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CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY

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

Drug Discovery Institute (M.B.T., C.A.K., B.P., P.W., B.W.D., A.P.D., C.E.R., J.S.L.), Departments of Pharmacology and Chemical Biology (M.B.T., C.A.K., A.P.D., J.S.L.), Chemistry (T.H.G., P.W.), Biological Sciences (F.L.X., W.S.S.), and Pharmaceutical Sciences (B.S.R., R.B., B.W.D.), and Center for Chemical Methodologies and Library Development (P.W.), University of Pittsburgh, Pittsburgh, Pennsylvania; and Medical Sciences Program, Indiana University, Bloomington, Indiana (J.R.S., C.E.W.)

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 µM, and inhibited [³H]vinblastine binding noncompetitively, with a K_i of 4.5 ± 0.6 µM. We also found noncompetitive inhibition of [³H]dolastatin 10 binding by disorazole C₁, with a K_i of 10.6 ± 1.5 µM, 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.

Address correspondence to: John S. Lazo, Department of Pharmacology and Chemical Biology, University of Pittsburgh Drug Discovery Institute, Biomedical Science Tower 3, Suite 10040, 3401 Fifth Avenue, University of Pittsburgh, Pittsburgh, PA 15260. E-mail: lazo{at}pitt.edu