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 thre e reaction conditions. In 1.0 M glutamate + 1.0 mM MgCl2, with a 37 de grees C reaction temperature, we could find no concentration of curaci n A that completely inhibited polymerization. A maximal inhibitory eff ect on turbidity development (about 50%) was observed with 5 mu M drug . Higher drug concentrations induced an abnormal polymerization reacti on, which at 40 mu M differed little from the control reaction except for slightly delayed depolymerization in response to reducing the temp erature to 0 degrees C. in 0.8 M glutamate (no MgCl2), with a 30 degre es C reaction temperature, complete inhibition did occur at 3-5 mu M d rug, but higher drug concentrations again induced an abnormal polymeri zation reaction. With 40 mu M curacin A this reaction was also similar to the control reaction, except that cold-induced depolymerization wa s slightly enhanced relative to the control. When polymerization was i nduced by microtubule-associated proteins, maximal inhibition of turbi dity development (about 70%) occurred with 2 mu M drug, with higher cu racin 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 cura cin A consisted of large aggregates of tightly coiled spiral fibers th at had a 2-3 filament substructure. The implications of these findings with curacin A are discussed in terms of the use of tubulin polymeriz ation assays as a screen for identifying new antimitotic drugs. (C) 19 95 Wiley-Fiss, Inc.