Microtubule Binding and Disruption and Induction of Premature Senescence by Disorazole C1 □S

Disorazoles comprise a family of 29 macrocyclic polyketides isolated from the fermentation broth of the myxobacterium Sorangium cellulosum. The major fermentation product, disorazole A1, was found previously to irreversibly bind to tubulin and to have potent cytotoxic activity against tumor cells, possibly because of its highly electrophilic epoxide moiety. To test this hypothesis, we synthesized the epoxide-free disorazole C1 and found it retained potent antiproliferative activity against tumor cells, causing prominent G2/M phase arrest and inhibition of in vitro tubulin polymerization. Furthermore, disorazole C1 produced disorganized microtubules at interphase, misaligned chromosomes during mitosis, apoptosis, and premature senescence in the surviving cell populations. Using a tubulin polymerization assay, we found disorazole C1 inhibited purified bovine tubulin polymerization, with an IC50 of 11.8 0.4 M, and inhibited [ H]vinblastine binding noncompetitively, with a Ki of 4.5 0.6 M. We also found noncompetitive inhibition of [H]dolastatin 10 binding by disorazole C1, with a Ki of 10.6 1.5 M, indicating that disorazole C1 bound tubulin uniquely among known antimitotic agents. Disorazole C1 could be a valuable chemical probe for studying the process of mitotic spindle disruption and its relationship to premature senescence. Natural products have provided a plethora of pharmacologically useful drugs and chemical probes. The disorazole polyene macrodiolides were first isolated from the myxobacterium Sorangium cellulosum in 1994 and characterized to have significant antifungal activity with no antibacterial activity (Jansen et al., 1994). Initial biochemical and pharmacological studies were restricted to the major fermentation product, disorazole A1 (Fig. 1), which blocks cell proliferation, causes G2/M phase arrest and loss of microtubules, and induces apoptosis. Moreover, it blocks in vitro polymerization of tubulin (Elnakady et al., 2004; Kopp et al., 2005). Disorazole A1 contains a highly electrophilic divinyl oxirane moiety that we hypothesized might mediate the mitotic arrest and inhibition of tubulin polymerization through covalent binding to tubulin (Wipf et al., 2006). The highly electrophilic divinyl oxirane of disorazole A1 is generally not viewed as a therapeutically desirable moiety; therefore, we synthesized the rare family member disorazole C1 (Fig. 1), which is devoid of reactive groups. It is remarkable that disorazole C1 retained antimitotic activity (Wipf and Graham, 2004; Wipf et al., 2006). Structural analogs suggested that the functional group array of disorazole C1 and its three-dimensional conformation were critical for biological activity, but little is known about its mechanism of action. In the current comprehensive report, we demonstrate that disorazole C1 has potent antiproliferative activity against a wide variety of human tumor cells, disrupts cellular microtubule integrity, blocks tubulin polymerization in vitro, binds tubulin in a unique manner, and causes apoptosis and premature cellular senesThis work was supported by the National Institutes of Health [Grants CA078039 and CA097190; SPORE in Head and Neck Cancer]; the Fiske Drug Discovery Fund; and the METACyt Initiative of Indiana University [partially funded through a grant from the Lilly Endowment, Inc.]. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.108.147330. □S The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. ABBREVIATIONS: DMSO, dimethyl sulfoxide; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; BrdU, 5-bromo-2 deoxyuridine; JG-03-14, 2,4-dibromo-5-carbethoxyoy-3-(3,4-dimethyoxyphenyl)pyrrole. 0022-3565/09/3283-715–722$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 328, No. 3 Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics 147330/3439487 JPET 328:715–722, 2009 Printed in U.S.A. 715 http://jpet.aspetjournals.org/content/suppl/2008/12/09/jpet.108.147330.DC1 Supplemental material to this article can be found at: at A PE T Jornals on D ecem er 1, 2017 jpet.asjournals.org D ow nladed from