Effect-directed analysis reveals inhibition of zebrafish uptake transporter Oatp1d1 by caulerpenyne, a major secondary metabolite from the invasive marine alga Caulerpa taxifolia

Abstract

Caulerpa taxifolia is a marine alga of tropical and subtropical distribution and a well-known invasive species in several temperate regions. Its invasiveness mainly stems from the production of secondary metabolites, some of which are toxic or repellent substances. In this study we investigated the possible inhibitory effects of C. taxifolia secondary metabolites on the activity of two zebrafish (Danio rerio) uptake transporters that transport organic anions (Oatp1d1) and cations (Oct1). Both transporters were transiently transfected and overexpressed in human embryonic kidney HEK293T cells. Transport activity assays using lucifer yellow (LY) and 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP+) as model substrates were applied for the determination of Oatp1d1 and Oct1 interactors. A two-step Effect-Directed Analysis (EDA) procedure was applied for the separation and identification of compounds. We identified caulerpenyne (CYN) as the major metabolite in C. taxifolia and reveal its potent inhibitory effect towards zebrafish Oatp1d1 as well as weak effect on zebrafish Oct1 transport. The observed effect was confirmed by testing CYN purified from C. taxifolia, resulting in an IC of 17.97 μM, and a weak CYN interaction was also determined for the zebrafish Oct1 transporter. Finally, using Michaelis-Menten kinetics experiments, we identified CYN as a non-competitive inhibitor of the zebrafish Oatp1d1. In conclusion, this study describes a novel mechanism of biological activity in C. taxifolia, shows that CYN was a potent non-competitive inhibitor of zebrafish Oatp1d1, and demonstrates that EDA can be reliably used for characterization of environmentally relevant complex biological samples.

Natural Products of Marine Macroalgae from South Eastern Australia, with Emphasis on the Port Phillip Bay and Heads Regions of Victoria

“…Sesquiterpenes, with acetoxy and aldehyde functionalities (21)(22)(23)(24), Figure 3, have been shown to be potent feeding deterrents and in some cases cytotoxic towards predatory species of herbivorous fish [29]. It has been suggested that caulerpenyne (24), an acyclic acetylenic sesquiterpene found in C. trifaria, as a minor secondary metabolite and in C. taxifolia, as a major secondary metabolite, is a key player in the cytotoxicity of Caulerpa algae towards herbivores [30].…”

“…This sesquiterpene appears to contribute to the invasiveness of the genus Caulerpa by means of its inhibition of the key organic anion transporters, Oatp1d1 and Oct1. These transporters play a role in the toxicity defense of a herbivorous predator of the genus Caulerpa, the zebra fish (Danio rerio) [30]. Although this particular mechanism of action has only been demonstrated towards zebrafish, it appears to provide some reasoning behind the apparent success of Caulerpa as an invasive species of algae [30].…”

“…These transporters play a role in the toxicity defense of a herbivorous predator of the genus Caulerpa, the zebra fish (Danio rerio) [30]. Although this particular mechanism of action has only been demonstrated towards zebrafish, it appears to provide some reasoning behind the apparent success of Caulerpa as an invasive species of algae [30]. Compound (23) was shown to display moderate anti-microbial activity against B. subtilis and the marine fungus Dreschleria haloides.…”

Molecular Interactions as Drivers of Changes in Marine Ecosystems

“…It was found to be neurotoxic [17], to act as tubulin assembly inhibitor [18], antiproliferative and pro-apoptotic agent [19,20], and as inhibitor of various enzymes, including alpha-amilase [21], lipoxygenase [22,23], and xanthine oxidase [24]. Among the actions of potential ecotoxicological interest, it has been described that 1 acts as an inhibitor of cytochrome P450 dependent activities [25], which are important for the clearance of xenobiotics, and a potent noncompetitive inhibitor of zebrafish Oatp1d1 [26], a protein with a crucial role in absorption, distribution, metabolism, and elimination processes [27].…”