Fig 8 - uploaded by Susan Bane
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![Stereoview of one of the tubulin-colchicine complexes from the crystal structure of tubulin/ colchicine:stathmin-like domain complex (PDB 1SA0, [110]). The backbone of the α-subunit is shown in light blue and the backbone of the β-subunit is colored yellow. DAMA-colchicine (Fig. 2) is colored by atom type (green-carbon, red-oxygen, blue-nitrogen, yellow-sulfur; hydrogen atoms are not shown). Cys-241 (left) and Cys-356 (right) are shown in black. The backbone of the β-subunit peptide 1-41 is shown in dark blue and the backbone of the β-subunit peptide 214-241 is shown in pink. To view this figure in color, see the insert and the companion CD-ROM.](profile/Susan-Bane/publication/226075450/figure/fig5/AS:1009266328604673@1617639252338/Stereoview-of-one-of-the-tubulin-colchicine-complexes-from-the-crystal-structure-of.png)
Stereoview of one of the tubulin-colchicine complexes from the crystal structure of tubulin/ colchicine:stathmin-like domain complex (PDB 1SA0, [110]). The backbone of the α-subunit is shown in light blue and the backbone of the β-subunit is colored yellow. DAMA-colchicine (Fig. 2) is colored by atom type (green-carbon, red-oxygen, blue-nitrogen, yellow-sulfur; hydrogen atoms are not shown). Cys-241 (left) and Cys-356 (right) are shown in black. The backbone of the β-subunit peptide 1-41 is shown in dark blue and the backbone of the β-subunit peptide 214-241 is shown in pink. To view this figure in color, see the insert and the companion CD-ROM.
Source publication
Colchicine is one of the oldest known antimicrotubule drugs. It exerts its biological effects by binding to a single site
on the β-subunit of the tubulin heterodimer. The resulting colchicine-tubulin complex substiochiometrically inhibits tubulin
assembly and suppresses microtubule dynamics. A large number of molecules with significant structural d...
Context in source publication
Context 1
... labeling performed with [ 3 H]colchicine identified two β-tubulin peptides: β1-41 and β214-241. Figure 8 shows a stereoview of the X-ray structure in which the two peptides are highlighted. The β214-241 peptide forms part of the colchicine-binding site, but the β1-41 peptide does not, although the C terminal amino acids of this peptide are in the vicinity of the colchicine-binding site. ...
Citations
... Colchicine, a highly toxic molecule extracted from Colchicum autumnale L. (commonly known as meadow saffron) is the first discovered microtubule destabilizing agent (Massarotti et al., 2012;Peterson and Mitchison, 2002;Weisenberg et al., 1968) which was confirmed by the crystal structure of colchicine structural analog, N-deacetyl-N-(2-mercaptoacetyl) colchicine (DAMA-colchicine) in complex with tubulin heterodimer (PDB entry: 1SA0; Ravelli et al., 2004) (Fig. 1). The binding of colchicine to tubulin substiochiometrically blocks tubulin assembly and diminishes microtubule disassembly (Susan, 2008). The classification of a microtubule assembly inhibiting substance as colchicine site ligand when introduced with [ 3 H] colchicine with an implicit assumption that colchicine site ligand occupies the same binding pocket of colchicine, formed the major roadblock for identifying the exact binding positions of new leads inside the colchicine binding domain. ...
... The classification of a microtubule assembly inhibiting substance as colchicine site ligand when introduced with [ 3 H] colchicine with an implicit assumption that colchicine site ligand occupies the same binding pocket of colchicine, formed the major roadblock for identifying the exact binding positions of new leads inside the colchicine binding domain. The pragmatic assumption of competitive inhibition taking place without explicit verification in this case (Susan, 2008;Hastie, 1991) was unraveled by the advent of numerous crystal structures of tubulin in complex with colchicine site molecules along with biochemical characterizations in eukaryotes. ...
... The structural location on H. contortus b-tubulin model showed that zones-1 and À2 amino acids were mostly overlapped whereas zone-3 residues were deeply buried. The non-similarity with corresponding SIFt pattern strongly suggests the existence of multiple pharmacophores (Susan, 2008) and differential modes of ligand binding mechanism (David-Pfeuty et al., 1979;Lin and Hamel, 1981). ...
The oxetane functional group offers a variety of potential advantages when incorporated within appropriate therapeutic agents as a ketone surrogate. OXi8006, a 2-aryl-3-aroyl-indole analogue, functions as a small-molecule inhibitor of tubulin polymerization that has a dual mechanism of action as both an antiproliferative agent and a tumor-selective vascular disrupting agent. Replacement of the bridging ketone moiety in OXi8006 with an oxetane functional group has expanded structure activity relationship (SAR) knowledge and provided insights regarding oxetane incorporation within this class of molecules. A new synthetic method using an oxetane-containing tertiary alcohol subjected to Lewis acid catalyzed conditions led to successful Friedel-Crafts alkylation and yielded fourteen new oxetane-containing indole-based molecules. This synthetic approach represents the first method to successfully install an oxetane ring at the 3-position of a 2-aryl-indole system. Several analogues showed potent cytotoxicity (micromolar GI50 values) against human breast cancer cell lines (MCF-7 and MDA-MB-231) and a pancreatic cancer cell line (PANC-1), although they proved to be ineffective as inhibitors of tubulin polymerization. Molecular docking studies comparing colchicine with the OXi8006-oxetane analogue 5m provided a rationale for the differential interaction of these molecules with the colchicine site on the tubulin heterodimer.
Tubulin, the basic component of microtubules, is present in most eukaryotic cells as multiple gene products, called isotypes. The major tubulin isotypes are highly conserved in terms of structure and drug binding capabilities. Tubulin isotype betaVI, however, is significantly divergent from the other isotypes in sequence, assembly properties, and function. It is the major beta-tubulin isotype of hematopoietic tissue and forms the microtubules of platelet marginal bands. The interaction of the major tubulin isotypes betaI, betaII, betaIII, and betaIotaV with antimicrotubule drugs has been widely studied, but little is known about the drug binding properties of tubulin isotype betaVI. In this investigation, we characterize the activity of various colchicine site ligands with tubulin isolated from Gallus gallus erythrocytes (CeTb), which is approximately 95% betaVI. Colchicine binding is thought to be a universal property of higher eukaryotic tubulin; however, we were unable to detect colchicine binding to CeTb under any experimental conditions. Podophyllotoxin and nocodazole, other colchicine site ligands with divergent structures, were able to inhibit paclitaxel-induced CeTb assembly. Surprisingly, the colchicine isomer allocolchicine also inhibited CeTb assembly and displayed measurable, moderate affinity for CeTb (K(a) = 0.18 x 10(5) M(-1) vs 5.0 x 10(5) M(-1) for bovine brain tubulin). Since allocolchicine and colchicine differ in their C ring structures, the two C ring colchicine analogues were also tested for CeTb binding. Kinetic experiments indicate that thiocolchicine and chlorocolchicine bind to CeTb, but very slowly and with low affinity. Molecular modeling of CeTb identified five divergent amino acid residues within 6 A of the colchicine binding site compared to betaI, betaII, and betaIV; three of these amino acids are also altered in betaIII-tubulin. Interestingly, the altered amino acids are in the vicinity of the A ring region of the colchicine binding site rather than the C ring region. We propose that the amino acid differences in the binding site constrict the A ring binding domain in CeTb, which interferes with the positioning of the trimethoxyphenyl A ring and prevents C ring binding site interactions from efficiently occurring. Allocolchicine is able to accommodate the altered binding mode because of its smaller ring size and more flexible C ring substituents. The sequence of the colchicine binding domain of CeTb isotype betaVI is almost identical to that of its human hematopoietic counterpart. Thus, through analysis of the interactions of ligands with CeTb, it may be possible to discover colchicine site ligands that specifically target tubulin in human hematopoietic cells.
