Interaction of a new bis-indol derivative, KAR-2 with tubulin and its antimitotic activity

Abstract

1. KAR-2 (3′'-(β-chloroethyl)-2′',4′'-dioxo-3,5′'-spiro-oxazolidino-4-deacetoxy-vinblastine), is a bis-indol derivative; catharantine is coupled with the vindoline moiety which contains a substituted oxazolidino group. Our binding studies showed that KAR-2 exhibited high affinity for bovine purified brain tubulin (K_d=3 μM) and it inhibited microtubule assembly at a concentration of 10 nM.

2. Anti-microtubular activity of KAR-2 was highly dependent on the ultrastructure of microtubules: while the single tubules were sensitive, the tubules cross-linked by phosphofructokinase (ATP: D-fructose-6-phosphate-1-phosphotransferase, EC 2.7.1.11) exhibited significant resistance against KAR-2.

3. The cytoplasmic microtubules of Chinese hamster ovary mammalian and Sf9 insect cells were damaged by 1 μg ml⁻¹ KAR-2, as observed by indirect immunofluorescence and transmission electron microscopy. Scanning electron microscopy revealed intensive surface blebbing on both types of cells in the presence of KAR-2.

4. KAR-2 was effective in the mouse leukaemia P338 test in vivo without significant toxicity. Studies on a primary cerebro-cortical culture of rat brain and differentiated PC12 cells indicated that the toxicity of KAR-2 was significantly lower than that of vinblastine. The additional property of KAR-2 that distinguishes it from bis-indol derivatives is the lack of anti-calmodulin activity.

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