14 research outputs found
Chemically diverse microtubule stabilizing agents initiate distinct mitotic defects and dysregulated expression of key mitotic kinases.
Microtubule stabilizers are some of the most successful drugs used in the treatment of adult solid tumors and yet the molecular events responsible for their antimitotic actions are not well defined. The mitotic events initiated by three structurally and biologically diverse microtubule stabilizers; taccalonolide AJ, laulimalide/fijianolide B and paclitaxel were studied. These microtubule stabilizers cause the formation of aberrant, but structurally distinct mitotic spindles leading to the hypothesis that they differentially affect mitotic signaling. Each microtubule stabilizer initiated different patterns of expression of key mitotic signaling proteins. Taccalonolide AJ causes centrosome separation and disjunction failure to a much greater extent than paclitaxel or laulimalide, which is consistent with the distinct defects in expression and activation of Plk1 and Eg5 caused by each stabilizer. Localization studies revealed that TPX2 and Aurora A are associated with each spindle aster formed by each stabilizer. This suggests a common mechanism of aster formation. However, taccalonolide AJ also causes pericentrin accumulation on every spindle aster. The presence of pericentrin at every spindle aster initiated by taccalonolide AJ might facilitate the maintenance and stability of the highly focused asters formed by this stabilizer. Laulimalide and paclitaxel cause completely different patterns of expression and activation of these proteins, as well as phenotypically different spindle phenotypes. Delineating how diverse microtubule stabilizers affect mitotic signaling pathways could identify key proteins involved in modulating sensitivity and resistance to the antimitotic actions of these compounds
Biological Characterization of an Improved Pyrrole-Based Colchicine Site Agent Identified through Structure-Based Design s
ABSTRACT A refined model of the colchicine site on tubulin was used to design an improved analog of the pyrrole parent compound, JG-03-14. The optimized compound, NT-7-16, was evaluated in biological assays that confirm that it has potent activities as a new colchicine site microtubule depolymerizer. NT-7-16 exhibits antiproliferative and cytotoxic activities against multiple cancer cell lines, with IC 50 values of 10-16 nM, and it is able to overcome drug resistance mediated by the expression of P-glycoprotein and the bIII isotype of tubulin. NT-7-16 initiated the concentration-dependent loss of cellular microtubules and caused the formation of abnormal mitotic spindles, leading to mitotic accumulation. The direct interaction of NT-7-16 with purified tubulin was confirmed, and it was more potent than combretastatin A-4 in these assays. Binding studies verified that NT-7-16 binds to tubulin within the colchicine site. The antitumor effects of NT-7-16 were evaluated in an MDA-MB-435 xenograft model and it had excellent activity at concentrations that were not toxic. A second compound, NT-9-21, which contains dichloro moieties in place of the 3,5-dibromo substituents of NT-7-16, had a poorer fit within the colchicine site as predicted by modeling and the Hydropathic INTeractions score. Biological evaluations showed that NT-9-21 has 10-fold lower potency than NT-7-16, confirming the modeling predictions. These studies highlight the value of the refined colchicine-site model and identify a new pyrrole-based colchicine-site agent with potent in vitro activities and promising in vivo antitumor actions
AMP-activated protein kinase fortifies epithelial tight junctions during energetic stress via its effector GIV/Girdin.
Loss of epithelial polarity impacts organ development and function; it is also oncogenic. AMPK, a key sensor of metabolic stress stabilizes cell-cell junctions and maintains epithelial polarity; its activation by Metformin protects the epithelial barrier against stress and suppresses tumorigenesis. How AMPK protects the epithelium remains unknown. Here, we identify GIV/Girdin as a novel effector of AMPK, whose phosphorylation at a single site is both necessary and sufficient for strengthening mammalian epithelial tight junctions and preserving cell polarity and barrier function in the face of energetic stress. Expression of an oncogenic mutant of GIV (cataloged in TCGA) that cannot be phosphorylated by AMPK increased anchorage-independent growth of tumor cells and helped these cells to evade the tumor-suppressive action of Metformin. This work defines a fundamental homeostatic mechanism by which the AMPK-GIV axis reinforces cell junctions against stress-induced collapse and also provides mechanistic insight into the tumor-suppressive action of Metformin
Structure–Activity Relationship and in Vitro and in Vivo Evaluation of the Potent Cytotoxic Anti-microtubule Agent <i>N</i>‑(4-Methoxyphenyl)‑<i>N</i>,2,6-trimethyl-6,7-dihydro‑5<i>H</i>‑cyclopenta[<i>d</i>]pyrimidin-4-aminium Chloride and Its Analogues As Antitumor Agents
A series
of 21 substituted cyclopenta[<i>d</i>]pyrimidines
were synthesized as an extension of our discovery of the parent compound
(±)-<b>1</b>·HCl as an anti-microtubule agent. The
structure–activity relationship indicates that the <i>N</i>-methyl and a 4<i>N</i>-methoxy groups appear
important for potent activity. In addition, the 6-substituent in the
parent analogue is not necessary for activity. The most potent compound <b>30</b>·HCl was a one to two digit nanomolar inhibitor of
most tumor cell proliferations and was up to 7-fold more potent than
the parent compound (±)-<b>1</b>·HCl. In addition, <b>30</b>·HCl inhibited cancer cell proliferation regardless
of Pgp or βIII-tubulin status, both of which are known to cause
clinical resistance to several anti-tubulin agents. In vivo efficacy
of <b>30</b>·HCl was demonstrated against a triple negative
breast cancer xenograft mouse model. Compound <b>30</b>·HCl
is water-soluble and easily synthesized and serves as a lead compound
for further preclinical evaluation as an antitumor agent
Janus Compounds, 5-Chloro-N4-methyl-N4-aryl-9H-pyrimido[4,5-b]indole-2,4-diamines, Cause Both Microtubule Depolymerizing and Stabilizing Effects
While evaluating a large library of compounds designed to inhibit microtubule polymerization, we identified four compounds that have unique effects on microtubules. These compounds cause mixed effects reminiscent of both microtubule depolymerizers and stabilizers. Immunofluorescence evaluations showed that each compound initially caused microtubule depolymerization and, surprisingly, with higher concentrations, microtubule bundles were also observed. There were subtle differences in the propensity to cause these competing effects among the compounds with a continuum of stabilizing and destabilizing effects. Tubulin polymerization experiments confirmed the differential effects and, while each of the compounds increased the initial rate of tubulin polymerization at high concentrations, total tubulin polymer was not enhanced at equilibrium, likely because of the dueling depolymerization effects. Modeling studies predict that the compounds bind to tubulin within the colchicine site and confirm that there are differences in their potential interactions that might underlie their distinct effects on microtubules. Due to their dual properties of microtubule stabilization and destabilization, we propose the name Janus for these compounds after the two-faced Roman god. The identification of synthetically tractable, small molecules that elicit microtubule stabilizing effects is a significant finding with the potential to identify new mechanisms of microtubule stabilization
Janus Compounds, 5-Chloro-N4-methyl-N4-aryl-9H-pyrimido[4,5-b]indole-2,4-diamines, Cause Both Microtubule Depolymerizing and Stabilizing Effects
While evaluating a large library of compounds designed to inhibit microtubule polymerization, we identified four compounds that have unique effects on microtubules. These compounds cause mixed effects reminiscent of both microtubule depolymerizers and stabilizers. Immunofluorescence evaluations showed that each compound initially caused microtubule depolymerization and, surprisingly, with higher concentrations, microtubule bundles were also observed. There were subtle differences in the propensity to cause these competing effects among the compounds with a continuum of stabilizing and destabilizing effects. Tubulin polymerization experiments confirmed the differential effects and, while each of the compounds increased the initial rate of tubulin polymerization at high concentrations, total tubulin polymer was not enhanced at equilibrium, likely because of the dueling depolymerization effects. Modeling studies predict that the compounds bind to tubulin within the colchicine site and confirm that there are differences in their potential interactions that might underlie their distinct effects on microtubules. Due to their dual properties of microtubule stabilization and destabilization, we propose the name Janus for these compounds after the two-faced Roman god. The identification of synthetically tractable, small molecules that elicit microtubule stabilizing effects is a significant finding with the potential to identify new mechanisms of microtubule stabilization
The Bat Flower: A Source of Microtubule-Destabilizing and -Stabilizing Compounds with Synergistic Antiproliferative Actions
The biosynthesis of secondary metabolites
provides higher plants
with mechanisms of defense against microbes, insects, and herbivores.
One common cellular target of these molecules is the highly conserved
microtubule cytoskeleton, and microtubule-targeting compounds with
insecticidal, antifungal, nematicidal, and anticancer activities have
been identified from plants. A new retro-dihydrochalcone, taccabulin
A, with microtubule-destabilizing activity has been identified from
the roots and rhizomes of <i>Tacca</i> species. This finding
is notable because the microtubule-stabilizing taccalonolides are
also isolated from these sources. This is the first report of an organism
producing compounds with both microtubule-stabilizing and -destabilizing
activities. A two-step chemical synthesis of taccabulin A was performed.
Mechanistic studies showed that taccabulin A binds within the colchicine
site on tubulin and has synergistic antiproliferative effects against
cancer cells when combined with a taccalonolide, which binds to a
different site on tubulin. Taccabulin A is effective in cells that
are resistant to many other plant-derived compounds. The discovery
of a natural source that contains both microtubule-stabilizing and
-destabilizing small molecules is unprecedented and suggests that
the synergistic action of these compounds was exploited by nature
long before it was discovered in the laboratory
The design and discovery of water soluble 4-substituted-2,6-dimethylfuro[2, 3-d]pyrimidines as multitargeted receptor tyrosine kinase inhibitors and microtubule targeting antitumor agents
The design, synthesis and biological evaluations of fourteen 4-substituted 2,6-dimethylfuro[2,3-d]pyrimidines are reported. Four compounds (11-13, 15) inhibit vascular endothelial growth factor receptor-2 (VEGFR-2), platelet-derived growth factor receptor β (PDGFR-β), and target tubulin leading to cytotoxicity. Compound 11 has nanomolar potency, comparable to sunitinib and semaxinib, against tumor cell lines overexpressing VEGFR-2 and PDGFR-β. Further, 11 binds at the colchicine site on tubulin, depolymerizes cellular microtubules and inhibits purified tubulin assembly and overcomes both βIII-tubulin and P-glycoprotein-mediated drug resistance, and initiates mitotic arrest leading to apoptosis. In vivo, its HCl salt, 21, reduced tumor size and vascularity in xenograft and allograft murine models and was superior to docetaxel and sunitinib, without overt toxicity. Thus 21 affords potential combination chemotherapy in a single agent. © 2014 Elsevier Ltd. All rights reserved
Design, Synthesis, and Preclinical Evaluation of 4‑Substituted-5-methyl-furo[2,3‑<i>d</i>]pyrimidines as Microtubule Targeting Agents That Are Effective against Multidrug Resistant Cancer Cells
The design, synthesis, and biological
evaluations of eight 4-substituted
5-methyl-furo[2,3-<i>d</i>]pyrimidines are reported. Synthesis
involved <i>N</i><sup>4</sup>-alkylation of <i>N</i>-aryl-5-methylfuro[2,3-<i>d</i>]pyrimidin-4-amines, obtained
from Ullmann coupling of 4-amino-5-methylfuro[2,3-<i>d</i>]pyrimidine and appropriate aryl iodides. Compounds <b>3</b>, <b>4</b>, and <b>9</b> showed potent microtubule depolymerizing
activities, while compounds <b>6</b>–<b>8</b> had
slightly lower potency. Compounds <b>4</b>, <b>6</b>, <b>7</b>, and <b>9</b> inhibited tubulin assembly with IC<sub>50</sub> values comparable to that of combretastatin A-4 (CA-4).
Compounds <b>3</b>, <b>4</b>, and <b>6</b>–<b>9</b> circumvented Pgp and βIII-tubulin mediated drug resistance,
mechanisms that can limit the efficacy of paclitaxel, docetaxel, and
the vinca alkaloids. In the NCI 60-cell line panel, compound <b>3</b> exhibited GI<sub>50</sub> values less than 10 nM in 47 of
the cell lines. In an MDA-MB-435 xenograft model, compound <b>3</b> had statistically significant antitumor effects. The biological
effects of <b>3</b> identify it as a novel, potent microtubule
depolymerizing agent with antitumor activity