Abstract
We have demonstrated that bis-indol Vinca alkaloids of anti-mitotic activities (vinblastine, vincristine and navelbine) bind to calmodulin in a Ca 2+-dependent manner. We designed direct binding tests (flourescence energy transfer and circular dichroism measurements) to quantify the interactions of bis-indol derivatives with calmodulin. The dissociation constants of calmodulin-navelbine and calmodulin-vinblastine complexes with 1:1 stoichiometry are 0.5 μM and 3 μM, respectively. These values indicate that the binding affinities of these Vinca alkaloids to calmodulin and tubulin are comparable. Immunological, enzyme kinetic and fluorescence anisotropy measurements showed that bis-indol alkaloids inhibit the interactions of calmodulin with target proteins. The results of indirect enzyme-linked immunosorbent assay showed that bis-indol alkaloids effectively antagonize with anti-calmodulin antibody for calmodulin binding (IC 50 = 90 μM and 430 μM for navelbine, vincristine and vinblastine, respectively). According to the fluorescence anisotropy and enzyme kinetic measurements, vinblastine, vincristine and navelbine, similarly to trifluoperazine, the classica calmodulin antagonist, compete with target enzyme [phosphofructokinase (ATP: D-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11)] for calmodulin binding and inhibit the calmodulin-mediated inactivation of the enzyme. Monomers, catharantine and vindoline, do not have an inhibitory effect either on immunocomplex formation or on calmodulin-enzyme interaction. Navelbine appeared in our tests as the most potent drug in inhibiting the association of calmodulin to target proteins in comparison to other bis-indol derivatives. Since navelbine and vinblastine posses identical vindoline moiety, although they differ in the catharantine part, the difference in anti-calmodulin potencies is suggested to reside predominantly on this portion of the molecules. These findings might establish the pharmacological importance of these activities in the specificity and toxicity of the drugs.
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More From: European Journal of Pharmacology: Molecular Pharmacology
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