Anti-calmodulin potency of indol alkaloids in in vitro systems.
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 (fluorescence 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 microM and 3 microM, 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 (IC50 = 90 microM, 400 microM, and 430 microM for navelbine, vincristine and vinblastine, respectively). According to the fluorescence anisotropy and enzyme kinetic measurements, vinblastine, vincristine and vinblastine, similarly to trifluoperazine, the classic calmodulin antagonist, compete with target enzyme [phosphofructokinase (ATP: D-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11)] for 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 possess 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.
Molnár
A
A
Institute of Enzymology, Hungarian Academy of Sciences, Budapest, Hungary.
Liliom
K
K
Orosz
F
F
Vértessy
B G
BG
Ovádi
J
J
D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000378:metabolism*; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D066298:In Vitro Techniques; D007700:Kinetics; D010732:Phosphofructokinase-1 / Q000378:metabolism; D014748:Vinca Alkaloids / Q000378:metabolism*
D000972:Antineoplastic Agents, Phytogenic; D002147:Calmodulin; D014748:Vinca Alkaloids; D010732:Phosphofructokinase-1; D002118:Calcium