CIL-102 binds to tubulin at colchicine binding site and triggers apoptosis in MCF-7 cells by inducing monopolar and multinucleated cells.

A plant dictamine analog, 1-[4-(furo[2,3-b]quinolin-4-ylamino)phenyl]ethanone (CIL-102) has been shown to exert potent anti-tumor activity. In this study, we examined the mode of interaction of CIL-102 with tubulin and unraveled the cellular mechanism responsible for its anti-tumor activity. CIL-102 bound to tubulin at a single site with a dissociation constant ~0.4 μM. Isothermal titration calorimetry revealed that CIL-102-tubulin interaction is highly enthalpy driven and that the binding affords a large negative heat capacity change (ΔC(p) = -790 cal mol(-1) K(-1)) with an enthalpy-entropy compensation. An analysis of the modified Dixon plot suggested that CIL-102 competitively inhibited the binding of podophyllotoxin, a colchicine-binding site agent, to tubulin. Computational modeling indicated that CIL-102 binds exclusively at the β-subunit of tubulin and that CIL-102 and colchicine partially share their binding sites on tubulin. It bound to tubulin reversibly and the binding was estimated to be ~1000 times faster than that of colchicine. CIL-102 potently inhibited the proliferation of MCF-7 cells, induced monopolar spindle formation and multi-nucleation. At half-maximal inhibitory concentration, the spindle microtubules were visibly depolymerized and disorganized. CIL-102 reduced the inter-polar distances of bipolar mitotic cells indicating that it impaired microtubule-kinetochore attachments. CIL-102-treatment induced apoptosis in MCF-7 cells in association with increased nuclear accumulation of p53 and p21 suggesting that apoptosis is triggered through a p53-p21 dependent pathway. The results indicated that CIL-102 exerted anti-proliferative activity by disrupting microtubule functions through tubulin binding and provided important insights into the differential mode of tubulin binding by CIL-102 and colchicine.