Effects of Azaspiracid-1, A Potent Cytotoxic Agent, on Primary Neuronal Cultures. A Structure-Activity Relationship Study Carmen Vale, ² K. C. Nicolaou, ‡,§ Michael O. Frederick, ‡ Bele ´n Go ´mez-Limia, ² Amparo Alfonso, ² Mercedes R. Vieytes, | and Luis M. Botana* ,² Departamento de Farmacologı ´a and Departamento de Fisiologı ´a, Facultad de Veterinaria, USC, Lugo, Spain, Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, and Department of Chemistry and Biochemistry, UniVersity of California, San Diego, La Jolla, California 92093 ReceiVed September 8, 2006 Azaspiracids (AZAs) are marine phycotoxins with an unknown mechanism of action, implicated in human intoxications. We investigated the effect of azaspiracid-1 (AZA-1) on the cytosolic calcium concentration ([Ca 2+ ] c ), intracellular pH (pH i ), and neuron viability in neuronal cultures. AZA-1 increased [Ca 2+ ] c and decreased neuronal viability. The effects of several fragments of the AZA-1 molecule (13 different chemical structures) were examined. The ent-ABCD-azaspiracid-1 (2) showed similar potency to AZA-1 (1) in increasing [Ca 2+ ] c but higher cytotoxity than AZA-1. The chemical structures containing only the ABCD or the ABCDE ring domains (3-8) caused a [Ca 2+ ] c increase but did not alter cell viability. The compounds containing only the FGHI ring domain of AZA-1 (9-14) did not modify the [Ca 2+ ] c or the cell viability. Therefore, the effect of AZA-1 on [Ca 2+ ] c depends on the presence of the ABCD or the ABCDE-ring structure, but the complete chemical structure is needed to produce neurotoxic effects. Introduction Phycotoxins constitute a rich source of active pharmacological tools, with a wide range of mechanisms of action. One of the recently isolated marine biotoxins that has created great concern with regard to seafood poisoning and human health is azaspi- racid-1 and its congeners. 1 Marine phycotoxins belonging to the azaspiracid family were first identified in The Netherlands in 1995 following cases of shellfish intoxication after consump- tion of mussels cultivated in Killary Harbor, Ireland. 1,2 Cases of shellfish intoxications associated with azaspiracids were recently reported in several European coastal countries, including Ireland, UK, Norway, Netherlands, France, Spain, and Italy. 1-6 The discovery of azaspiracids and its recognized health hazard led to the declaration of a new toxic syndrome, named azaspiracid poisoning (AZP). Eleven different members of the azaspiracid family of compounds have been described. AZA-1 (azaspiracid), AZA-2 (8-methylazaspiracid), and AZA-3 (22- demethylazaspiracid) are the predominant azaspiracids in nature. Other azaspiracids (i.e., AZA-4 through AZA-11) differ by the presence or lack of methyl and hydroxyl groups on the azaspiracid structure. 2,7,8 The toxic episodes caused by AZAs show gastrointestinal illnesses. Following human consumption of AZA-contaminated shellfish, there is generally a rapid onset of symptoms very similar to those of diarrheic shellfish poisoning, including nausea, vomiting, severe diarrhea, and stomach cramps. 9 In mice and rats, intraperitoneal injections induce neurological symptoms resembling paralytic shellfish poisoning with progressive pa- ralysis, fatigue, difficulty breathing, and subsequent death as soon as 35 min after injection. 3,10 The AZAs are characterized by unusual structural motifs including spiro-ring assemblies, a secondary amino group, and a carboxylic acid moiety, making them unique within the nitrogen-containing marine toxins. So far, there is no information about the cellular target, the mechanism of action, or the toxicity in nervous tissues of the AZAs. Previous studies in our laboratory have focused on the effects of AZA-1 on neuroblas- toma cells and human lymphocytes. This previous work suggested that the toxin reduces cellular F-actin in a nonapo- ptotic manner. 11 A high cytotoxicity of AZA-1 has been recently shown in several cell lines, 12 and even more recently it has been described that this toxin inhibits the electrical activity of neuronal networks; 13 however the mechanisms of action of these toxins are still unknown. Because these toxins are a serious threat to human health and could offer a new therapeutic strategy to modify the function of neuronal systems, we decided to explore the possible effects of their action on nervous tissue as well as investigate the parts of the molecule that could account for this effect. Primary cultures of cerebellar granule cells (CGC) were used as the cellular model, since these cells constitute one of the most reliable models for the study of neural function and pathology. 14,15 Cytosolic calcium concentration ([Ca 2+ ] c ) and intracellular pH (pH i ) are two of the primary events in the cellular response to external stimuli used to study the mechanism of action and the toxicological effects of toxins. Therefore, in this work we studied the effects of AZA-1 on the [Ca 2+ ] c , intracellular pH and neuronal viability. In addition, the effects of the different domains of the AZA-1 molecule were also examined (see Figure 1) in order to elucidate the active part of the AZA-1 structure. Results and Discussion The AZA-1 structure (1), as well as the truncated fragments (2-14) of the molecule examined in this study, is shown in Figure 1. The chemical structure of the natural azaspiracid analogues is summarized in Scheme 1. Effects of AZA-1 (1) on [Ca 2+ ] c and pH i in Neuronal Cerebellar Granule Cells. Previous studies in our laboratory had shown that natural AZA-1 (1) increased the [Ca 2+ ] c without modifying pH i levels in human lymphocytes. 11 The effects of * Corresponding author. Tel: 34-982 252 242. Fax : 34-982 252 242. E-mail: Luis.Botana@lugo.usc.es. ² Departamento de Farmacologı ´a, USC. ‡ The Scripps Research Institute. § University of California. | Departamento de Fisiologı ´a, USC. 356 J. Med. Chem. 2007, 50, 356-363 10.1021/jm061063g CCC: $37.00 © 2007 American Chemical Society Published on Web 12/13/2006