Molecular modelling and competition binding study of Br-noscapine and colchicine provide insight into noscapinoid-tubulin binding site

Abstract

We have previously discovered the tubulin-binding anti-cancer properties of noscapine and its derivatives (noscapinoids). Here, we present three lines of evidence that noscapinoids bind at or near the well studied colchicine binding site of tubulin: (1) in silico molecular docking studies of Br-noscapine and noscapine yield highest docking score with the well characterised colchicine-binding site from the co-crystal structure; (2) the molecular mechanics-generalized Born/surface area (MM-GB/SA) scoring results ΔΔG(bind-cald) for both noscapine and Br-noscapine (3.915 and 3.025 kcal/mol) are in reasonably good agreement with our experimentally determined binding affinity (ΔΔG(bind-Expt) of 3.570 and 2.988 kcal/mol, derived from K(d) values); and (3) Br-noscapine competes with colchicine binding to tubulin. The simplest interpretation of these collective data is that Br-noscapine binds tubulin at a site overlapping with, or very close to colchicine-binding site of tubulin. Although we cannot rule out a formal possibility that Br-noscapine might bind to a site distinct and distant from the colchicine-binding site that might negatively influence the colchicine binding to tubulin.

Figure 1

Inhibition of tubulin-colchicine fluorescence by Br-noscapine and comparision with noscapine. A, B, and C show the molecular structures of colchicine, noscapine (Nos) and Br-noscapine (BNos), respectively. The fluorescence emission of tubulin-colchicine complex is inhibited by Br-noscapine in a concentration dependent manner ranging from 25 μM to 100 μM (D). However, noscapine inhibited tubulin-colchicine fluorescence modestly even at concentration as high as 100 uM (>10%, D). E, the percentage inhibition of fluorescence from colchicine-tubulin complex (it can be viewed as the inhibition of colchicine binding to tubulin).

Figure 2

Superimposition of docked configurations onto the crystal structure of colchicine (red stick). The RMSD (heavy atoms from core rings) = 0.19 –1.06 Å.

Figure 3. Comparison of colchicine, noscapine, and Br-noscapine with the colchicine binding site of tubulin

(A) Showing the localization of docked configurations of these ligands primarily in β-tubulin near the α- tubulin/β-tubulin interface. The amino acid residues involved in the interaction of Br-noscapine at the binding site is represented in (B).

Figure 4. Results of Glide XP docking of colchicine, noscapine, and Br-noscapine into the colchicine binding site of tubulin

2D representations of the binding site with amino acid residues involved in the interaction of (A) colchicine and (B) Br-noscapine reveal identical sets of amino acids. (C) 3D representations of the binding site amino acids within a 4Å distances from superimposed conformations of docked noscapine (blue) and Br-noscapine (green). The co-crystal structure of colchicine is represented in red color. Some of the amino acids (Leu248, Cys241, Val318, Ala316 and Val238) are not shown for better clarity. (D) Overlapped docking poses of colchicine, noscapine, and Br-noscapine obtained form Glide docking onto co-crystal of colchicine within the colchicine binding site of tubulin. Tubulin is represented as Macromodel surface according to residue charge (electropositive charge, blue; neutral, yellow) as implemented in Maestro.

Figure 5

Overlapped docking poses of colchicine obtained from (a) Glide docking (RMSD = 0.760 Å) and (b) QPLD (RMSD = 0.595 Å). In this figure the conformation of colchicine in the co-crystal structure is represent in red color. Comparison between force field charges and quantum mechanical charges for colchicine, Br-noscapine and noscapine from standard Glide docking and QPLD docking.