Elucidation of the Tubulin-targeted Mechanism of Action of 9-(3-pyridyl) Noscapine.

We have recently reported the synthesis and antiproliferative potential of a series of biaryl type α-noscapine congeners. Among them, 9-(3-pyridyl) noscapine 3f (9-PyNos, henceforth), which was synthesized by adding pyridine unit to the tetrahydroisoquinoline part of natural α-noscapine core, was found to be the most effective one to inhibit proliferation of a variety of cancer cell lines. However, details of its interactions with its cellular target, tubulin, remain poorly understood. In this report, we examined the nature of interactions of 9-PyNos with tubulin based on the methodologies of spectrofluorimetry, circular dichroism, and turbidimetry techniques. Far-UV circular dichroism spectra indicated perturbation of tubulin secondary structure in the presence of 9-PyNos, not amounting, however, to the perturbation induced by noscapine. The noscapinoid nevertheless altered the surface configuration of the protein considerably, as indicated by an anilinonaphthalene sulphonate binding assay, and promoted colchicine binding to tubulin, the latter indicating its adjacent binding site with colchicine. 9-PyNos however, did not alter microtubule assembly considerably. Investigating the possible reason behind this apparent lack of strong inhibition of microtubule assembly, we found that the binding interactions of tubulin with 9-PyNos do not involve modification of cysteine residues of tubulin. Taken together, our data suggest that the antiproliferative mechanism of action of 9-PyNos involves disruption of structural integrity of tubulin without strong inhibition of tubulin assembly.