A Structural Basis for the Reduced Toxicity of Dinophysistoxin-2 Jason Huhn, † Philip D. Jeffrey, † Kristofer Larsen, ‡,§ Thomas Rundberget, ‡ Frode Rise, § Neil R. Cox, | Vickery Arcus, |,⊥ Yigong Shi,* ,†,# and Christopher O. Miles* ,‡,|,∇ Department of Molecular Biology, Lewis Thomas Laboratory, Princeton UniVersity, Washington Road, Princeton, New Jersey 08544, The National Veterinary Institute, PB 750 Sentrum, NO-0106 Oslo, Norway, Department of Chemistry, UniVersity of Oslo, P.O. Box 1033 Blindern, NO-0315 Oslo, Norway, AgResearch Ltd., Ruakura Research Centre, PriVate Bag 3123, Hamilton 3240, New Zealand, Department of Biology, The UniVersity of Waikato, PriVate Bag 3105, Hamilton, New Zealand, and Marine Institute, RinVille, Oranmore, Co. Galway, Ireland ReceiVed May 11, 2009 Okadaic acid (OA), dinophysistoxin-1 (DTX-1), and dinophysistoxin-2 (DTX-2) are algal toxins that can accumulate in shellfish and cause diarrhetic shellfish poisoning. Recent studies indicate that DTX-2 is about half as toxic and has about half the affinity for protein phosphatase 2A (PP2A) as OA. NMR structural studies showed that DTX-1 possessed an equatorial 35-methyl group but that DTX-2 had an axial 35-methyl group. Molecular modeling studies indicated that an axial 35-methyl could exhibit unfavorable interactions in the PP2A binding site, and this has been proposed as the reason for the reduced toxicity of DTX-2. Statistical analyses of published data indicate that the affinity of PP2A for DTX-1 is 1.6-fold higher, and for DTX-2 is 2-fold lower, than for OA. We obtained X-ray crystal structures of DTX-1 and DTX-2 bound to PP2A. The crystal structures independently confirm the C-35 stereochemistries determined in the earlier NMR study. The structure for the DTX-1 complex was virtually identical to that of the OA-PP2A complex, except for the presence of the equatorial 35-methyl on the ligand. The favorable placement of the equatorial 35-methyl group of DTX-1 against the aromatic π-bonds of His191 may account for the increased affinity of PP2A toward DTX-1. In contrast, the axial 35-methyl of DTX-2 caused the side chain of His191 to rotate 140° so that it pointed toward the solvent, thereby opening one end of the hydrophobic binding cage. This rearrangement to accommodate the unfavorable interaction from the axial 35-methyl of DTX-2 reduces the binding energy and appears to be responsible for the reduced affinity of PP2A for DTX-2. These results highlight the potential of molecular modeling studies for understanding the relative toxicity of analogues once the binding site at the molecular target has been properly characterized. Introduction Okadaic acid (OA) 1 (1) (Figure 1) was first isolated from the sponges Halichondria okadai and Halichondria melanodocia (1) and shown to be a potent new type of protein phosphatase (PP1 and PP2A) inhibitor (2). Subsequent studies have shown that OA, the closely related analogues dinophysistoxins-1 (DTX- 1) (2)(3) and -2 (DTX-2) (3)(4), and their derivatives are responsible for causing a form of food poisoning known as diarrhetic shellfish poisoning (DSP) (5). DSP occurs around the world and is caused by the consumption of filter-feeding shellfish that have consumed microalgae containing okadaic analogues (6). Heterotrophic and mixotrophic dinoflagellates of the genus Dinophysis have long been known to contain okadaic analogues (3), although only recently have suitable culture methods been developed (7) that have allowed the production of these toxins by Dinophysis to be demonstrated (8). Recently, DTX-2 was found to be significantly less toxic in a mouse bioassay and a weaker inhibitor of PP2A than OA (9). This finding is of considerable public health and regulatory significance because these compounds were previously assumed to be equipotent. The stereochemistries of DTX-1 and DTX-2 have been studied in detail by NMR, and their 35-methyl groups were found to differ in their stereochemistries at C-35 (10, 11), a finding that was supported by subsequent synthetic studies (12). The availability of a crystal structure of OA bound to the core enzyme of PP2A (13) facilitated molecular modeling studies of the binding of OA, DTX-1, and DTX-2 to PP1 and PP2A (10). These studies predicted that the axial 35-methyl * To whom correspondence should be addressed. Tel: +47 2321-6226. Fax: +47 2321-6201. E-mail: chris.miles@vetinst.no (C.O.M.). Tel: +86 10-6279-6163. Fax: +86 10-6279-2736. E-mail: shi-lab@tsinghua.edu.cn (Y.S.). † Princeton University. ‡ National Veterinary Institute. § University of Oslo. | AgResearch Ltd. ⊥ The University of Waikato. # Present address: Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 10084, China. ∇ Marine Institute. 1 Abbreviations: DTX, dinophysistoxin; MRM, multiple reaction moni- toring; PP, protein phosphatase; OA, okadaic acid; TEF, toxic equivalence factor. Figure 1. Structures of OA (1), DTX-1 (2), and DTX-2 (3)(10, 11). DTX-2 was previously usually depicted as 4. Chem. Res. Toxicol. 2009, 22, 1782–1786 1782 10.1021/tx9001622 CCC: $40.75  2009 American Chemical Society Published on Web 10/21/2009