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.

Introduction

Tubulin is the major protein component of microtubules, a cytoskeletal organelle that plays an essential role in cell division, intracellular transport, cell motility, and shape maintenance (1). During mitosis, microtubules are required for mitotic spindle formation and chromosomal separation. Hence, targeting tubulin in rapidly dividing tumor cells is a sound strategy for cancer therapy (2). Approved drugs that affect microtubule dynamics include paclitaxel (Taxol™) and vinblastine. Their use, however, is limited by poor therapeutic window, multiple drug resistance, low solubility, and general systemic toxicity. Hence, a considerable challenge to discover and develop novel small molecule inhibitors of tubulin dynamics exists for the pharmaceutical industry. Several small heterocycle-based compounds appeared recently and showed some unique characteristics; as a representative example, D-24851 demonstrated anti-tumor effect in xenograft models (3). Indole-based D-24851 did not bind to any of the characterized binding sites on tubulin. Moreover, in a variety of tests, this phase II drug candidate did not show neurotoxicity, a common side-effect of antimitotic agents.

Evaluation of antimitotic agents displaying desirable efficacy and safety profiles similar to D24851 is rather lengthy. This multistep procedure includes studies of the in vitro effect on tubulin polymerization, cytotoxicity and cell cycle effects, and a panel of functional assays. In this paper, we report on a physiologically relevant one-pot procedure for the assessment of an antimitotic activity of compound libraries. Simplicity of the experimental protocol, relevance of the model, and the potential for both medium-throughput and structure-activity relationship studies are the distinctive features of the developed screening protocol.

The sea urchin embryo has long been used as a model organism for the developmental biological studies (4,5,6,7,8). A number of factors make this system suited for conducting a wide range of biological tests. These include straightforward artificial spawning, fertilization and rearing, rapid synchronous development, embryo optical transparency, and well understood embryogenesis. As a result, sea urchin embryos have been successfully used in studies of the effects of various antiproliferative agents (9,10,11,12).

Important to our objectives, there are two distinct processes directly connected to microtubule dynamics during early sea urchin embryogenesis. These are cleavage (i.e., a series of successive mitotic cycles occurring within approximately 30-min intervals) and ciliary swimming of hatched embryos within 9–12 h after fertilization. Both processes can be easily monitored and quantified, yielding the opportunity to screen compound libraries for the molecules affecting microtubule structure and function.

In our initial set of experiments, we investigated the effect of a potent tubulin-destabilizing drug D-24851 (3) on the embryonic development of sea urchin Paracentrotus lividus from the fertilized egg until the beginning of active feeding (mid-pluteus stage). Subsequently, we proposed a protocol for parallel screening of small molecules for tubulin destabilizing activity using the sea urchin embryos. The assay procedure was further validated by testing a series of diverse antiproliferative agents that affect different intracellular targets.

Materials and Methods

Adult sea urchins P. lividus were collected from the Mediterranean Sea at the Cyprus coast and kept in an aerated seawater tank. Gametes were obtained by intracelomic injection of 0.5 M KC1. Eggs were washed with filtered sea water and fertilized by adding drops of diluted sperm. Embryos were cultured at room temperature under gentle agitation with a motor-driven plastic paddle (60 rpm) in filtered sea water up to the beginning of active feeding (mid-pluteus stage). The embryos were observed with a Biolam LOMO light microscope (LOMO PLC, St. Petersburg, Russia). Electronic images were obtained using digital camera Olympus C4000 digital camera with a microscope adaptor 10× (Optica M, St. Petersburg, Russia).

Test articles were obtained as follows. D-24851 [N-(pyridin-4-yl)-(1-[4-chlorbenzyl]-indol-3-yl)-glyoxyl-amid], SK2 [(2,3-di-hydro-benzo[1,4]dioxin-6-yl)-(5-[2-([pyridin-3-ylmethyl]-amino)-phenyl]-[1,3,4]oxadiazol-2-yl)-amine], SK3 [benzo(1,3) dioxol-5-yl-(5-[2-([pyridin-4-ylmethyl]-amino)-phenyl]-[1,3,4] oxadiazol-2-yl)-amine], and SK4 [(2,3-dihydro-benzo[1,4]dioxin-6-yl)-(5-[2-([pyridin-4-ylmethyl]-amino)-phenyl]- [1,3,4]oxadiazol-2-yl)-amine] were synthesized at Zelinsky Institute of Organic Chemistry (Moscow, Russia) as described elsewhere (3,13). Acrylamide, apigenin, 3′-azido-3′-deoxythymidine (azidothymidine), colchicine, cytocha-lasin D, dolastatin 15, 5-fluorouracyl, hydroxyurea, nocodazole, paclitaxel, and roscovitine were obtained from Sigma-Aldrich Chemie GmbH (Steinheim, Germany), 4-phenylbutyric acid was obtained from Acros Organics (Geel, Belgium), and 2-methoxyestradiol was obtained from Koch-Light Laboratories Ltd. (Colnbrook, Buckinghamshire, UK). Anti-tumor medicines mitoxantrone (Mitoxantrone-Lans™; Lans Pharm, Russia), mitomycin C (Mitomycin-Vero™; Veropharm, Russia), and vinblastine (Vinblastin-Richter™; Gedeon Richter, Budapest, Hungary) were purchased at pharmacies as commercial preparative forms. Combretastatin A-4 disodium phosphate was a gift of OXiGENE (Waltham, MA, USA). Ruboxyl [14-(1-oxyl-2,2,6,6,-tetramethylpiperidyl-4)-acetoxyrubo-mycin hydrochloride] was a gift of Dr. Vladimir A. Serezhenkov, Institute of Chemical Physics, Russian Academy of Sciences, Moscow, Russia. Structures of noncommercial compounds ruboxyl, SK2, SK3, and SK4 are presented in Figure 1.