Characterization of tubulin-alkaloid interactions by enzyme-linked immunosorbent assay

An indirect enzyme-linked immunosorbent assay has been developed to characterize the interactions of drugs (bisindol alkaloids and colchicine) with immobilized tubulin. The binding of polyclonal antibodies raised in rabbits to tubulin heterodimers and monoclonal antibodies against the C-terminal regions of alpha- and beta-tubulin subunits is tested at various concentrations of the drugs. The results of the displacement experiments showed that (i) the monomeric alkaloids compete with neither polyclonal nor monoclonal antibodies for tubulin binding; (ii) the dimer alkaloids displace the polyclonal but not the monoclonal antibodies from tubulin; and (iii) the inhibitory potencies of the bisindol alkaloids of different chemical structures are different. A new semisynthetic derivative of bisindol alkaloids, KAR-2, was found to be a powerful ligand in inhibiting both tubulin polymerization and immunocomplex formation. Colchicine did not inhibit binding of the antibodies to the immobilized tubulin. Competitive-displacement experiments were also designed to test the anti-tubulin activity of drugs in solution. The results suggest that while bisindol alkaloids interact with tubulin bound on surface or in solution, colchicine binds exhaustively to tubulin in solution and enhances the affinity of polyclonal antibodies probably via long-distance interactions between the binding domains in tubulin.