Silicon oxide microchips functionalized with fluorescent probes for quantitative real-time glutathione sensing in living cells.

Glutathione (GSH) plays a vital role in the regulation of intracellular functions which alterations in physiological glutathione levels are associated to various diseases. Molecular bioimaging is a sensitive method for GSH detection, but challenges persist in the development of fluorescent probes, mainly concerning long-term tracking of intracellular GSH concentration because of aggregation of molecular probes and their washout in cells. Engineered nanomaterials have shown great promise for increasing the disease diagnosis accuracy.

Tannic Acid and Ethyl Gallate Potentialize Paclitaxel Effect on Microtubule Dynamics in Hep3B Cells.

Among broad-spectrum anticancer agents, paclitaxel (PTX) has proven to be one of the most effective against solid tumors for which more specific treatments are lacking. However, drawbacks such as neurotoxicity and the development of resistance reduce its therapeutic efficacy. Therefore, there is a need for compounds able to improve its activity by synergizing with it or potentiating its effect, thus reducing the doses required.

Synthesis and Structure-Activity Relationship Studies of C(13)-Desmethylene-(-)-Zampanolide Analogs.

We describe the synthesis and biochemical and cellular profiling of five partially reduced or demethylated analogs of the marine macrolide (-)-zampanolide (ZMP). These analogs were derived from 13-desmethylene-(-)-zampanolide (DM-ZMP), which is an equally potent cancer cell growth inhibitor as ZMP. Key steps in the synthesis of all compounds were the formation of the dioxabicyclo[15.

Intracellular Mechanical Drugs Induce Cell-Cycle Altering and Cell Death.

Current advances in materials science have demonstrated that extracellular mechanical cues can define cell function and cell fate. However, a fundamental understanding of the manner in which intracellular mechanical cues affect cell mechanics remains elusive. How intracellular mechanical hindrance, reinforcement, and supports interfere with the cell cycle and promote cell death is described here.

Integrating magnetic capabilities to intracellular chips for cell trapping.

Current microtechnologies have shown plenty of room inside a living cell for silicon chips. Microchips as barcodes, biochemical sensors, mechanical sensors and even electrical devices have been internalized into living cells without interfering their cell viability. However, these technologies lack from the ability to trap and preconcentrate cells in a specific region, which are prerequisites for cell separation, purification and posterior studies with enhanced sensitivity.

A Method for the Stereoselective Construction of the Hemiaminal Center in Zampanolides.

We have developed a new method for the stereoselective establishment of the -acyl hemiaminal moiety in zampanolide-type structures that involves the reaction of ()-sorbamide () with BINAL-H and subsequent amide transfer from a putative aluminum carboximidoate complex to the aldehyde moiety of a dactylolide precursor, such as or . The method has enabled the efficient synthesis of 13-desmethylene-(-)-zampanolide (), which was found to be an equipotent cell growth inhibitor as the natural product (-)-zampanolide ().

Synthesis, Microtubule-Binding Affinity, and Antiproliferative Activity of New Epothilone Analogs and of an EGFR-Targeted Epothilone-Peptide Conjugate.

A new simplified, epoxide-free epothilone analog was prepared incorporating an N-(2-hydroxyethyl)-benzimidazole side chain, which binds to microtubules with high affinity and inhibits cancer cell growth in vitro with nM potency. Building on this scaffold, a disulfide-linked conjugate with the purported EGFR-binding (EGFR, epidermal growth factor receptor) peptide GE11 was then prepared. The conjugate retained significant microtubule-binding affinity, in spite of the size of the peptide attached to the benzimidazole side chain.

Gallic acid sensitizes paclitaxel-resistant human ovarian carcinoma cells through an increase in reactive oxygen species and subsequent downregulation of ERK activation.

Paclitaxel (PTX) is currently used as a front-line chemotherapeutic agent for several types of cancer, including ovarian carcinoma; however, PTX-resistance frequently arises through multiple mechanisms. The development of new strategies using natural compounds and PTX in combination has been the aim of several prior studies, in order to enhance the efficacy of chemotherapy. In this study, we found the following: (i) gallic acid (GA), a phenolic compound, potentiated the capacity of PTX to decrease proliferation and to cause G2/M cycle arrest in the PTX-resistant A2780AD ovarian cancer cell line; (ii) GA exerted a pro-oxidant action by increasing the production of reactive oxygen species (ROS), and co-treatment with the antioxidant agent N‑acetyl-L‑cysteine (NAC) prevented GA+PTX-induced cell proliferation inhibition and G2/M phase arrest; (iii) PTX stimulated ERK phosphorylation/activation, and co-treatment with the MEK/ERK inhibitor PD98049 potentiated the proliferation inhibition and G2/M phase arrest; (iv) and finally, GA abrogated the PTX-induced stimulation of ERK phosphorylation, a response that was prevented by co-treatment with NAC.

Modification of C-seco taxoids through ring tethering and substituent replacement leading to effective agents against tumor drug resistance mediated by βIII-Tubulin and P-glycoprotein (P-gp) overexpressions.

In our efforts to improve the efficacy of taxane-based microtubule (MT) stabilizing agents against tumor drug resistance mediated by multiple mechanisms, two clinically relevant factors were focused: i.e., P-glycoprotein and βIII-tubulin overexpression.

Structural Basis of Microtubule Stabilization by Discodermolide.

Microtubule-stabilizing agents (MSAs) are widely used in chemotherapy. Using X-ray crystallography we elucidated the detailed binding modes of two potent MSAs, (+)-discodermolide (DDM) and the DDM-paclitaxel hybrid KS-1-199-32, in the taxane pocket of β-tubulin. The two compounds bind in a very similar hairpin conformation, as previously observed in solution.

Structural Determinants of the Dictyostatin Chemotype for Tubulin Binding Affinity and Antitumor Activity Against Taxane- and Epothilone-Resistant Cancer Cells.

A combined biochemical, structural, and cell biology characterization of dictyostatin is described, which enables an improved understanding of the structural determinants responsible for the high-affinity binding of this anticancer agent to the taxane site in microtubules (MTs). The study reveals that this macrolide is highly optimized for MT binding and that only a few of the structural modifications featured in a library of synthetic analogues resulted in small gains in binding affinity. The high efficiency of the dictyostatin chemotype in overcoming various kinds of clinically relevant resistance mechanisms highlights its potential for therapeutic development for the treatment of drug-resistant tumors.

Aggregated Compound Biological Signatures Facilitate Phenotypic Drug Discovery and Target Elucidation.

Predicting the cellular response of compounds is a challenge central to the discovery of new drugs. Compound biological signatures have risen as a way of representing the perturbation produced by a compound in the cell. However, their ability to encode specific phenotypic information and generating tangible predictions remains unknown, mainly because of the inherent noise in such data sets.

Structural and Biochemical Characterization of the Interaction of Tubulin with Potent Natural Analogues of Podophyllotoxin.

Four natural analogues of podophyllotoxin obtained from the Mexican medicinal plant Bursera fagaroides, namely, acetyl podophyllotoxin (2), 5'-desmethoxy-β-peltatin A methyl ether (3), 7',8'-dehydro acetyl podophyllotoxin (4), and burseranin (5), have been characterized, and their interactions with tubulin have been investigated. Cytotoxic activity measurements, followed by immunofluorescence microscopy and flow cytometry studies, demonstrated that these compounds disrupt microtubule networks in cells and cause cell cycle arrest in the G2/M phase in the A549 cell line. A tubulin binding assay showed that compounds 1-4 were potent assembly inhibitors, displaying binding to the colchicine site with Kb values ranging from 11.

Synthesis and Anti-Proliferative Activity of Sulfanyltriazolylnaphthalenols and Sulfanyltriazolylnaphthalene-1,4-diones.

A series of new sulfanyltriazolylnaphthalenols (10a-f and 13a-f) and sulfanyltriazolylnaphthalene-1,4-diones (14a-f) were synthesized and evaluated against a panel of cancer cell lines. Among the tested compounds, 10b and 10d showed the best anti-proliferative activity with GI50 values ranging from 2.72 to 10 and 3.

The Mechanism of the Interactions of Pironetin Analog/Combretastatin A-4 Hybrids with Tubulin.

We here report an investigation of the interactions with tubulin of two types of molecules of a hybrid structural type consisting in a combretastatin A-4 moiety and a simplified pironetin fragment. The cytotoxicities of the molecules on two reference tumoral cell lines were measured. In addition, the effects of the compounds on the cell cycle and on microtubule assembly were observed.

Synthesis and biological evaluation of new oxadiazoline-substituted naphthalenyl acetates as anticancer agents.

A series of new oxadiazoline-substituted naphthalenyl acetates 3a-e and oxadiazoline-substituted 4-methoxynaphthalenyl acetates 7b-e were synthesized and tested by the National Cancer Institute (NCI) for their in vitro anticancer activity. The two derivatives bearing acetoxy groups at the 1 and 3 positions of the phenyl ring 3c and 7c were the most active showing significant anticancer activity against all tested cancer cell lines, with GI50 values ranging from 0.175 to 3.

Design and synthesis of pironetin analogue/colchicine hybrids and study of their cytotoxic activity and mechanisms of interaction with tubulin.

We here report the synthesis of a series of 12 hybrid molecules composed of a colchicine moiety and a pironetin analogue fragment. The two fragments are connected through an ester-amide spacer of variable length. The cytotoxic activities of these compounds and their interactions with tubulin have been investigated.

The impact of cyclopropane configuration on the biological activity of cyclopropyl-epothilones.

Two cis-12,13-cyclopropyl-epothilone B variants have been synthesized, differing only in the configuration of the stereocenters at C12 and C13. The syntheses were based on a common allylic alcohol intermediate that was converted into the corresponding diastereomeric hydroxymethyl-cyclopropanes by means of stereoselective Charette cyclopropanations. Macrocyclizations were accomplished through ring-closing metathesis (RCM).

Heparin modulates the mitogenic activity of fibroblast growth factor by inducing dimerization of its receptor. a 3D view by using NMR.

In vitro mitogenesis assays have shown that sulfated glycosaminoglycans (GAGs; heparin and heparan sulfate) cause an enhancement of the mitogenic activity of fibroblast growth factors (FGFs). Herein, we report that the simultaneous presence of FGF and the GAG is not an essential requisite for this event to take place. Indeed, preincubation with heparin (just before FGF addition) of cells lacking heparan sulfate produced an enhancing effect equivalent to that observed when the GAG and the protein are simultaneously added.

Synthesis and biological evaluation of truncated α-tubulin-binding pironetin analogues lacking alkyl pendants in the side chain or the dihydropyrone ring.

The preparation of several new truncated analogues of the natural dihydropyrone pironetin is described. They differ from the natural product mainly in the suppression of some of the alkyl pendants in either the side chain or the dihydropyrone ring. Their cytotoxic activity and their interactions with tubulin have been investigated.

Synthesis and biological evaluation of colchicine B-ring analogues tethered with halogenated benzyl moieties.

Deacetylcolchicine was reacted with substituted benzyl halides to provide a library of compounds for biological analysis. Compound 7 (3,4-difluorobenzyl-N-aminocolchicine) was shown to possess cytotoxicity in cancer cell lines in the low nanomolar range. Significantly, it showed no loss of activity in the resistant A2780AD ovarian carcinoma cell line known to overexpress the ABCB1 drug transporter and was also unaffected by overexpression of class III β-tubulin in HeLa transfected cells.

Synthesis and biological evaluation of colchicine C-ring analogues tethered with aliphatic linkers suitable for prodrug derivatisation.

Colchicine was modified at the 10-OCH(3) position of the C-ring by reaction with heterocyclic amines or commercially available amines to afford a library of target colchicinoids in high yields (62-99%). Molecular modeling revealed that the incorporation of the linker groups led to a reduction in entropy and therefore binding affinity when compared with colchicine. Some colchicinoids were shown to be equicytotoxic with colchicine when evaluated in the DLD-1 colon cancer cells and retained activity in resistant A2780AD or HeLa cells with mutant Class III β-tubulin.

Fatty acids revert the inhibition of respiration caused by the antidiabetic drug metformin to facilitate their mitochondrial β-oxidation.

While metformin has been widely used to treat type 2 diabetes for the last fifty years, its mode of action remains unclear. Hence, we investigated the short-term alterations in energy metabolism caused by metformin administration in 3T3-L1 adipocytes. We found that metformin inhibited mitochondrial respiration, although ATP levels remained constant as the decrease in mitochondrial production was compensated by an increase in glycolysis.

Development of chromanes as novel inhibitors of the uncoupling proteins.

The uncoupling proteins (UCPs) are mitochondrial carriers that modulate the energetic efficiency and, as a result, can lower superoxide levels. Here, we describe the discovery of a small-molecule inhibitor of the UCPs. Screening of potential UCP1 regulators led to the identification of chromane derivatives that inhibit its proton conductance.

Cyclosporine A-induced nitration of tyrosine 34 MnSOD in endothelial cells: role of mitochondrial superoxide.

Aims: Cyclosporine A (CsA) has represented a fundamental therapeutic weapon in immunosuppression for the past three decades. However, its clinical use is not devoid of side effects, among which hypertension and vascular injury represent a major drawback. Endothelial cells are able to generate reactive oxygen and nitrogen species upon exposure to CsA, including formation of peroxynitrite.