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Two related compounds, 1,8-anilinonaphthalenesulfonate (1,8-ANS) and bis(1,8-anilinonaphthalenesulfonate) (Bis-ANS), are useful fluorescent probes for hydrophobic areas on protein molecules. Using fluorescence, we examined the binding of these compounds to bovine brain tubulin and found that Bis-ANS and 1,8-ANS bound to tubulin with Ki values of 2 and 25 microM, respectively. Bis-ANS potently inhibited the polymerization of tubulin into microtubules in vitro. In the presence of microtubule-associated protein 2, half-maximal inhibition of assembly was obtained at 3 microM Bis-ANS. In the presence of tau protein, half-maximal inhibition was obtained at 15 microM Bis-ANS. Surprisingly, 1,8-ANS, even at 200 microM, did not inhibit assembly. Scatchard analysis indicated one binding site for Bis-ANS on tubulin. Previous reports of 1,8-ANS binding to tubulin may have been influenced by the presence of Bis-ANS which until recently was a common contaminant of commercial supplies. Because of its intense fluorescence in addition to its potent inhibitory effects, Bis-ANS appears to be a useful probe to study microtubule assembly and other interactions involving tubulin.

A new class of antimitotic agents, derivatives of 2-styrylquinazolin-4(3H)-one (SQZ), was recently described [J. Med. Chem. 33:1721-1728 (1990)]. Because they appeared to interact at a new ligand binding site on tubulin, we attempted to determine their mechanism of action as inhibitors of tubulin polymerization. Although in initial studies inhibition of colchicine binding was negligible, substantial and competitive inhibition of this reaction could be demonstrated with very short incubation times (less than 5 min), provided that a relatively low colchicine to tubulin ratio was used. The initial apparent failure to inhibit colchicine binding resulted from extremely rapid binding to tubulin and dissociation from tubulin by the SQZ derivatives, in comparison with the slow, temperature-dependent, poorly reversible binding of colchicine. The most inhibitory of the SQZ derivatives in the colchicine binding assay was 6-methyl-2-styrylquinazolin-4(3H)-one (NSC 379310), and its interaction with tubulin, particularly as an inhibitor of colchicine binding, was compared with that of 2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone (MTPT), because the binding parameters of MTPT with tubulin have been well described. The data indicate that NSC 379310 binds to tubulin and dissociates from the protein about 3 times as rapidly as MTPT. The other SQZ derivatives with equal or greater potency as inhibitors of tubulin polymerization but apparently less potency as inhibitors of colchicine binding presumably bind to and/or dissociate from tubulin even more rapidly than does NSC 379310.

Derivatives of 6-benzyl-1,3-benzodioxole are known to bind to tubulin and inhibit tubulin polymerization. For a better understanding of the mechanism of action of the 6-benzyl-1,3-benzodioxole derivatives, we have examined their effect on the alkylation of tubulin sulfhydryls by iodo[14C]acetamide and N,N'-ethylene(bis)iodoacetamide. We have found that the 6-benzyl-1,3-benzodioxole derivatives with an intact dioxole ring affect alkylation to an extent proportional to their ability to inhibit tubulin polymerization. Those derivatives with the strongest resemblance to podophyllotoxin have the weakest effects. However, derivatives with a disrupted dioxole ring show little or no ability to inhibit polymerization, but their effect on alkylation is directly related to the degree of resemblance they bear to the trimethoxy ring of podophyllotoxin. It thus appears that the relatively simple approach of using alkylating agents can generate a significant amount of information on the mechanism by which various drugs interact with tubulin.

Nuclear magnetic resonance was used to measure spin-lattice water proton relaxation times (T1) during the first cell cycle in sea-urchin zygotes of packed Strongylocentrotus purpuratus. Following insemination there was a 90% increase in the T1 value. The increase in T1 at fertilization could be accounted for by the accumulation of extracellular fluid between the egg surface and the fertilization envelope. The T1 value then remained without change during the first cell cycle, except at metaphase when there was a significant 13% decrease. The lowered T1 values measured at metaphase were not related to a change in the water content of the packed cells, which remained fairly constant throughout the cell cycle. High hydrostatic pressure, low temperature and colchicine (agents that depolymerize mitotic apparatus microtubules) did not affect the T1 values in fertilized eggs. Treatment in vitro of a microtubule protein preparation with low temperature and colchicine resulted in an increased T1, which accompanied the depolymerization of microtubule protein. Since depolymerization of the microtubules associated with the mitotic apparatus by high pressure, colchicine or low temperature does not alter the T1 of water protons in the cell, it is proposed that the increased state of ordered water molecules at metaphase is maintained by non-microtubular factor(s) of the metaphase egg.

Pulse-labeling studies demonstrate that tubulin synthesized in the neuron cell body (soma) moves somatofugally within the axon (at a rate of several millimeters per day) as a well-defined wave corresponding to the slow component of axonal transport. A major goal of the present study was to determine what proportion of the tubulin in mature motor axons is transported in this wave. Lumbar motor neurons in 9-wk-old rats were labeled by injecting [35S]methionine into the spinal cord 2 wk after motor axons were injured (axotomized) by crushing the sciatic nerve. Immunoprecipitation with mAbs which recognize either class II or III beta-tubulin were used to analyze the distributions of radioactivity in these isotypes in intact and axotomized motor fibers 5 d after labeling. We found that both isotypes were associated with the slow component wave, and that the leading edge of this wave was enriched in the class III isotype. Axotomy resulted in significant increases in the labeling and transport rates of both isotypes. Immunohistochemical examination of peripheral nerve fibers demonstrated that nearly all of the class II and III beta-tubulin in nerve fibers is located within axons. Although the amounts of radioactivity per millimeter of nerve in class II and III beta-tubulin were significantly greater in axotomized than in control nerves (with increases of +160% and +58%, respectively), immunoassay revealed no differences in the amounts of these isotypes in axotomized and control motor fibers. We consider several explanations for this paradox; these include the possibility that the total tubulin content is relatively insensitive to changes in the amount of tubulin transported in the slow component wave because this wave represents the movement of only a small fraction of the tubulin in these motor fibers.

Antitumor active hydroxyquinoline derivatives such as (I) ‐ (III) are synthesized.