Sea Urchin Embryo as a Model Organism for the Rapid Functional Screening of Tubulin Modulators

Abstract

Identification of antimitotic molecules that affect tubulin dynamics is a multistep procedure. It includes in vitro tubulin polymerization assay, studies of a cell cycle effect, and general cytotoxicity assessment. To simplify this lengthy screening protocol, we have introduced and validated an assay system based on the sea urchin embryos. The proposed two-step procedure involves the fertilized egg test for mitotic arrest and the behavioral assessment of a free-swimming blastula. In order to validate the assay, we have analyzed the effect of a panel of known antiproliferative agents on the sea urchin embryo. For all tubulin destabilizing drugs, we observed rapid spinning and lack of forward movement of an embryo. Both effects are likely to result from the in vivo microtubule disassembly caused by test molecules. Notably, the described assay yields rapid information on antiproliferative, antimitotic, cytotoxic, and tubulin destabilizing activities of the molecules along with their solubility and permeability potential. Moreover, measured potencies of the test articles correlated well with the reported values in both in vitro and cell based assays.

Novel derivatives of 1,3,4-oxadiazoles are potent mitostatic agents featuring strong microtubule depolymerizing activity in the sea urchin embryo and cell culture assays

“…In this paper, we report on the development of a robust and high-yielding synthetic procedure to access novel 1,3,4-oxadiazole derivatives 1-25 that specifically affect tubulin dynamics (Table 1). Furthermore, in order to evaluate antimitotic activity of these small molecules we introduced reliable phenotypic assay based on the sea urchin embryo [22,23]. This in vivo screening yielded a rapid ''one pot'' assessment of anti-proliferative, anti-mitotic, and cytotoxic effects of the oxadiazoles 1-25 on a living organism.…”

Evaluation of drug toxicity profiles based on the phenotypes of ascidian Ciona intestinalis

“…As shown in Table 1, MTCs in Ciona roughly corresponded to concentrations of each drug similar to toxic effects experienced by zebrafish. Paclitaxel shows no toxic effect on zebrafish larvae near MTC, but can inhibit cleavage of fertilized eggs in sea urchin at 10 mM [20]. Nevertheless, MTCs were validated in other species, and toxicity profiles at MTCs may be caused by sufficient inhibition of the targets.…”

Synthesis and antimitotic properties of ortho-substituted polymethoxydiarylazolopyrimidines

“…The targeted diarylazolopyrimidines 7-9 and by-product 6 were evaluated for antimitotic activity in a phenotypic sea urchin embryo assay. 34 The assay provides rapid information on antiproliferative, embryotoxic, and microtubule destabilizing activities of tested molecules. The results are presented in Table 1 with CA4 as a positive control.…”

“…34 Fertilized eggs and hatched blastulae were exposed to 2-fold decreasing concentrations of compounds. Duplicate measurements showed no differences in effective threshold concentration (EC) values.…”

“…6- (3-Hydroxy-4-methoxyphenyl)-7-(3,4,5-trimethoxyphenyl)[1,2,4 Phenotypic sea urchin embryo assay. 34 Adult sea urchins, Paracentrotus lividus L. (Echinidae), were collected from the Mediterranean Sea on the Cyprus coast and kept in an aerated seawater tank. Gametes were obtained by intracoelomic injection of 0.5 M KCl.…”