Axel H. Schönthal

Department of Molecular Microbiology and Immunology, K. Norris Jr. Comprehensive Cancer Center, University of Southern California, 2011 Zonal Ave., HMR-405, Los Angeles, CA 90033

Received 8/25/98 Accepted 12/2/98

3. NATURAL PRODUCT INHIBITORS OF PHOSPHATASES

One compound that has proven to be extremely useful for the study of gene regulation by phosphatases is okadaic acid, a complex polyether derivative of a 38-carbon fatty acid. It is synthesized by marine dinoflagellates and accumulates in filter feeding organisms such as shellfish or the black sponge Halichondria okadaii from which it was first isolated (9, 10). Okadaic acid is a recognized threat to human health through its ability to cause diarrhetic shellfish poisoning. Its only cellular targets that could be identified so far are certain members of the serine/threonine protein phosphatase family, including PP2A (see table 1). Okadaic acid binds to the catalytic subunit and inhibits its enzymatic activity (11). Because of this specific repression of phosphatase activity, okadaic acid quickly became a ubiquitous tool to investigate the cellular functions of the respective okadaic acid-sensitive phosphatases (12).

Okadaic acid inhibits different phosphatases differentially, i.e. the concentration of the drug that inhibits phosphatase activity by 50% (IC50) varies greatly among the different members of this enzyme family (see table 1). Cellular effects that are observed in response to low concentrations of okadaic acid are often contributed to the inhibition of PP2A, as this particular phosphatase is inhibited at subnanomolar concentrations of the drug. However, this conclusion could be misleading, because there are other, less abundant phosphatases that are also affected by low okadaic acid concentrations, such as PP4 and PP5 (see table 1). Furthermore, PCR analysis indicated there are more phosphatases of this type yet to be discovered (25). Therefore, the cellular or molecular consequences of okadaic acid treatment, even at low concentrations, cannot be ascribed unequivocally to the inhibition of one particular phosphatase (12, 26).

In addition to okadaic acid, several other naturally occurring compounds have been found that are also able to inhibit phosphatase activity (see table 2). Although these inhibitors constitute a structurally diverse group of toxins that are produced by different organisms, computational analysis revealed that many of them possess similar three-dimensional motifs that are involved in binding to the phosphatase catalytic subunit (14, 27, 28). As is the case with okadaic acid, the inhibitory potency of the different compounds varies greatly among the different types of phosphatases. For example, the IC50 of tautomycin is approximately 10-fold higher for PP2A than for PP1, whereas the reverse is true for microcystin; fostriecin inhibits PP2A >10,000-fold more potently than PP1 (see refs. in table 2). Because of these differential effects, the combinatorial use of various phosphatase inhibitors may prove helpful to further narrow the list of candidate phosphatases that may be involved in the cellular processes under investigation (29).

It has to be kept in mind, however, that there is significant variation among the published IC50 values, which are dependent on the concentration of phosphatase as well as on the type of substrate used (17). Furthermore, these values are derived from measurements performed in vitro, i.e. by the use of cellular lysates or purified enzymes. To study the role of phosphatases in signal transduction pathways and gene regulation, these inhibitors need to be added to cells in culture. In this case, however, the efficient concentrations are significantly higher, and therefore the IC50 values do not reliably apply to these cell culture conditions. For instance, the published IC50 for okadaic acid is around 1 nM with respect to PP2A activity in vitro (when added to diluted cellular lysate) (11, 15, 30), whereas in cell culture (when added to growing cells) the IC50 was found to be 30 nM and nearly 1 microM for NIH3T3 fibroblasts and MCF-7 breast cancer cells, respectively (45, 46).

Another caveat has to be considered when interpreting results obtained with the use of phosphatase inhibitors. It cannot be completely excluded that these compounds exhibit certain effects on cellular processes due to their potential interaction with yet unknown, non-phosphatase targets. Okadaic acid has been extensively studied, and no other cellular targets have been identified so far. However, some of the other phosphatase inhibitors do affect the activity of non-phosphatase proteins; most notably fostriecin, which inhibits partially purified type II topoisomerase, although at much higher concentrations (47).

Potential unknown targets of phosphatase inhibitors might contribute to the paradox that some of these compounds have tumor promoting activity, whereas others exhibit antitumor activity. Alternatively, or in addition, it may be of importance which combination of the various phosphatases is targeted by a certain inhibitor. Okadaic acid, calyculin A, microcystin-LR, and nodularin are potent tumor promoters or liver carcinogens (48-52), whereas tautomycin has not been found to promote tumors on mouse skin or rat glandular stomach (37). Moreover, fostriecin, cantharidin, and cantharidin derivatives have demonstrated antitumor properties (40, 53-55). Fostriecin in particular exhibits antitumor activity against a wide spectrum of tumor cells in vitro, and is under evaluation as an antitumor drug in clinical trials (56-58).