LIMITATIONS IN THE USE OF TUBULIN POLYMERIZATION ASSAYS AS A SCREEN FOR THE IDENTIFICATION OF NEW ANTIMITOTIC AGENTS - THE POTENT MARINE NATURAL PRODUCT CURACIN-A AS AN EXAMPLE

Curacin A is a newly isolated lipid natural product that binds in the colchicine site of tubulin and inhibits mitosis. We have examined its effects on tubulin polymerization, studied by turbidimetry, under three reaction conditions. In 1.0 M glutamate + 1.0 mM MgCl2, with a 37 degrees C reaction temperature, we could find no concentration of curacin A that completely inhibited polymerization. A maximal inhibitory effect on turbidity development (about 50%) was observed with 5 mu M drug. Higher drug concentrations induced an abnormal polymerization reaction, which at 40 mu M differed little from the control reaction except for slightly delayed depolymerization in response to reducing the temperature to 0 degrees C. in 0.8 M glutamate (no MgCl2), with a 30 degrees C reaction temperature, complete inhibition did occur at 3-5 mu M drug, but higher drug concentrations again induced an abnormal polymerization reaction. With 40 mu M curacin A this reaction was also similar to the control reaction, except that cold-induced depolymerization was slightly enhanced relative to the control. When polymerization was induced by microtubule-associated proteins, maximal inhibition of turbidity development (about 70%) occurred with 2 mu M drug, with higher curacin A concentrations inducing abnormal polymerization reactions that reached about 60% of the control turbidity readings. Under all three reaction conditions the polymer induced by high concentrations of curacin A consisted of large aggregates of tightly coiled spiral fibers that had a 2-3 filament substructure. The implications of these findings with curacin A are discussed in terms of the use of tubulin polymerization assays as a screen for identifying new antimitotic drugs. (C) 1995 Wiley-Fiss, Inc.