NBD-isocolcemid-tubulin interaction: a novel one-step reaction involving no conformational adjustment of reactants.

Isocolcemid, a colcemid analogue in which the positions of the C-ring methoxy and carbonyl are exchanged, is virtually inactive in binding to tubulin and inhibiting the formation of microtubule assembly. We have found that the substitution of a NBD group in the side chain of the B-ring of isocolcemid can reverse the effect of these structural alterations (at the C-ring) and the newly synthesized NBD-isocolcemid restores the lost biological activity. It inhibits microtubule assembly with an IC(50) of 12 microM and competes efficiently with [(3)H]colchicine, for binding to tubulin. NBD-isocolcemid has two binding sites on tubulin; one is characterized by fast binding, whereas the binding to the other site is slow. These two sites are independent and unrelated to each other. Colchicine and its analogues compete with NBD-isocolcemid for the slow site. Association and dissociation rate constants for the fast site, obtained from the stopped-flow measurements, are (7.37 +/- 0. 70) x 10(5) M(-1) s(-1) and 7.82 +/- 2.74 s(-1), respectively. While the interaction of colchicine and its analogues with tubulin involves two steps, NBD-isocolcemid binding to tubulin at the slow site has been found to be a one-step reaction. This is evident from the linear dependence of the observed rate constant (k(obs)) with both NBD-isocolcemid and tubulin concentrations. The interaction of NBD-isocolcemid with tubulin does not involve the conformational change of NBD-isocolcemid, as is evident from the unchanged CD spectra of the drug. The absence of enhanced GTPase activity of tubulin and the native-like protease cleavage pattern of the NBD-isocolcemid-tubulin complex suggest an unaltered conformation of tubulin upon NBD-isocolcemid binding to it as well. Implications of this on the mechanism of polymerization inhibition have been discussed.

Molecular dynamics simulation of colchicinoids.

Colchicine, a tricyclic alkaloid, has a remarkable range of biological activities. It binds with tubulin and prevents the formation of microtubules. This compound consists of a six membered aromatic ring (A ring), a seven membered troponoid ring (C ring) and another seven membered aliphatic ring (B ring). Using molecular mechanics and molecular dynamics simulations as tools, conformational analysis of colchicine and its several important analogs were done. Molecular mechanics studies show that conformational space of these molecules have one low energy region. Taking the low energy minima as the starting conformation, molecular dynamics simulation for 100 pico seconds is done for each of the analogs and molecular dynamics simulation in solution is done for three representative compounds colchicine,isocolchicine and A-C compound. Internal coordinate trajectories show that the value of the dihedral angle C9-C7-C1-C14 (phi), (C7-C1 bond connects the A and C ring), is within 40 degrees to 50 degrees for all the compounds with fluctuations less than 15 degrees. These calculations indicate that there is an overall similarity in the dynamically averaged structure of all the drugs. The A ring and B ring of the compounds are more or less rigid. The C ring is somewhat flexible, the average conformation and motional properties show overall similarity. The potential energy curve and dynamics behaviour of colchicine and isocolchicine suggests that the difference in binding property of colchine and isocolchicine may originate from the positional difference of carbonyl oxygen and methoxy group of C ring, which is the only difference in the structures of the two compounds and this has no effect on the motional property and average conformations of these two compounds. From our study it is proposed that the movements occuring at various positions of the drug molecules are significantly correlated. It is suggested that such correlated motion may play an important role in the biological property of these compounds.

N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)colcemid, a probe for different classes of colchicine-binding site on tubulin.

The nature of binding of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-colcemid (NBD-colcemid), an environment-sensitive fluorescent analogue of colchicine, to tubulin was tested. This article reports the first fluorometric study where two types of binding site of a colchicine analogue on tubulin were detected. Binding of NBD-colcemid to one of these sites equilibrates slowly. NBD-colcemid competes with colchicine for this site. Binding of NBD-colcemid to this site also causes inhibition of tubulin self-assembly. In contrast, NBD-colcemid binding to the other site is characterised by rapid equilibration and lack of competition with colchicine. Nevertheless, binding to this site is highly specific for the colchicine nucleus, as alkyl-NBD analogues have no significant binding activity. Fast-reaction-kinetic studies gave 1.76 x 10(5) M-1 s-1 for the association and 0.79 s-1 for the dissociation rate constants for the binding of NBD-colcemid to the fast site of tubulin. The association rate constants for the two phases of the slow site are 444.4 M-1 s-1 and 11.67 M-1 s-1 [corrected], respectively. These two sites may be related to the two sites of colchicine reported earlier, with binding characteristics altered by the increased hydrophobic nature of NBD-colcemid.

A fluorescent analog of colcemid, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-colcemid, as a probe for the colcemid-binding sites of tubulin and microtubules.

The synthesis and biological testing of the fluorescent analog of colcemid, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-colcemid (NBD-colcemid), are here described. NBD-colcemid exhibited a visible absorption maximum at 465 nm and fluoresced in the range of 520-540 nm, highly in environments of low polarity, whereas only slightly in aqueous solution. The addition of NBD-colcemid to bovine brain tubulin was accompanied by a striking enhancement of fluorescence. The fluorescent titration study suggested a stoichiometric binding of NBD-colcemid to tubulin. Assembled microtubules were directly visualized after mixing with NBD-colcemid using a fluorescence microscope. NBD-colcemid reversibly disrupted the metaphase spindles of sea urchin eggs as well as unlabeled colcemid. However, even when the birefringence of spindles was mostly lost, self-quenching properties of the NBD fluorescence allowed tubulin and its oligomers aggregated in higher concentrations in eggs to be visualized under a fluorescence microscope. The results suggest a wide applicability of NBD-colcemid as a fluorescent probe for studying the interactions of colcemid with tubulin and microtubules, as well as for localizing other colcemid-binding structures within cells.

Synthesis and biological effects of novel thiocolchicines. 3. Evaluation of N-acyldeacetylthiocolchicines, N-(alkoxycarbonyl) deacetylthiocolchicines, and O-ethyldemethylthiocolchicines. New synthesis of thiodemecolcine and antileukemic effects of 2-demethyl- and 3-demethylthiocolchicine.

Novel and known analogues of thiocolchicine were evaluated in vitro in a tubulin binding assay and in vivo in mice for acute toxicity and in the P388 lymphocytic leukemia assay. This evaluation included N-acyldeacetylthiocolchicines, N-(alkoxycarbonyl)deacetylthiocolchicines, thiodemecolcine and its methyl carbamate, and O-ethyl ethers of demethylthiocolchicines. Selective ether cleavage of thiodemecolcine with concentrated sulfuric acid at 50 degree C afforded the 2-demethyl congener, characterized as its N,O-diacetyl derivative. Several of the compounds showed high potency in the tubulin binding assay, matching the potency of colchicine. Several N-(alkoxycarbonyl)deacetylcolchicines (carbamates) exhibited strong binding affinity to tubulin but had only weak activities against the P388 tumor system, suggesting that other factors besides tubulin binding may be important for the biological effects. The compounds potent in the tubulin binding assay and in the P388 leukemia assay in mice were generally also toxic to mice in the acute toxicity test, showing thus a similar behavior of thiocolchicines to that observed earlier with colchicines. A considerable amount of data collected for 2-demethyl- and 3-demethylthiocolchicine suggests that the latter represents a broad-spectrum antitumor agent of considerable promise and possibly a less toxic substitute for colchicine.