Current Medicinal Chemistry - Volume 9, Issue 22, 2002

As the first recognized member of the “okadaic acid class” of phosphatase inhibitors, the marine natural product okadaic acid is perhaps the most well-known member of a diverse array of secondary metabolites that have emerged as valuable probes for studying the roles of various cellular protein serine / threonine phosphatases. This review provides a historical perspective on the role that okadaic acid has played in stimulating a broad spectrum of modern scientific research as a result of the natural product's ability to bind to and inhibit important classes of protein serine / threonine phosphatases. The relationships between the structure and biological activities of okadaic acid are briefly reviewed, as well as the structural information regarding the particular cellular receptors protein phosphatases 1 (PP1) and 2A. Laboratory syntheses of okadaic acid and its analogs are thoroughly reviewed. Finally, an interpretation of the critical contacts observed between okadaic acid and PP1 by X-ray crystallography is provided, and specific molecular recognition hypotheses that are testable via the synthesis and assay of non-natural analogs of okadaic acid are suggested.

The protein serine / threonine phosphatases constitute a unique class of enzymes that are critical for cell regulation, as they must counteract the activities of thousands of protein kinases in human cells. Uncontrolled inhibition of phosphatase activity by toxic inhibitors can lead to widespread catastrophic effects. Over the past decade, a number of natural product toxins have been identified that specifically and potently inhibit protein phosphatase-1 and -2A. Amongst these are the cyanobacteria-derived cyclic heptapeptide microcystin-LR and the polyether fatty acid okadaic acid from dinoflagellate sources.The molecular mechanism underlying potent inhibition of protein phosphatase-1 by these toxins is becoming clear through insights gathered from diverse sources. These include:1. Comparison of structure-activity relationships amongst the different classes of toxins.2. Delineation of the structural differences between protein phosphatase-1 and -2A that account for their differing sensitivity to toxins, particularly okadaic acid and microcystin-LR.3. Determination of the crystal structure of protein phosphatase-1 with microcystin-LR, okadaic acid and calyculin bound.4. Site-specific mutagenesis and biochemical analysis of protein phosphatase-1 mutants.Taken together, these data point to a common binding site on protein phosphatase-1 for okadaic acid, microcystin-LR and the calyculins. However, careful analysis of these data suggest that each toxin binds to the common binding site in a subtly different way, relying on distinct structural interactions such as hydrophobic binding, hydrogen bonding and electrostatic interactions to different degrees.The insights derived from studying the molecular enzymology of protein phosphatase-1 may help explain the different sensitivities of other structurally conserved protein serine / theonine phosphatases to toxin inhibition. Furthermore, studies on the binding of structurally diverse toxins at the active site of protein phosphatase-1 are leading to a clearer understanding of potential enzyme-substrate interactions in this important class of cell regulatory proteins.

The serine / threonine phosphatases are inhibited by a variety of natural toxins, including the microcystins and nodularins. Progress in understanding the details of the biosynthetic origin and the binding of these compounds is discussed, as is the progress made in synthesizing the members of these families. Additionally, the work by several groups to either synthesize simplified analogues that are still potent, or introduce selectivity for PP1 over PP2A are discussed. Finally, the properties of a series of five new truncated analogues are examined.

A review of the current status of the chemistry and biology of fostriecin (CI-920) is provided. Fostriecin is a structurally unique, naturally-occurring phosphate monoester that exhibits potent and efficacious antitumor activity. Initially it was suggested that its activity could be attributed to a direct, albeit weak, inhibition of the enzyme topoisomerase II. However, recent studies have shown that fostriecin inhibits the mitotic entry checkpoint through the much more potent and selective inhibition of protein phosphatase 2A (PP2A) and protein phosphatase 4 (PP4). In fact, it is the most selective small molecule inhibitor of a protein phosphatase disclosed to date. The contribution, if any, that topoisomerase II versus PP2A / PP4 inhibition makes to fostriecin's antitumor activity has not yet been fully defined. Initial phase I clinical trials with fostriecin never reached dose-limiting toxicity or therapeutic dose levels and were halted due to its storage instability and unpredictable chemical purity. Hence, the total synthesis of fostriecin has been pursued in order to confirm its structure and stereochemistry, to provide access to quantities of the pure natural product, and to access key partial structures or simplified / stable analogs. Several additional natural products have been isolated which contain similar structural features (phospholine, phoslactomycins, phosphazomycin, leustroducsins, sultriecin, and cytostatin), and some exhibit comparable biological properties.

The recent progress in synthetic and SAR studies of the specific protein phosphatase inhibitors tautomycin and tautomycetin is reviewed. This article covers the total synthesis of tautomycin and synthetic studies of the spiroketal and the anhydride segments and tautomycetin, and SAR studies on PP inhibition and apoptosis-inducing activity of tautomycin.

Reversible phosphorylation is a key mechanism for regulating the biological activity of many human proteins that affect a diverse array of cellular processes, including protein-protein interactions, gene transcription, cell-cycle progression and apoptosis. Once viewed as simple house keeping enzymes, recent studies have made it eminently clear that, like their kinase counterparts, protein phosphatases are dynamic and highly regulated enzymes. Therefore, the development of compounds that alter the activity of specific phosphatases is rapidly emerging as an important area in drug discovery. Because >98% of protein phosphorylation occurs on serine and threonine residues, the identification of agents that alter the activity of specific serine / threonine phosphatases seems especially promising for drug development in the future. This review is focused on the enzymes encoded by the PPP-gene family, which includes PP1, PP2A, PP2B, PP4, PP5, PP6 and PP7. The structure / functions of human phosphatases will be addressed briefly, as will the natural product inhibitors of PP1-PP6 (e.g. okadaic acid, microcystins, nodularin, cantharidin, calyculin A, tautomycin, and fostriecin). The development of chimeric antisense oligonucleotides that support RNAase H mediated degradation of the targeted mRNA has resulted in compounds capable of specifically suppressing the expression of PP5 (ISIS 15534) and PP1gamma 1 (ISIS 14435) in human cells. Such compounds have already proven useful for the validation of drug targets, and if difficulties associated with systemic delivery of antisense oligonucleotides can be overcome, antisense is poised to have a major impact on the clinical management of many human disorders.