Highly dynamic mitotic-spindle microtubules are among the most successful targets for anticancer therapy. Microtubule-targeted drugs, including paclitaxel and Vinca alkaloids, were previously considered to work primarily by increasing or decreasing the cellular microtubule mass. Although these effects might have a role in their chemotherapeutic actions, we now know that at lower concentrations, microtubule-targeted drugs can suppress microtubule dynamics without changing microtubule mass; this action leads to mitotic block and apoptosis. In addition to the expanding array of chemically diverse antimitotic agents, some microtubule-targeted drugs can act as vascular-targeting agents, rapidly depolymerizing microtubules of newly formed vasculature to shut down the blood supply to tumours.

Microtubules as a target for anticancer drugs.

Exceptional advances in molecular biology and genetic research have expedited cancer drug development tremendously. The declared paradigm is the development of 'personalized and tailored drugs' that precisely target the specific molecular defects of a cancer patient. It is therefore appropriate to revisit the intellectual foundations of the development of such agents, as many have shown great clinical success. One hundred years ago, Paul Ehrlich, the founder of chemotherapy, received the Nobel Prize for Physiology or Medicine. His postulate of creating 'magic bullets' for use in the fight against human diseases inspired generations of scientists to devise powerful molecular cancer therapeutics.

Paul Ehrlich's magic bullet concept: 100 years of progress.

Since its approval by the FDA in 1992 for the treatment of ovarian cancer, the use of Taxol has dramatically increased. Although treatment with Taxol has led to improvement in the duration and quality of life for some cancer patients, the majority eventually develop progressive disease after initially responding to Taxol treatment. Drug resistance represents a major obstacle to improving the overall response and survival of cancer patients. This review focuses on mechanisms of Taxol resistance that occur directly at the microtubule, such as mutations, tubulin isotype selection and post-translational modifications, and also at the level of regulatory proteins. A review of tubulin structure, microtubule dynamics, the mechanism of action of Taxol and its binding site on the microtubule are included, so that the reader can evaluate Taxol resistance in context.

Mechanisms of Taxol resistance related to microtubules.

BMS-247550, a novel epothilone derivative, is being developed by Bristol-Myers Squibb Company (BMS) as an anticancer agent for the treatment of patients with malignant tumors. BMS-247550 is a semisynthetic analogue of the natural product epothilone B and has a mode of action analogous to that of paclitaxel ( i.e. , microtubule stabilization). In vitro , it is twice as potent as paclitaxel in inducing tubulin polymerization. Like paclitaxel, BMS-247550 is a highly potent cytotoxic agent capable of killing cancer cells at low nanomolar concentrations. Importantly, BMS-247550 retains its antineoplastic activity against human cancers that are naturally insensitive to paclitaxel or that have developed resistance to paclitaxel, both in vitro and in vivo . Tumors for which BMS-247550 demonstrated significant antitumor activity encompass both paclitaxel-sensitive and -refractory categories, i.e. , ( a ) paclitaxel-resistant: HCT116/VM46 colorectal (multidrug resistant), Pat-21 breast and Pat-7 ovarian carcinoma (clinical isolates; mechanisms of resistance not fully known), and A2780Tax ovarian carcinoma (tubulin mutation); ( b ) paclitaxel-insensitive: Pat-26 human pancreatic carcinoma (clinical isolate) and M5076 murine fibrosarcoma; and ( c ) paclitaxel sensitive: A2780 ovarian, LS174T, and HCT116 human colon carcinoma. In addition, BMS-247550 is p.o. efficacious against preclinical human tumor xenografts grown in immunocompromised mice or rats. Schedule optimization studies indicate that BMS-247550 is efficacious when administered frequently (every 2 days × 5) or intermittently (every 4 days × 3 or every 8 days × 2). These efficacy data demonstrate that BMS-247550 has the potential to surpass Taxol® in both clinical efficacy and ease of use ( i.e. , less frequent treatment schedule and/or oral administration).

https://clincancerres.aacrjournals.org/content/clincanres/7/5/1429.full.pdf

BMS-247550: a novel epothilone analog with a mode of action similar to paclitaxel but possessing superior antitumor efficacy.

Recent preclinical studies have shown that frequent administration in vivo of low doses of chemotherapeutic drugs (“metronomic” dosing) can affect tumor endothelium and inhibit tumor angiogenesis, reducing significant side effects (e.g., myelosuppression) involving other tissues, even after chronic treatment. This suggests that activated endothelial cells may be more sensitive, or even selectively sensitive, to protracted (“high-time”) low-dose chemotherapy compared with other types of normal cells, thus creating a potential therapeutic window. To examine this hypothesis, we assessed the effects of several different chemotherapeutic drugs—namely paclitaxel, 4-hydroperoxycyclophosphamide, BMS-275183 (an oral taxane), doxorubicin, epothilone B (EpoB) and its analogue 5-methylpyridine EpoB—on human microvascular or macrovascular endothelial cells, fibroblasts, and drug-sensitive or multidrug-resistant breast cancer cell lines in cell culture, using both short-term (24 h) versus long-term (144 h), continuous exposures, where drug-containing medium was replaced every 24 h. Whereas little differential and only weak effects were observed using the short-term exposure, a striking trend of comparative vascular endothelial cell hypersensitivity was induced using the continuous long-term exposure protocol. Potent differential growth inhibition effects as well as induction of apoptosis were observed with IC50 values in the range of 25–143 pm for paclitaxel, BMS-275183, EpoB, and 5-methylpyridine-EpoB. In contrast, the IC50 values for tumor cells and fibroblasts tested were in the range of 500 pm to >1 nm for these drugs. Similar differential IC50 values were noted using 4-hydroperoxycyclophosphamide. The results are consistent with the possibility that continuous low-dose therapy with various chemotherapeutic drugs may have a highly selective effect against cycling vascular endothelial cells, and may be relevant to the use of continuous or frequent administration of low doses of certain types of drugs as an optimal way of delivering antiangiogenic therapy.

/pdf/protracted-low-dose-effects-on-human-endothelial-cell-2sjv80komo.pdf

Protracted low-dose effects on human endothelial cell proliferation and survival in vitro reveal a selective antiangiogenic window for various chemotherapeutic drugs.

The epothilones are naturally occurring antimitotic drugs that share with the taxanes a similar mechanism of action without apparent structural similarity. Although photoaffinity labeling and electron crystallographic studies have identified the taxane-binding site on β-tubulin, similar data are not available for epothilones. To identify tubulin residues important for epothilone binding, we have isolated two epothilone-resistant human ovarian carcinoma sublines derived in a single-step selection with epothilone A or B. These epothilone-resistant sublines exhibit impaired epothilone- and taxane-driven tubulin polymerization caused by acquired β-tubulin mutations (β274Thr→Ile and β282Arg→Gln) located in the atomic model of αβ-tubulin near the taxane-binding site. Using molecular modeling, we investigated the conformational behavior of epothilone, which led to the identification of a common pharmacophore shared by taxanes and epothilones. Although two binding modes for the epothilones were predicted, one mode was identified as the preferred epothilone conformation as indicated by the activity of a potent pyridine-epothilone analogue. In addition, the structure–activity relationships of multiple taxanes and epothilones in the tubulin mutant cells can be fully explained by the model presented here, verifying its predictive value. Finally, these pharmacophore and activity data from mutant cells were used to model the tubulin binding of sarcodictyins, a distinct class of microtubule stabilizers, which in contrast to taxanes and the epothilones interact preferentially with the mutant tubulins. The unification of taxane, epothilone, and sarcodictyin chemistries in a single pharmacophore provides a framework to study drug–tubulin interactions that should assist in the rational design of agents targeting tubulin.

/pdf/a-common-pharmacophore-for-epothilone-and-taxanes-molecular-4psssytdbq.pdf

A common pharmacophore for epothilone and taxanes: Molecular basis for drug resistance conferred by tubulin mutations in human cancer cells

The efficacy of anticancer therapy is limited by the development of drug resistance. While the role of p53 in the intrinsic sensitivity of human cancer cells to paclitaxel (PTX) remains controversial, its role in acquired paclitaxel resistance has never been addressed. In this study we examined the p53 status of three paclitaxel selected human ovarian carcinoma sublines, resistant to paclitaxel due to acquired beta-tubulin mutations which impair paclitaxel's interaction with tubulin. In contrast to parental cells which have wt p53, in all PTX-resistant sublines p53 was functionally inactive. Two of the resistant sublines expressed high levels of transcriptionally inactive p53 protein, each with a distinct point mutation in codons 236 and 239 of the DNA binding domain. The third subline presented a novel p53 pseudo-null phenotype as a result of markedly decreased wt p53 mRNA expression. Introduction of ectopic wt p53 had no effect on PTX sensitivity in both parental and resistant cells, while it induced p21WAF1/CIP1, demonstrating an intact p53 pathway. While PTX resistance is primarily conferred by the tubulin mutations, the loss of functional p53 observed in all clones, suggests that this loss may facilitate the development of resistance potentially by providing a clonal advantage which promotes the isolation of paclitaxel resistant cells.

/pdf/paclitaxel-selects-for-mutant-or-pseudo-null-p53-in-drug-iz78bj7bu5.pdf

George Poy

Papers

A common pharmacophore for epothilone and taxanes: Molecular basis for drug resistance conferred by tubulin mutations in human cancer cells

Paclitaxel selects for mutant or pseudo-null p53 in drug resistance associated with tubulin mutations in human cancer