Colchicine binding

Experiment 7. Alkaloid colchicine binds tubulin in cell structures. 

The 14C-chloroacetate of 7V-acetylcolchinol 199 and the 14C-isothiocyanate 200 were also found to react covalently with tubulin, but in a non-specific manner167, contrary to compounds 197 and 198 which react covalently with the colchicine binding site on tubulin with a -subunit a-subunit marking ratio169 of about 4 1. 

Colchicine (6) was isolated by Pelletier and Caventou in 1820 and is the main alkaloid of the poisonous meadow saffron plant (Colchicum autum-nale L.) [12-16]. Following some considerable debate over colchicine s structure [17-20] and its successful synthesis [21-26], colchicine was found to bind to tubulin at what is referred to as the colchicine binding site [1,27]. 

Combretastatins are a class of compounds originally derived from the African Willow tree (Combretum caffrum) and are powerful reversible inhibitors of tubulin polymerization. This class of molecules has been shown to bind to the colchicine binding site of tubulin, by the same mode of action as mentioned above (Sect. 1.2). Combretastatins consist of a ris-slilbcnc core structure. To date, there have been several compounds that have shown promise as potential anticancer drugs. However, development of these compounds as anticancer agents is limited by issues of chemical stability, bioavailibilty, toxicity, and solubility. 

The disappearance of tritiated vindesine from the blood of rats has been reported to be biphasic, with half-life estimates of 15 min (distribution) and 10 hr (elimination) (59) it is likely that the prolonged elimination phase represents a hybrid between the second elimination phase described above for vincristine and the prolonged third phase evident on inspection of log concentration-time plots for vincristine in the rat. Biliary excretion contributes heavily to the elimination of vindesine in the rat. The bioavailability of vindesine in the rat appears to be very poor. The distribution of vincristine to different tissues in the mouse has been correlated with the estimated concentration of tubulin in the tissues (40). Tubulin concentration was measured by the capacity of a tissue to bind colchicine (40) comparable relationships between tissue concentrations of vincristine and colchicine binding capacity were observed for the dog and the monkey, but it should be emphasized that the correlations were based on the assumption that tissue tubulin content is closely similar in the mouse, dog, and monkey. 

Cortese F, BhattacharyyaB, Wolff J, Podophyllo toxin as a probe for the colchicine binding site of tubulin, /5zh/ Chem 252 1134—1140, 1977. 

ITP = Inhibition of tubulin polymerization. b ICB = Inhibition of colchicine binding. 

In looking at these conclusions, one could speculate that rings A and C in these spindle toxins are most important, and they may bind to different loci on the colchicine binding site. It is suspected that two sites of tubulin which respectively bind to rings A and C of colchicine and its bioactive analogs are part of a polypeptide segment which is arranged in a helix, as shown in Fig. 28. 

Radiolabeled 3-demethyl-3-chloroacetylthiocolchicine with a l4C label in the chloroacetyl moiety (DCTC) was found to be a potent inhibitor of tubulin polymerization and of colchicine binding to tubulin. The reaction was 80-90% inhibited in the presence of saturating, amounts of known antitubulin compounds such as podophyllotoxin, combretastatin A-4, and colchicine itself. The tubulin /3 subunit was labeled 5-6 times faster than the a subunit. Cyanogen bromide digestion of the /3 subunit which had reacted covalently with DCTC indicated that at least three positions in /3-tubulin had reacted with DCTC. Purification and amino acid sequencing of these peptides are in progress (138). 

Small molecules modulating microtubule assembly have played major roles as tools in microtubule research, in a manner closely related to their chemotherapeutic interest [1], Tubulin was first purified in the last century as the colchicine-binding protein proposed to be the subunit of cellular microtubules [2], More recently, a colchicine derivative was employed to help crystallization and determine the structure of tubulin by X-ray diffraction [3], The colchicine, vinblastine [4] and paclitaxel [5] sites are main drug binding sites of tubulin, to which many other substances bind. The discovery of microtubule stabilization by paclitaxel [6] prompted its clinical development [7] and a burst of research on new MSAs, as well as the generalized use of paclitaxel or docetaxel as convenient reagents to assemble (see Fig. 1), stabilize or detect microtubules in the laboratory. One example is the development... 

Fig. 4 Diagram of the crystal structure of the T2R complex showing the binding sites of MT-destabilizing drugs (PDB entry 1Z2B) [13]. Protein subunits are represented as ribbons. RB3-SLD is colored orange, a-tubulin is purple, and p-tubulin is green. Small-molecule ligands are represented as spheres (vinblastine orange, colchicine red, GTP yellow, and GDP magenta). Colchicine binds to the p-subunit at the intradimer interface. Vinblastine binds at the interdimer interface...

The colchicine binding site in tubulin was initially identified using a colchicine derivative bearing a sulfhydryl group, permitting unambiguous orientation of the ligand in the electron density map [15]. We have also determined the structure of... 

Molecular Modeling on Compounds Targeting the Colchicine Binding Site... 

In 2000, Macdonald and co-workers reported CoMFA-based 3D-QSAR models able to describe the binding of different structural types of combretastatins to the colchicine binding site [26], The study resulted in the first significantly predictive models for the class of combretastatins. 

Table 2 Summary of hydrogen bond interactions that modeling studies suggested to occur between different CSI and residues of the colchicine binding site...

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