Structure-based design and synthesis of novel furan-diketopiperazine-type derivatives as potent microtubule inhibitors for treating cancer

Plinabulin, a synthetic analog of the marine natural product “diketopiperazine phenylahistin,” displayed depolymerization effects on microtubules and targeted the colchicine site, which has been moved into phase III clinical trials for the treatment of non-small cell lung cancer (NSCLC) and the prevention of chemotherapy-induced neutropenia (CIN). To develop more potent anti-microtubule and cytotoxic derivatives, the co-crystal complexes of plinabulin derivatives were summarized and analyzed. We performed further modifications of the tert-butyl moiety or C-ring of imidazole-type derivatives to build a library of molecules through the introduction of different groups for novel skeletons. Our structure–activity relationship study indicated that compounds 17o (IC₅₀ = 14.0 nM, NCI-H460) and 17p (IC₅₀ = 2.9 nM, NCI-H460) with furan groups exhibited potent cytotoxic activities at the nanomolar level against various human cancer cell lines. In particular, the 5-methyl or methoxymethyl substituent of furan group could replace the alkyl group of imidazole at the 5-position to maintain cytotoxic activity, contradicting previous reports that the tert-butyl moiety at the 5-position of imidazole was essential for the activity of such compounds. Immunofluorescence assay indicated that compounds 17o and 17p could efficiently inhibit microtubule polymerization. Overall, the novel furan-diketopiperazine-type derivatives could be considered as a potential scaffold for the development of anti-cancer drugs.     

Sustained activation of p34(cdc2) is required for noscapine-induced apoptosis.

Mitotic arrest and subsequent apoptosis has been observed in many types of cells treated with anti-microtubule agents. However, the molecular mechanisms underlying the two events as well as their relationship are not well understood; on the contrary, there has been increasing evidence indicating that anti-microtubule agents might induce apoptosis via signaling pathways independent of mitosis. In this study, we found that apoptosis induced by noscapine, an anti-microtubule drug previously shown to cause both mitotic arrest and apoptotic cell death, was blocked by inhibiting p34(cdc2) activity with olomoucine in FM3A murine mammary carcinoma cells or by reducing the level and activity of p34(cdc2) in a mutant cell line FT210 derived from FM3A. Furthermore, transfection of the mutant FT210 cells with wild-type p34(cdc2) restored their ability to undergo mitotic arrest and then apoptosis in response to noscapine. Thus, we conclude that sustained activation of the p34(cdc2) kinase during mitotic arrest is required for subsequent apoptosis induced by noscapine, establishing a link between the two events.     

Antitumor activity of phenylahistin in vitro and in vivo.

Phenylahistin is a new cell cycle inhibitor produced by Aspergillus ustus. Since phenylahistin was produced as a scalemic mixture of (-)-phenylahistin and its enantiomer, we separated each enantiomer and evaluated their antitumor activity in vitro. (-)-Phenylahistin exhibited antitumor activity against 8 tumor cell lines with IC50 values ranging from 1.8 x 10(-7) to 3.7 x 10(-6), while (+)-phenylahistin exhibited 33-100-fold less potent activity than (-)-phenylahistin did. (-)-Phenylahistin also showed antitumor activity against P388 leukemia and Lewis lung carcinoma cells in vivo.     

Actin- and microtubule-targeting bioprobes: their binding sites and inhibitory mechanisms

Actin filaments and microtubules play important biological functions in mammalian cells, such as mitosis, cytokinesis, cell signaling, intracellular transport, and cell motility. Therefore, small molecules that interact with these cytoskeletons are expected to be useful not only as antitumor agents, but also as tools for understanding a wide variety of the cellular functions of cytoskeletons. A large number of compounds have been reported as anti-microtubule or anti-actin agents, but only a few compounds have been clarified as to their binding sites on target molecules and their inhibition mechanisms. Here, I describe our recent research into anti-actin and anti-microtubule natural products. Some inhibitors contain active moieties, such as alpha,beta-unsaturated delta-lactone or allely epoxide, in their structure, and covalently bind to their target molecules. Furthermore, some compounds show new inhibition mechanisms by binding on novel sites in target molecules.     

Structure–activity relationship studies of thalidomide analogs with a taxol-like mode of action

Anti-microtubule agents such as paclitaxel and docetaxel have played an important role in the treatment of cancer for many years. Recently, a small molecule that has a taxol-like mode of action (5HPP-33) was reported. Herein, the detailed structure–activity relationship (SAR) studies of 5HPP-33 analogs that are substituted at the isoindole and phenyl rings are described. Bulky substitutions (such as di-isopropyl groups) on the phenyl ring result in the isoindole and phenyl rings being perpendicular to each other. It was found that this conformation is critical for anti-microtubule activity. These studies have provided valuable information, which will be helpful in the design of more potent analogs.     

Effects of estramustine, a new anti-microtubule drug, on the induction of casein gene expression by prolactin

Estramustine, a new anti-microtubule drug, was added to the culture medium of rabbit mammary explants with lactogenic hormones. In the absence of the drug, prolactin with insulin and cortisol stimulated DNA synthesis and it induced beta-casein and beta-casein mRNA accumulation in the tissue. As opposed to other anti-microtubule agents such as colchicine, estramustine was unable to prevent prolactin actions. An examination of the mammary cells by immunofluorescence revealed that the microtubule network was significantly altered under the influence of estramustine. These data indicate that the integrity of microtubules is not required for prolactin to deliver its message to the mammary cell. These data also suggest that other anti-microtubule drugs such as colchicine which prevent prolactin action act through their binding to tubulin molecule unrelated to microtubule structures.     

Synthesis and structure-activity relationships of benzophenone-bearing diketopiperazine-type anti-microtubule agents

KPU-105 (4), a potent anti-microtubule agent that contains a benzophenone was derived from the diketopiperazine-type vascular disrupting agent (VDA) plinabulin 3, which displays colchicine-like tubulin depolymerization activity. To develop derivatives with more potent anti-microtubule and cytotoxic activities, we further modified the benzophenone moiety of 4. Accordingly, we obtained a 4-fluorobenzophenone derivative 16j that inhibited tumor cell growth in vitro with a subnanomolar IC(50) value against HT-29 cells (IC(50)=0.5 nM). Next, the effect of 16j on mitotic spindles was evaluated in HeLa cells. Treatment with 3nM of 16j partially disrupted the interphase microtubule network. By contrast, treatment with the same concentration of CA-4 barely affected the microtubule network, indicating that 16j exhibited more potent anti-mitotic effects than did CA-4.     

4'-Alkoxyl substitution enhancing the anti-mitotic effect of 5-(3',4',5'-substituted)anilino-4-hydroxy-8-nitroquinazolines as a novel class of anti-microtubule agents

Mitosis inhibitors are powerful anticancer drugs. Based on a novel anti-microtubule agent of 5-(4'-methoxy)anilino-4-hydroxy-8-nitroquinazoline, a series of 5-(3',4',5'-substituted)anilino-4-hydroxy-8- nitroquinazolines were designed and synthesized to investigate the effect of the substitution on the inhibitory activity against mitotic progression of tumor cells. The large alkoxyl substitution on the 4'-position of 5-anilino ring is beneficial for the potency. The 5-(3',4',5'-trimethoxy)anilino-8-nitroquinazoline (1h) displays an overwhelming activity in arresting the cells at the G2/M phase, providing a promising new template for further development of potent microtubule-targeted anti-mitotic drugs.     

Effect of antimicrotubule agents on terminal glycosyltransferases and other enzymes associated with rat liver subcellular fractions.

Previous studies have shown that anti microtubule agents disrupt Golgi complexes in hepatocytes and other cells, causing breakdown or vesiculation of Golgi cisternal membranes. Whether this change in the structure of the Golgi membranes is associated with changes in Golgi membrane function is not known. The present study was initiated to investigate this issue; i.e., to determine whether anti-microtubule agents that cause structural changes in Golgi membranes in vivo would, at the same time, affect characteristic enzyme functions of Golgi membranes. To this end, colchicine was given to young rats in vivo and various hepatic subcellular membranes were subsequently isolated and utilized for enzyme assays. Initially it was shown that colchicine (2.5 mg/kg body wt.) given for 5h significantly decreased the activities of the Golgi membrane associated enzymes galactosyl-, sialyl- and N-acetylglucosaminyl-transferases. More detailed experiments indicated that low doses of colchicine (0.8 mg/kg body wt.), although less effective than higher doses, decreased the activities of the terminal glycosylating enzymes maximally at 5h, with partial and complete recovery at 12 and 24h respectively. Treatment in vivo of rats with vinblastine (20 mg/kg body wt.) for 5h mimicked the action of colchicine. Two microsomal glycosylating enzymes (mannosyl and N acetylglucosaminyl transferases) were unaffected by the treatment with colchicine, as were various hepatic 'marker' enzymes such as 5' nucleotidase, glucose 6 phosphatase and succinate: 2-(p iodophenyl)-3-(p nitrophenyl)-5-phenyltetrazolium reductase (succinate dehydrogenase; EC 1.3.99.1), which were found to be enriched in plasma membrane, endoplasmic-reticulum and mitochondrial-membrane fractions respectively. These results show that anti-microtubule agents specifically suppress the activity of Golgi-associated glycosyltransferases in liver. Although it seems likely that these changes are related to the previously observed structural changes in hepatocyte Golgi complexes after colchicine treatment, to what extent the results are linked to the interaction of colchicine with microtubule protein remains to be clarified.     

Correlation of betacyanin synthesis with cell division in cell suspension cultures of Phytolacca americana

In suspension cultures of Phytolacca americana , betacyanin accumulation was reduced when cell division was inhibited by treatment with various inhibitors of DNA synthesis or anti-microtubule drugs. Aphidicolin (APC), an inhibitor of DNA synthesis, reduced the incorporation of radioactivity from labeled tyrosine into betacyanin, but the incorporation of radioactivity from labeled 3,4-dihydroxyphenylalanine (DOPA) into betacyanin was not affected by similar treatments. Propyzamide, another anti-microtubule drug, reduced incorporation of radioactivity from tyrosine and DOPA into betacyanin. However, the rate of incorporation from DOPA was higher than that from tyrosine. The results suggest that inhibition of betacyanin accumulation in Phytolacca americana cells by APC and propyzamide is due to suppression of the reaction converting tyrosine to DOPA, which may be closely related to cell division.     

Expression Levels of a Kinesin-13 Microtubule Depolymerase Modulates the Effectiveness of Anti-Microtubule Agents

Background

Chemotheraputic drugs often target the microtubule cytoskeleton as a means to disrupt cancer cell mitosis and proliferation. Anti-microtubule drugs inhibit microtubule dynamics, thereby triggering apoptosis when dividing cells activate the mitotic checkpoint. Microtubule dynamics are regulated by microtubule-associated proteins (MAPs); however, we lack a comprehensive understanding about how anti-microtubule agents functionally interact with MAPs. In this report, we test the hypothesis that the cellular levels of microtubule depolymerases, in this case kinesin-13 s, modulate the effectiveness of the microtubule disrupting drug colchicine.

Methodology/Principal Findings

We used a combination of RNA interference (RNAi), high-throughput microscopy, and time-lapse video microscopy in Drosophila S2 cells to identify a specific MAP, kinesin-like protein 10A (KLP10A), that contributes to the efficacy of the anti-microtubule drug colchicine. KLP10A is an essential microtubule depolymerase throughout the cell cycle. We find that depletion of KLP10A in S2 cells confers resistance to colchicine-induced microtubule depolymerization to a much greater extent than depletion of several other destabilizing MAPs. Using image-based assays, we determined that control cells retained 58% (±2%SEM) of microtubule polymer when after treatment with 2 µM colchicine for 1 hour, while cells depleted of KLP10A by RNAi retained 74% (±1%SEM). Likewise, overexpression of KLP10A-GFP results in increased susceptibility to microtubule depolymerization by colchicine.

Conclusions/Significance

Our results demonstrate that the efficacy of microtubule destabilization by a pharmacological agent is dependent upon the cellular expression of a microtubule depolymerase. These findings suggest that expression levels of Kif2A, the human kinesin-13 family member, may be an attractive biomarker to assess the effectiveness of anti-microtubule chemotherapies. Knowledge of how MAP expression levels affect the action of anti-microtubule drugs may prove useful for evaluating possible modes of cancer treatment.     

Microtubules, but not actin microfilaments, regulate vacuole motility and morphology in hyphae of Pisolithus tinctorius

While it is now recognised that transport within the endomembrane system may occur via membranous tubules, spatial regulation of this process is poorly understood. We have investigated the role of the cytoskeleton in regulating the motility and morphology of the motile vacuole system in hyphae of the fungus Pisolithus tinctorius by studying (1) the effects of anti-microtubule (oryzalin, nocodazole) and anti-actin drugs (cytochalasins, latrunculin) on vacuolar activity, monitored by fluorescence microscopy of living cells; and (2) the ultrastructural relationship of microtubules, actin microfilaments, and vacuoles in hyphae prepared by rapid-freezing and freeze-substitution. Anti-microtubule drugs reduced the tubular component of the vacuole system in a dose-dependent and reversible manner, the extent of which correlated strongly with the degree of disruption of the microtubule network (monitored by immunofluorescence microscopy). The highest doses of anti-microtubule drugs completely eliminated tubular vacuoles, and only spherical vacuoles were observed. In contrast, anti-actin drugs did not reduce the frequency of tubular vacuoles or the motility of these vacuoles, even though immunofluorescence microscopy confirmed perturbation of microfilament organisation. Electron microscopy showed that vacuoles were always accompanied by microtubules. Bundles of microtubules were found running in parallel along the length of tubular vacuoles and individual microtubules were often within one microtubule diameter of a vacuole membrane. Our results strongly support a role for microtubules, but not actin microfilaments, in the spatial regulation of vacuole motility and morphology in fungal hyphae.     

Vascular disrupting agent drug classes differ in effects on the cytoskeleton

Vascular disrupting agents (VDAs), anti-cancer drugs that target established tumor blood vessels, fall into two main classes: microtubule targeting drugs, exemplified by combretastatin A4 (CA4), and flavonoids, exemplified by 5,6-dimethylxanthenone-4-acetic acid (DMXAA). Both classes increase permeability of tumor vasculature in mouse models, and DMXAA in particular can cause massive tumor necrosis. The molecular target of CA4 is clearly microtubules. The molecular target(s) of DMXAA remains unclear. It is thought to promote inflammatory signaling in leukocytes, and has been assumed to not target microtubules, though it is not clear from the literature how carefully this assumption has been tested. An earlier flavone analog, flavone acetic acid, was reported to promote mitotic arrest suggesting flavones might possess anti-microtubule activity, and endothelial cells are sensitive to even mild disruption of microtubules. We carefully investigated whether DMXAA directly affects the microtubule or actin cytoskeletons of endothelial cells by comparing effects of CA4 and DMXAA on human umbilical vein endothelial cells (HUVEC) using time-lapse imaging and assays for cytoskeleton integrity. CA4 caused retraction of the cell margin, mitotic arrest and microtubule depolymerization, while DMXAA, up to 500 μM, showed none of these effects. DMXAA also had no effect on pure tubulin nucleation and polymerization, unlike CA4. We conclude that DMXAA exhibits no direct anti-microtubule action and thus cleanly differs from CA4 in its mechanism of action at the molecular level.     

Tau protein is involved in the apoptotic process induced by anti-microtubule agents on neuroblastoma cells

Paclitaxel and docetaxel are potent anti-microtubule and antimitotic agents that induce apoptosis in bone marrow-derived cells and epithelial cells. This study examined apoptosis induced by anti-microtubule agents in the neuroblastoma SK-N-SH cell line with a special focus on tau protein which is one of the main Microtubule-Associated- Proteins (MAPs) in neuronal cells. In time, treatment with 1 M paclitaxel successively induced formation of bundles, then pseudo-asters concomitantly with mitotic block and phosphorylation of bcl-2 (48 h), then phosphorylation of tau and externalization of phosphatidylserine at the early phase of apoptosis (72 h) and finally DNA fragmentation (96 h). Similar results were obtained with 0.5 M vinorelbine. Paclitaxel induced a lower increase in tau phosphorylation in differentiated SK-N-SH/RA+ cells which are less sensitive to apoptosis. Moreover, doxorubicin whose mechanism of action is independent of microtubules also induced immunostaining of tau at 72 h treatment. In conclusion, our results on neuroblastoma cells show that overexpression of hyperphosphorylated tau is involved in the apoptotic process induced by anti-microtubule agents and may be extended to others cytostatic drugs. Thus, tau protein may play a role in the cellular events observed in neuroblastoma cells undergoing apoptosis.     

Anti-microtubule ‘plinabulin’ chemical probe KPU-244-B3 labeled both α- and β-tubulin

Plinabulin (1, NPI-2358), a potent microtubule-targeting agent derived from the natural diketopiperazine ‘phenylahistin’ with a colchicine-like tubulin depolymerization activity, is an anticancer agent undergoing Phase II clinical trials in four countries including the United States. In order to understand the precise binding mode of plinabulin with tubulin, a new bioactive biotin-tagged photoaffinity probe 4 (KPU-244-B3) was designed and synthesized. Probe 4 showed significant binding affinity to tubulin in a binding assay, and selectively bound to tubulin in an HT-1080 cell lysate without photo-irradiation. In a tubulin photoaffinity labeling study, probe 4 labeled both α- and β-tubulin subunits and these interactions were competitively inhibited by plinabulin during photo-irradiation. These results suggest that plinabulin binds in the boundary region between α- and β-tubulin near the colchicine binding site, and not inside the colchicine binding cavity.     

The role of microtubules in guard cell function

Guard cells are able to sense a multitude of environmental signals and appropriately adjust the stomatal pore to regulate gas exchange in and out of the leaf. The role of the microtubule cytoskeleton during these stomatal movements has been debated. To help resolve this debate, in vivo stomatal aperture assays with different microtubule inhibitors were performed. We observed that guard cells expressing the microtubule-binding green fluorescent fusion protein (green fluorescent protein::microtubule binding domain) fail to open for all major environmental triggers of stomatal opening. Furthermore, guard cells treated with the anti-microtubule drugs, propyzamide, oryzalin, and trifluralin also failed to open under the same environmental conditions. The inhibitory conditions caused by green fluorescent protein::microtubule binding domain and these anti-microtubule drugs could be reversed using the proton pump activator, fusicoccin. Therefore, we conclude that microtubules are involved in an upstream event prior to the ionic fluxes leading to stomatal opening. In a mechanistic manner, evidence is presented to implicate a microtubule-associated protein in this putative microtubule-based signal transduction event.     

Effects of anti-microtubule agents on microtubule organization in cells lacking the kinesin-13 MCAK

Dynamic microtubules are necessary for proper mitotic spindle assembly and chromosome segregation during mitosis. Members of the kinesin superfamily of molecular motor proteins are important to spindle function. Of particular interest is the Kinesin-13 family member MCAK, which acts to regulate microtubule dynamics during spindle assembly and to ensure proper attachments of chromosomes to spindle microtubules. The unique ability of MCAK to regulate microtubule dynamics makes it a potential target for development of new drugs that alter spindle function. Here, we knocked down MCAK via RNAi in normal and malignant cell lines and found that the two tested malignant cell lines were acutely sensitive to MCAK knockdown, while the tested normal cells were less sensitive. In addition, we looked at the effect of combining MCAK knockdown and drug treatment with paclitaxel or vinblastine to identify spindle assembly defects. We found that MCAK knockdown increased the morphological defects of the microtubule cytoskeleton in HeLa cells caused by anti-microtubule drugs. Our studies support the idea that MCAK would be a good target for new chemotherapeutic development and may be particular useful in combination therapies with currently available anti-microtubule agents.     

DIATOM MINERALIZATION OF SILICIC ACID. VII. INFLUENCE OF MICROTUBULE DRUGS ON SYMMETRY AND PATTERN FORMATION IN VALVES OF NAVICULA SAPROPHILA DURING MORPHOGENESIS1

Microtubule involvement in diatom valve symmetry and pattern formation was investigated using cells synchronized subsequent to mitosis and cytokinesis but prior to cell wall formation. Two analog drug pairs, each consisting of an active and an inactive microtubule drug, were used to distinguish inhibitory effects related or unrelated to microtubule disruption. The active anti-microtubule drug of each analog pair produced significantly higher percentages of aberrant valves than did the respective inactive analogs. High frequencies of aberrant valves also were caused by N-isophenlpropylcarbamate, which disorganizes rather than disrupts microtubules. Valves could be placed into different classes based upon characteristic aberrations. Formation of these classes was not random but was instead a function of both the drug and the drug concentration. The central nodule and the raphe were the principal valve components affected by anti-microtubule drugs. Stria alterations appeared as a secondary result of alterations in the central nodule/raphe. Valve aberrations occurred at very low drug concentrations in the range 1 × 10^?6 to 1 × 10^?9M.     

NMK-BH2, a novel microtubule-depolymerising bis (indolyl)-hydrazide-hydrazone,induces apoptotic and autophagic cell death in cervical cancer cells by binding to tubulin at colchicine – site

BackgroundMicrotubules, the key components of the eukaryotic cytoskeleton and mitotic spindle, are one of the most sought-after targets for cancer chemotherapy, especially due to their indispensible role in mitosis. Cervical cancer is a prevalent malignancy among women of developing countries including India. In spite of the remarkable therapeutic advancement, the non-specificity of chemotherapeutic drugs adversely affect the patients' survival and well-being, thus, necessitating the quest for novel indole-based anti-microtubule agent against cervical cancer, with high degree of potency and selectivity.MethodsFor in vitro studies, we used MTT assay, confocal microscopy, fluorescence microscopy, flow cytometry and Western blot analysis. Study in cell free system was accomplished by spectrophotometry, fluorescence spectroscopy and TEM and computational analysis was done by AutodockTools 1.5.6.ResultsNMK-BH2 exhibited significant and selective anti-proliferative activity against cervical cancer HeLa cells (IC₅₀ = 1.5 μM) over normal cells. It perturbed the cytoskeletal and spindle microtubules of HeLa cells leading to mitotic block and cell death by apoptosis and autophagy. Furthermore, NMK-BH2 targeted the tubulin-microtubule system through fast and strong binding to the αβ-tubulin heterodimers at colchicine-site.ConclusionThis study identifies and characterises NMK-BH2 as a novel anti-microtubule agent and provides insights into its key anti-cancer mechanism through two different cell death pathways: apoptosis and autophagy, which are mutually independent.General significanceIt navigates the potential of the novel bis (indolyl)-hydrazide-hydrazone, NMK-BH2, to serve as lead for development of new generation microtubule-disrupting chemotherapeutic with improved efficacy and remarkable selectivity towards better cure of cervical cancer.