Microtubule binding and disruption and induction of premature senescence by disorazole C(1).

Disorazoles comprise a family of 29 macrocyclic polyketides isolated from the fermentation broth of the myxobacterium Sorangium cellulosum. The major fermentation product, disorazole A(1), 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 C(1) and found it retained potent antiproliferative activity against tumor cells, causing prominent G(2)/M phase arrest and inhibition of in vitro tubulin polymerization. Furthermore, disorazole C(1) 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 C(1) inhibited purified bovine tubulin polymerization, with an IC(50) of 11.8 +/- 0.4 microM, and inhibited [3H]vinblastine binding noncompetitively, with a K(i) of 4.5 +/- 0.6 microM. We also found noncompetitive inhibition of [3H]dolastatin 10 binding by disorazole C(1), with a K(i) of 10.6 +/- 1.5 microM, indicating that disorazole C(1) bound tubulin uniquely among known antimitotic agents. Disorazole C(1) could be a valuable chemical probe for studying the process of mitotic spindle disruption and its relationship to premature senescence.

Cellular analysis of disorazole C and structure-activity relationship of analogs of the natural product.

Structure-activity analyses of synthetic disorazole C(1) and eight of its analogs indicate that the presence of a vinyl oxirane moiety or a tetraene sequence is not necessary for potent cytotoxic and antimitotic properties. Using an automated multiparameter fluorescence-based cellular assay to simultaneously probe the effects of disorazole analogs on cellular microtubules, mitotic arrest, and cytotoxicity, we found that disorazole C(1) enhanced the mitotic index and chromatin condensation and arrested cells in the G2/M phase of the cell cycle. All structural analogs and synthesis precursors of disorazole C(1) were at least two orders of magnitude less potent than the parent compound, thus indicating that both the functional group array and the three-dimensional conformation of the parent compound are critical for interaction with the biological target. We conclude that disorazole C(1) is a potent inducer of mitotic arrest and hypothesize that this biological activity may be mediated by microtubule perturbation.

Dual specificity protein phosphatases: therapeutic targets for cancer and Alzheimer's disease.

The complete sequencing of the human genome is generating many novel targets for drug discovery. Understanding the pathophysiological roles of these putative targets and assessing their suitability for therapeutic intervention has become the major hurdle for drug discovery efforts. The dual-specificity phosphatases (DSPases), which dephosphorylate serine, threonine, and tyrosine residues in the same protein substrate, have important roles in multiple signaling pathways and appear to be deregulated in cancer and Alzheimer's disease. We examine the potential of DSPases as new molecular therapeutic targets for the treatment of human disease.

Regulation of Cdc25A half-life in interphase by cyclin-dependent kinase 2 activity.

Cdc25A regulates cell cycle progression, has oncogenic and anti-apoptotic activity, and is over-expressed in many human tumors. Phosphorylation by Chk1 and Cds1/Chk2 down-regulates Cdc25A levels in response to genotoxic stresses. Nevertheless, it remains unclear whether Chk1 and Cds1/Chk2 are uniquely responsible for regulating Cdc25A stability during interphase or if other kinase activities contribute. Here we report that treatment of HeLa cells with the cyclin-dependent kinase inhibitor roscovitine caused a concentration- and time-dependent increase in Cdc25A protein levels. Transfection with dominant-negative Cdk mutants demonstrated that only a Cdk2 mutant increased Cdc25A protein levels; Cdk1 and Cdk3 mutants had no effect. The increased Cdc25A protein levels were the result of an increase in the half-life of the protein; no increase in Cdc25A mRNA levels was observed. These results demonstrate Cdk2 kinase activity contributes to the labile nature of Cdc25A during interphase and redefine the nature of the Cdc25A-Cdk2 autoamplification feedback loop.

Dual-specificity phosphatases as targets for antineoplastic agents.

Dual-specificity protein phosphatases are a subclass of protein tyrosine phosphatases that are uniquely able to hydrolyse the phosphate ester bond on both a tyrosine and a threonine or serine residue on the same protein. Dual-specificity phosphatases have a central role in the complex regulation of signalling pathways that are involved in cell stress responses, proliferation and death. Although this enzyme family is increasingly the target of drug discovery efforts in pharmaceutical companies, a summary of the salient developments in the biology and medicinal chemistry of dual-specificity phosphatases has been lacking. We hope that this comprehensive overview will stimulate further progress in the development of small-molecule inhibitors that could form the basis for a new class of target-directed therapeutic agents.