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.

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Dinophysistoxin-3 (DTX3, 3), the principal toxin of scallops implicated in diarrhetic shellfish poisoning, is a mixture of 7-0-acyl esters of 35-methylokadaic acid. To investigate the effects of unsaturation in the acyl moieties on biological activities, three demethyl analogs of DTX3, 7-0-palmitoyl, 7-0-linoleoyl, and 7-0-docosahexaenoyl okadaic acid, were prepared and tested for mouse lethality, cytotoxicity, and diarrheagenicity.The activities of okadaic acid were generally decreased by acylation of 7-OH. The decrease was most significant in the mouse lethality, moderate in cytotoxicity, and only slight in the fluid accumulating potency in mouse intestinal loops. Diarrheagenicity measured by suckling mouse assays was affected little by the acylation. Potency of analogs genellaly increased in parallel with the unsaturation in the acyls. Diarrheticshellfish poisoning (DSP)1 "3) is a gastroenteritis caused by ingestion of the shellfish containing dinoflagellate toxins. The worldwide occurrence and the high morbidity rate of the poisoning pose serious threats to both human health and shellfish industries.Guided by a mouse lethality test we isolated three different groups of polyether compounds and analyzed their structures. One group consists of okadaic acid (OA, 1),4' 5) and its derivatives named dinophysistoxin-1 (DTX1 , 2)6) and dinophysistoxin-3 (DTX3, 3).7) The other two groups consist of macrolides of the pectenotoxin family7'8) and a newly isolated toxin named yessotoxin.9) Among these toxins OA, DTX1and DTX3are the most important in causing diarrheal symptoms.10)ll) Moreover, recent reports by Fujiki et al.12' l3) indicate that OA and DTX1 possibly promote tumor formation.