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.

The influence of natural products upon drug discovery.

Cyclin-dependent kinases (cdk) play an essential role in the intracellular control of the cell division cycle (cdc). These kinases and their regulators are frequently deregulated in human tumours. Enzymatic screening has recently led to the discovery of specific inhibitors of cyclin-dependent kinases, such as butyrolactone I, flavopiridol and the purine olomoucine. Among a series of C2, N6, N9-substituted adenines tested on purified cdc2/cyclin B, 2-(1-ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine (roscovitine) displays high efficiency and high selectivity towards some cyclin-dependent kinases. The kinase specificity of roscovitine was investigated with 25 highly purified kinases (including protein kinase A, G and C isoforms, myosin light-chain kinase, casein kinase 2, insulin receptor tyrosine kinase, c-src, v-abl). Most kinases are not significantly inhibited by roscovitine. cdc2/cyclin B, cdk2/cyclin A, cdk2/cyclin E and cdk5/p35 only are substantially inhibited (IC50 values of 0.65, 0.7, 0.7 and 0.2 microM, respectively). cdk4/cyclin D1 and cdk6/cyclin D2 are very poorly inhibited by roscovitine (IC50 > 100 microM). Extracellular regulated kinases erk1 and erk2 are inhibited with an IC50 of 34 microM and 14 microM, respectively. Roscovitine reversibly arrests starfish oocytes and sea urchin embryos in late prophase. Roscovitine inhibits in vitro M-phase-promoting factor activity and in vitro DNA synthesis in Xenopus egg extracts. It blocks progesterone-induced oocyte maturation of Xenopus oocytes and in vivo phosphorylation of the elongation factor eEF-1. Roscovitine inhibits the proliferation of mammalian cell lines with an average IC50 of 16 microM. In the presence of roscovitine L1210 cells arrest in G1 and accumulate in G2. In vivo phosphorylation of vimentin on Ser55 by cdc2/cyclin B is inhibited by roscovitine. Through its unique selectivity for some cyclin-dependent kinases, roscovitine provides a useful antimitotic reagent for cell cycle studies and may prove interesting to control cells with deregulated cdc2, cdk2 or cdk5 kinase activities.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1997.t01-2-00527.x

Biochemical and Cellular Effects of Roscovitine, a Potent and Selective Inhibitor of the Cyclin‐Dependent Kinases cdc2, cdk2 and cdk5

Natural products to drugs: natural product derived compounds in clinical trials

Microbes have made a phenomenal contribution to the health and well-being of people throughout the world. In addition to producing many primary metabolites, such as amino acids, vitamins and nucleotides, they are capable of making secondary metabolites, which constitute half of the pharmaceuticals on the market today and provide agriculture with many essential products. This review centers on these beneficial secondary metabolites, the discovery of which goes back 80 years to the time when penicillin was discovered by Alexander Fleming.

/pdf/microbial-drug-discovery-80-years-of-progress-2u89dilx0f.pdf

Microbial drug discovery: 80 years of progress

Tubulin polymerization into microtubules is a dynamic process, with the equilibrium between growth and shrinkage being essential for many cellular processes. The antineoplastic agent taxol hyperstabilizes polymerized microtubules, leading to mitotic arrest and cytotoxicity in proliferating cells. Using a sensitive filtration-calorimetric assay to detect microtubule nucleating activity, we have identified epothilones A and B as compounds that possess all the biological effects of taxol both in vitro and in cultured cells. The epothilones are equipotent and exhibit kinetics similar to taxol in inducing tubulin polymerization into microtubules in vitro (filtration, light scattering, sedimentation, and electron microscopy) and in producing enhanced microtubule stability and bundling in cultured cells. Furthermore, these 16-membered macrolides are competitive inhibitors of [3H]taxol binding, exhibiting a 50% inhibitory concentration almost identical to that of taxol in displacement competition assays. Epothilones also cause cell cycle arrest at the G2-M transition leading to cytotoxicity, similar to taxol. In contrast to taxol, epothilones retain a much greater toxicity against P-glycoprotein-expressing multiple drug resistant cells. Epothilones, therefore, represent a novel structural class of compounds, the first to be described since the original discovery of taxol, which not only mimic the biological effects of taxol but also appear to bind to the same microtubule-binding site as taxol.

https://cancerres.aacrjournals.org/content/canres/55/11/2325.full.pdf

Epothilones, a New Class of Microtubule-stabilizing Agents with a Taxol-like Mechanism of Action

At therapeutic concentrations, the antineoplastic agent taxol selectively perturbs mitotic spindle microtubules. Taxol has recently been shown to induce apoptosis, similar to the mechanism of cell death induced by other antineoplastic agents. However, taxol has shown efficacy against drug-refractory cancers, raising the possibility that this pharmacological agent may trigger an alternative apoptotic pathway. The kinetics and IC50 of mitotic (M) block, aberrant mitosis, and cytotoxicity following taxol treatment were analyzed in human cell lines as well as normal mouse embryo fibroblasts (MEFs) and MEFs derived from p53-null mice. Apoptosis was followed by DNA gel electrophoresis and by in situ DNA end-labeling (TUNEL). Taxol induced two forms of cell cycle arrest: either directly in early M at prophase or, for those cells progressing through aberrant mitosis, arrest in G1 as multimininucleated cells. TUNEL labeling revealed that DNA nicking occurred within 30 min of the arrest in prophase. In contrast, G1-arrested, multimininucleated cells became TUNEL positive only after several days. In the subset of cells that became blocked directly in prophase, both wt p53-expressing and p53-null MEFs responded similarly to taxol, showing rapid onset of DNA nicking and apoptosis. However, p53-null MEFs progressing through aberrant mitosis failed to arrest in the subsequent G1 phase or to become TUNEL positive, and remained viable. Taxol induces two forms of cell cycle arrest, which in turn induce two independent apoptotic pathways. Arrest in prophase induces rapid onset of a p53-independent pathway, whereas G1-block and the resulting slow (3–5 days) apoptotic pathway are p53 dependent.

/pdf/taxol-induced-mitotic-block-triggers-rapid-onset-of-a-p53-3n671yljk2.pdf

Taxol-induced mitotic block triggers rapid onset of a p53-independent apoptotic pathway.

The past few years have witnessed the regulatory approvals of the anticancer microtubule stabilising taxane drugs, Taxol® and Taxotere®, which are rapidly gaining acceptance as important antineoplastic agents with potential against numerous solid tumour malignancies. Despite a basic understanding of the biochemical target of taxanes dating back nearly 20 years, new classes of tubulin-binding microtubule polymerisation enhancers were only reported in the last two years. Epothilones and discodermolide are newly discovered compounds, which are structurally distinct from the taxanes, but which possess similar tubulin polymerising and cell biological effects. In the first studies reported, these compounds displayed similar or greater potencies than taxanes, and the epothilones may represent an advance over the taxanes in retaining toxicity against various taxane-resistant cell lines. This review summarises the data published on epothilones and discodermolide and proposes further steps that could establish thes...

Epothilones: novel microtubule-stabilising agents

Ion-exchange chromatography.

A method of determining tubulin protein polymerization activity in a test sample is disclosed. The method comprises: combining the test sample with a polymerizable tubulin protein; contacting said test sample and polymerizable tubulin protein with a material which traps polymerized tubulin protein; measuring the amount of polymerised tubulin protein trapped by said material, and comparing the amount of polymerized tubulin protein trapped by said material to a standard. A method of determining tubulin protein polymerization inhibition in a test sample is also disclosed, which comprises: combining said test sample with a polymerizable tubulin protein and a pre-determined amount of a compound which causes tubulin protein polymerization to form a mixture; contacting said mixture with a material which traps polymerized tubulin protein; measuring the amount of polymerized tubulin protein trapped by said material, and comparing the amount of polymerized tubulin protein trapped by said material to a standard.

Daniel M. Bollag

Papers

Epothilones, a New Class of Microtubule-stabilizing Agents with a Taxol-like Mechanism of Action

Taxol-induced mitotic block triggers rapid onset of a p53-independent apoptotic pathway.

Epothilones: novel microtubule-stabilising agents

Ion-exchange chromatography.

Process for assessing tubulin protein polymerization