The effects of 6-benzyl-1,3-benzodioxole derivatives on the alkylation of tubulin.

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.

The effect of 2-(4-methyl-1-piperazinylmethyl) acrylophenone dihydrochloride on the alkylation of tubulin.

2-(4-Methyl-1-piperazinylmethyl) acrylophenone dihydrochloride (MPMAP) is a novel inhibitor of microtubule assembly in vitro and in vivo whose molecular mechanism of action has not been investigated (M. L. Mallevais, A. Delacourte, I. Lesieur, D. Lesieur, M. Cazin, C. Brunet, and M. Luyckx (1984) Biochimie 66, 477-482). We have examined the effect of MPMAP on the alkylation of tubulin by iodo[14C]acetamide and N,N'-ethylenebis(iodoacetamide) (EBI). MPMAP is a very potent inhibitor of tubulin alkylation by iodo[14C]acetamide. MPMAP gives half-maximal inhibition at a concentration of 15 microM. MPMAP also inhibits the alkylation of denatured tubulin and of aldolase, implying that it reacts strongly with sulfhydryl groups. MPMAP does not, however, interfere with formation by EBI of a crosslink between cysteines 239 and 354 in the beta subunit of tubulin, suggesting that these sulfhydryls are located in a cleft in the tubulin molecule.

beta 2-Tubulin, a form of chordate brain tubulin with lesser reactivity toward an assembly-inhibiting sulfhydryl-directed cross-linking reagent.

Beta 1 and beta 2 are the designations given to two forms of beta-tubulin that have different electrophoretic mobilities on discontinuous polyacrylamide gels in the presence of sodium dodecyl sulfate [Little, M. (1979) FEBS Lett. 108, 283-286]. Beta 1 and beta 2 constitute respectively 75% and 25% of the total beta-tubulin in bovine brain. Although beta 1 appears to be ubiquitous in animals, beta 2 has so far only been found in the brains of cows, pigs, deer, rats, chicks, and dogfish but not in squid brain. Beta 2 is not found in bovine kidneys, in porcine lungs, or in any nonchordate tubulin that has been examined. When tubulin is reacted with the sulfhydryl-directed reagent N,-N'-ethylenebis(iodoacetamide) (EBI), beta 1, but not beta 2, is converted to a faster moving form, beta. The yield of beta 2 in this reaction is not altered by the presence of drugs. When [14C]EBI is used as a probe, most of the label is incorporated into beta 1 rather than beta 2. Tubulin molecules that have reacted with EBI to form beta are much less likely to polymerize into microtubules than are molecules that have not formed beta. In view of the observation that only beta 1, and not beta 2, can form beta, it is possible that beta 1 represents a form of tubulin whose assembly may be regulated by a mechanism involving sulfhydryls. In contrast, beta 2 may represent a form of tubulin whose assembly is regulated by some other mechanism.