Inhibitory effect of okadaic acid derivatives on protein phosphatases. A study on structure-affinity relationship

Abstract

The effect of structural modifications of okadaic acid (OA), a polyether C38 fatty acid, was studied on its inhibitory activity toward type 1 and type 2A protein phosphatases (PP1 and PP2A) by using OA derivatives obtained either by isolation from natural sources or by chemical processes. The dissociation constant (Ki) for the interaction of OA with PP2A was estimated to be 30 (26-33) nM [median (95% confidence limits)]. The OA derivatives used and their affinity for PP2A, expressed as Ki (in brackets) were as follows: 35-methyl-OA (DTX1) [19 (12-25) pM], OA-9,10-episulphide (acanthifolicin) [47 (25-60) pM], 7-deoxy-OA [69 (31-138) pM], 14,15-dihydro-OA [315 (275-360) pM], 2-deoxy-OA [899 (763-1044) pM], 7-O-palmitoyl-OA [greater than 100 nM], 7-O-palmitoyl-DTX1 [greater than 100 nM], methyl okadate [much greater than 100 nM], 2-oxo-decarboxy-OA [much greater than 100 nM] and the C-15-C-38 fragment of OA [much greater than 100 nM]. The sequence of the affinity of these derivatives for PP1 was essentially the same as that observed with PP2A, although the absolute values of Ki were very different for the enzymes. The inhibitory effect of OA on PP2A was reversed by applying a murine monoclonal antibody against OA, which recognizes modifications of the 7-hydroxyl group of the OA molecule. It has been shown by n.m.r. spectroscopy and X-ray analysis that one end (C-1-C-24) of the OA molecule assumes a circular conformation. The present results suggest the importance of the conformation for the inhibitory action of OA on the protein phosphatases. The ratios of the Ki values for PP1 to that for PP2A, which were within the range 10(3)-10(4), tended to be smaller for the derivatives with lower affinity, indicating that the structural changes in OA impaired the affinity for PP2A more strongly than that for PP1.

Crystal Structure of the Tumor-promoter Okadaic Acid Bound to Protein Phosphatase-1

“…The importance of the hydrogen bond between Tyr-272 and the acid group of OA is exemplified by the observation that esterification or removal of the acidic moiety in OA results in elimination of its inhibitory activity and by the fact that mutation of Tyr-272 to Phe results in a 50-fold increase in the K i value (20,22). Despite the intimate interaction of the C-2 hydroxyl group of OA with the Arg-96 side chain, removal of the hydroxyl group results in only a 7-fold increase in the K i value (24). In contrast, mutation of Arg-221 to Ser confers resistance to inhibition by OA, underlining the importance of the interaction between the Arg and the C-24 hydroxyl of OA (25).…”

The methyl ester of okadaic acid is more potent than okadaic acid in disrupting the actin cytoskeleton and metabolism of primary cultured hepatocytes

“…Usually, MeOk has been considered as an inactive molecule because of its low activity in inhibiting PPs (Nishiwaki et al, 1990;Takai et al, 1992). However, recent studies indicated that MeOk induced reorganization of the actin cytoskeleton of human neuroblastoma cells (Vilarino et al, 2008), and although the potency of MeOk was lower than that of OA, its mechanism of action was unknown.…”

“…Methyl okadaate (MeOk) is a methyl ester derivative of the OA, artificially produced in the laboratory, but also found in shellfish, and even in naturally collected or cultured dinoflagellates from the genera Prorocentrum and Dinophysis (Hu et al, 1992;Vale and Sampayo, 1999;Suzuki et al, 2004;Suarez-Gomez et al, 2005). Usually, MeOk has been considered as an inactive molecule because of its low activity in inhibiting PPs (Nishiwaki et al, 1990;Takai et al, 1992). However, recent studies indicated that MeOk induced reorganization of the actin cytoskeleton of human neuroblastoma cells (Vilarino et al, 2008), and although the potency of MeOk was lower than that of OA, its mechanism of action was unknown.…”

The mitotic serine/threonine kinase Aurora2/AIK is regulated by phosphorylation and degradation

“…Interestingly, a high concentration of okadaic acid was needed to aect Aurora2 activity. At this concentration okadaic acid inhibits both protein phosphatases PP2A and PP1 together, while at lower concentrations only PP2A would be inhibited (Takai et al, 1992).…”