioactivity. These compounds are potent myotonic/cardiotonic agents with modulatory effects on sodium channels (6). Pumiliotoxin B (323A) from the neotropical frog (see Fig. 1) Dendrobates pumilio has marked myotonic and cardiotonic activity (9). Pumiliotoxin B (323A) releases stored calcium ions, thereby potentiating muscle contraction. It also inhibits the return of calcium ions, prolonging the contractions (6). Pumiliotoxin A (307A) is much less potent as a positive ionotropic agent (9). Interestingly, pumiliotoxin 251D acts as a cardiac depressant (9). The observed bioactivity is very

Despite their ubiquity, the natural source of pumiliotoxins has remained a mystery. closely linked to the structure of the alkaloid. This structure–activity relationship has been an invaluable mechanistic probe for studying the functioning of sodium channels (6, 9). Considering the possible medical applications and the toxicity of the pumiliotoxins, it is no wonder that these alkaloids have generated much chemical and biological interest; however, despite their ubiquity, their natural source has remained a mystery. It has been demonstrated that frogs can sequester alkaloids, including pu-

miliotoxins, from their diet (10–12). Given that ants comprise a large part of the frog diet, parallels and overlaps between ant chemistry and frog chemistry are unavoidable. In the early 1970s, the alkaloids were elucidated in the myrmicine ants Monomorium pharaonis, a common pest species in Europe, and Solenopsis invicta, the imported fire ant in the United States (13–15). These compounds are components of the venom glands of these ants, and in fire ants their antibiotic activity, toxicity, repellency, and pheromonal roles have been investigated (16, 17). Additionally, their ‘‘species-specific’’ nature has been used as a taxonomic character in the large myrmicine genera Monomorium and Solenopsis (18–21). Over the past 20 years, ant venom alkaloids have been detected in frog skin extracts, and compounds previously detected in frogs have been found in ants. For example, simple pyrrolidines and piperidines as well as indolizidines and pyrrolizidines, all described previously in ants, were detected in frog skin extracts (22, 23). Conversely, decahydroquinolines previously described in frogs were detected in a group of small ants called thief ants [Solenopsis (Diplorhoptrum) spp.] (24). Most convincingly, a mixture of two decahydroquinolines and a quinolizidine was found in both an ant and a frog, and of many possible stereoisomers, these compounds even had the same stereochemistry in both animals (25). Although a dietary origin of the pumiliotoxin A alkaloids has been established, finding pumiliotoxins 307A and See companion article on page 8045. *To whom correspondence should be addressed. E-mail: [email protected]. © 2004 by The National Academy of Sciences of the USA

PNAS 兩 May 25, 2004 兩 vol. 101 兩 no. 21 兩 7841–7842

323A in ants is a remarkable result and the first step toward establishing their origin. The genera Brachymyrmex and Paratrechina are tiny ants in Formicidae, a subfamily not known for producing alkaloids. Additionally, in this case the glandular sources, if any, of the pumiliotoxins are not known. In fact, there were no detectable alkaloids of any kind in collections of Brachymyrmex depilis, a species known across the southern United States, from California or in collections of a related species, Brachymyrmex obscurior, from Puerto Rico (T.H.J., G. C. Snelling, J. Torres, and R. R. Snelling, unpublished data). Indeed, Soparito et al. (4) note the irregularity and seasonality of pumiliotoxins in Brachymyrmex cf. depilis. These results suggest that ants might not be the primary source for the pumiliotoxins. It is possible that these al-

kaloids are produced by microbial symbionts, some of which are widely found in ants (26). For example, ants in the pseudomyrmicine genus Tetraponera somewhat inconsistently produce interesting tricyclic alkaloids, ‘‘tetraponerines’’ (27), and it may be that the nitrogen-fixing bacteria found in a specialized pouch in the gut of these ants account for the compounds (28). On the other hand, the interspecific flow of plant alkaloids from plant through aphids to beetles has been demonstrated, and the pumiliotoxins may simply be of dietary origin in Brachymyrmex and aphid-tending Paratrechina (29, 30). In contrast with the acetate-derived myrmicine venom alkaloids, the branched-carbon ‘‘isoprenoid’’ structures of the pumiliotoxins lend support to this alternative. In a larger view, the results of Saporito et al. (4) demonstrate the piv-

otal role of chemical ecology in natural products chemistry, wherein the imperative for increased research has become clear. This necessity is driven by the need for new therapeutic drugs, most of which are either derived from natural sources or are synthetic derivatives of natural compounds (31), and by the threat to biodiversity in many parts of the world, potentially leading to the loss of novel compounds that are expressions of species that might be lost (32). It is clear by the work of Saporito et al. (4) that new biological perspectives and modern analytical instrumentation now provide an unimaginable ability to uncover the roles and structures of those compounds regulating living systems (33).

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We gratefully acknowledge Ralph A. Saporito for providing the figure.

Smith and Jones