The Spongistatins, Potently Cytotoxic Inhibitors of Tubulin Polymerization, Bind in a Distinct Region of the Vinca Domain

Abstract

The highly cytotoxic, sponge-derived, antimitotic macrolide polyether spongistatin 1 has been previously shown to inhibit microtubule assembly, the binding of vinblastine and GTP to tubulin, and displacement of GDP bound in the exchangeable site of tubulin. We have now examined in detail inhibition by spongistatin 1 of both [3H]vinblastine and [3H]dolastatin 10 binding to tubulin. We found spongistatin 1 to be a noncompetitive inhibitor of the binding of both radiolabeled drugs to tubulin, in contrast to competitive patterns obtained with vincristine versus [3H]vinblastine and with a chiral isomer of dolastatin 10 versus [3H]dolastatin 10. Since dolastatin 10 is itself a noncompetitive inhibitor of vinca alkaloid binding to tubulin, this implies at least three distinct binding sites for the structurally complex and diverse natural products that interfere with each others binding to tubulin and with nucleotide exchange. Spongistatin 1, in contrast to both vinca alkaloids and peptide antimitotic agents like dolastatin 10, does not induce formation of a GTP-independent, morphologically distinctive polymer ("aggregate"). We also examined eight compounds closely related structurally to spongistatin 1 (spongistatins 2-9). The most distinctive in their properties were spongistatins 6 and 8. These two compounds, despite activity comparable to spongistatin 1 as inhibitors of tubulin polymerization and [3H]vinblastine binding, had much reduced activity as inhibitors of nucleotide exchange and [3H]dolastatin 10 binding. Spongistatins 1 and 6 were compared for effects on dolastatin 10-induced aggregate formation in conjunction with effects on [3H]dolastatin 10 binding. Spongistatin 6 was about 4-fold less active than spongistatin 1 as an inhibitor of aggregation and over 20-fold less active as an inhibitor of dolastatin 10 binding.

Interaction of cryptophycin 1 with tubulin and microtubules

“…Moreover, it inhibits VLB binding to tubulin but not binding of colchicine. Thus, cryptophycin 1 belongs to an ever growing list of compounds that apparently interact with the Vinca domain in tubulin, a list which includes maytansine, dolastatin 10, phomopsin A, halichondrin B (for a review, see [14]), and spongistatins [15]. Although cryptophycin 1 does appear to bind to the Vinca site, it bears no structural similarity to the Vinca alkaloids and it is possible that the cryptophycins bind to a site on tubulin which overlaps the Vinca alkaloid site.…”

Direct Photoaffinity Labeling by Dolastatin 10 of the Amino-terminal Peptide of β-Tubulin Containing Cysteine 12

“…Materials-Purified bovine brain tubulin with nonradiolabeled GDP (26) or [8][9][10][11][12][13][14] C]GDP (27) Synthesis of H]Dolastatin 10 -Dolastatin 10 was first converted to 5-bromo-dolastatin 10. The dolastatin 10 (30 mg) was dissolved in 0.2 ml of acetic acid with 40 mg of silver trifluoroacetate.…”

“…All vinca domain drugs that have been examined inhibit the formation of the Cys-12/Cys-211 cross-link, with vinblastine having the weakest effect (2,11,12). Second, all compounds that inhibit vinca alkaloid binding to tubulin also inhibit nucleotide exchange on ␤-tubulin (7,(13)(14)(15). Vinblastine, however, only weakly inhibits nucleotide exchange (16), and rhizoxin is moderately inhibitory (7).…”

(−)-Doliculide, a New Macrocyclic Depsipeptide Enhancer of Actin Assembly

“…Drug effects on growth were evaluated by an increase in cell protein as described previously (21). Cells were grown at 37°C in a humidified 5% CO 2 atmosphere.…”