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Syntheses of 3-[(Alkylamino)methylene]-6-methylpyridine-2,4(1H,3H)-diones, 3-Substituted 7-Methyl-2H-pyrano[3,2-c]pyridine-2,5(6H)-dione Fluorescence Probes, and Tetrahydro-1H,9H-2,10-dioxa-9-azaanthracen-1-ones.
Synthesis (Stuttg)
PUBLISHED: 09-02-2014
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Various condensation and ring-closing reactions were used for the syntheses of 3-[(alkylamino)methylene]-6-methylpyri-dine-2,4(1H,3H)-diones, bicyclic pyridinones, and tricyclic morpholinopyrones. For instance, 3-[(dialkylamino)methylene]-6-methylpyridine-2,4(1H,3H)-diones were synthesized from the condensation of dialkylamines and 3-formyl-4-hydroxy-6-methylpyridin-2(1H)-one. 3-Formyl-4-hydroxy-6-methylpyridin-2(1H)-one, derived from 3-formyl-4-hydroxy-6-methylpyridin-2(1H)-one, was used to construct a number of bicyclic pyridinones via a one-pot Knoevenagal and intramolecular lactonization reaction. Tricyclic morpholinopyrones were assembled from a dialkylation reaction involving a dinucleophile, 3-amino-4-hydroxy-6-methyl-2H-pyran-2-one, and a dielectrophile, trans-3,6-dibromocyclohexene. Depending on the reaction conditions, isomers of the tricyclic molecules can be selectively produced, and their chemical structures were unequivocally determined using single-crystal X-ray analyses and 2D COSY spectroscopy. The fluorescently active bicyclic pyridinone compounds show longer absorption (368-430 nm; maximum) and emission wavelengths (450-467 nm) than those of 7-amino-4-methylcoumarin (AMC; ?abs,max = 350 nm; ?em = 430 nm) suggesting these molecules, such as 3-(2-aminoacetyl)-7-methyl-2H-pyrano[3,2-c]pyridine-2,5(6H)-dione, can be employed as fluorescence activity based probes for tracing biological pathways.
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Syntheses, neural protective activities, and inhibition of glycogen synthase kinase-3? of substituted quinolines.
Bioorg. Med. Chem. Lett.
PUBLISHED: 05-03-2014
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A new series of fifteen 5-, 6-, and 8-appended 4-methylquinolines were synthesized and evaluated for their neural protective activities. Selected compounds were further examined for their inhibition of glycogen synthase kinase-3? (GSK-3?) and protein kinase C (PKC). Two most potent analogs, compounds 3 and 10, show nanomolar protective activities in amyloid ?-induced MC65 cells and enzymatic inhibitory activities against GSK-3?, but poor PKC inhibitory activities. Using normal mouse model, the distribution of the most potent analog 3 in various tissues and possible toxic effects in the locomotors and inhibition of liver transaminases activities were carried out. No apparent decline of locomotor activity and no inhibition of liver transaminases were found. The compound appears to be safe for long-term use in Alzheimer's disease mouse model.
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Relative Binding Enthalpies from Molecular Dynamics Simulations Using a Direct Method.
J Chem Theory Comput
PUBLISHED: 03-07-2014
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The potential for reliably predicting relative binding enthalpies, ??E, from a direct method utilizing molecular dynamics is examined for a system of three phosphotyrosyl peptides binding to a protein receptor, the Src SH2 domain. The binding enthalpies were calculated from the potential energy differences between the bound and the unbound end-states of each peptide from equilibrium simulations in explicit water. The statistical uncertainties in the ensemble-mean energy values from multiple, independent simulations were obtained using a bootstrap method. Simulations were initiated with different starting coordinates as well as different velocities. Statistical uncertainties in ??E are 2 to 3 kcal/mol based on calculations from 40, 10 ns trajectories for each system (three SH2-peptide complexes or unbound peptides). Uncertainties in relative component energies, comprising solute-solute, solute-solvent and solvent-solvent interactions, are considerably larger. Energy values were estimated from an unweighted ensemble averaging of multiple trajectories with the a priori assumption that all trajectories are equally likely. Distributions in energy-rmsd space indicate that the trajectories sample the same basin and the difference in mean energy values between trajectories is due to sampling of alternative local regions of this superbasin. The direct estimate of relative binding enthalpies is concluded to be a reasonable approach for well-ordered systems with ??E values greater than ?3 kcal/mol, although the approach would benefit from future work to determine properly distributed starting points that would enable efficient sampling of conformational space using multiple trajectories.
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Inhibition of long chain fatty acyl-CoA synthetase (ACSL) and ischemia reperfusion injury.
Bioorg. Med. Chem. Lett.
PUBLISHED: 01-06-2014
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Various triacsin C analogs, containing different alkenyl chains and carboxylic acid bioisoteres including 4-aminobenzoic acid, isothiazolidine dioxide, hydroxylamine, hydroxytriazene, and oxadiazolidine dione, were synthesized and their inhibitions of long chain fatty acyl-CoA synthetase (ACSL) were examined. Two methods, a cell-based assay of ACSL activity and an in situ [(14)C]-palmitate incorporation into extractable lipids were used to study the inhibition. Using an in vivo leukocyte recruitment inhibition protocol, the translocation of one or more cell adhesion molecules from the cytoplasm to the plasma membrane on either the endothelium or leukocyte or both was inhibited by inhibitors 1, 9, and triacsin C. The results suggest that inhibition of ACSL may attenuate the vascular inflammatory component associated with ischemia reperfusion injury and lead to a decrease of infarct expansion.
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Quantitative electrochemical detection of cathepsin B activity in complex tissue lysates using enhanced AC voltammetry at carbon nanofiber nanoelectrode arrays.
Biosens Bioelectron
PUBLISHED: 01-02-2014
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The proteolytic activity of a cancer-related enzyme cathepsin B is measured with alternating current voltammetry (ACV) using ferrocene (Fc) labeled tetrapeptides attached to nanoelectrode arrays (NEAs) fabricated with vertically aligned carbon nanofibers (VACNFs). This combination enables the use of high AC frequencies (~1kHz) with enhanced electrochemical signals. The specific proteolysis of the Fc-peptide by cathepsin B produces decay in the ACV peak current versus the reaction time. The exponential component of the raw data can be extracted and defined as the "extracted proteolytic signal" which allows consistent quantitative analyses using a heterogeneous Michaelis-Menten model. A "specificity constant" kcat/KM = (3.68 ± 0.50) × 10(4)M(-1)s(-1) for purified cathepsin B was obtained. The detections of cathepsin B activity in different concentrations of whole lysate of human breast tissue, tissue lysate spiked with varied concentrations of cathepsin B, and the tissue lysate after immunoprecipitation showed that there is ~13.4 nM higher cathepsin B concentration in 29.1 µg mL(-1) of whole tissue lysate than the immunoprecipitated sample. The well-defined regular VACNF NEAs by e-beam lithography show a much faster kinetics for cathepsin B proteolysis with kcat/KM = 9.2 × 10(4)M(-1)s(-1). These results illustrate the potential of this technique as a portable multiplex electronic system for cancer diagnosis by rapid protease profiling of serum or blood samples.
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Design, synthesis, and bioevaluation of viral 3C and 3C-like protease inhibitors.
Bioorg. Med. Chem. Lett.
PUBLISHED: 08-20-2013
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A class of tripeptidyl transition state inhibitors containing a P1 glutamine surrogate, a P2 leucine, and a P3 arylalanines, was found to potently inhibit Norwalk virus replication in enzyme and cell based assays. An array of warheads, including aldehyde, ?-ketoamide, bisulfite adduct, and ?-hydroxyphosphonate transition state mimic, was also investigated. Tripeptidyls 2 and 6 possess antiviral activities against noroviruses, human rhinovirus, severe acute respiratory syndrome coronavirus, and coronavirus 229E, suggesting a broad range of antiviral activities.
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Electrochemical Protease Biosensor Based on Enhanced AC Voltammetry Using Carbon Nanofiber Nanoelectrode Arrays.
J Phys Chem C Nanomater Interfaces
PUBLISHED: 07-02-2013
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We report an electrochemical method for measuring the activity of proteases using nanoelectrode arrays (NEAs) fabricated with vertically aligned carbon nanofibers (VACNFs). The VACNFs of ~150 nm in diameter and 3 to 5 ?m in length were grown on conductive substrates and encapsulated in SiO2 matrix. After polishing and plasma etching, controlled VACNF tips are exposed to form an embedded VACNF NEA. Two types of tetrapeptides specific to cancer-mediated proteases legumain and cathepsin B are covalently attached to the exposed VACNF tip, with a ferrocene (Fc) moiety linked at the distal end. The redox signal of Fc can be measured with AC voltammetry (ACV) at ~1 kHz frequency on VACNF NEAs, showing distinct properties from macroscopic glassy carbon electrodes due to VACNFs unique interior structure. The enhanced ACV properties enable the kinetic measurements of proteolytic cleavage of the surface-attached tetrapeptides by proteases, further validated with a fluorescence assay. The data can be analyzed with a heterogeneous Michaelis-Menten model, giving "specificity constant" kcat /Km as (4.3 ± 0.8) × 10(4) M(-1)s(-1) for cathepsin B and (1.13 ± 0.38) × 10(4) M(-1)s(-1) for legumain. This method could be developed as portable multiplex electronic techniques for rapid cancer diagnosis and treatment monitoring.
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Macrocyclic inhibitors of 3C and 3C-like proteases of picornavirus, norovirus, and coronavirus.
Bioorg. Med. Chem. Lett.
PUBLISHED: 04-02-2013
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The design, synthesis, and in vitro evaluation of the first macrocyclic inhibitor of 3C and 3C-like proteases of picornavirus, norovirus, and coronavirus are reported. The in vitro inhibitory activity (50% effective concentration) of the macrocyclic inhibitor toward enterovirus 3C protease (CVB3 Nancy strain), and coronavirus (SARS-CoV) and norovirus 3C-like proteases, was determined to be 1.8, 15.5 and 5.1 ?M, respectively.
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Transport by OATP1B1 and OATP1B3 enhances the cytotoxicity of epigallocatechin 3-O-gallate and several quercetin derivatives.
J. Nat. Prod.
PUBLISHED: 01-17-2013
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Organic anion transporting polypeptides (OATPs) 1B1 and 1B3 are transporters that are expressed selectively in human hepatocytes under normal conditions. OATP1B3 is also expressed in certain cancers. Flavonoids such as green tea catechins and quercetin glycosides have been shown to modulate the function of some OATPs. In the present study, the extent to which six substituted quercetin derivatives (1-6) affected the function of OATP1B1 and OATP1B3 was investigated. Uptake of the radiolabeled model substrates estradiol 17?-glucuronide, estrone 3-sulfate, and dehydroepiandrosterone sulfate (DHEAS) was determined in the absence and presence of compounds 1-6 using Chinese hamster ovary (CHO) cells stably expressing either OATP1B1 or OATP1B3. Several of compounds 1-6 inhibited OATP-mediated uptake of all three model substrates, suggesting that they could also be potential substrates. Compound 6 stimulated OATP1B3-mediated estradiol 17?-glucuronide uptake by increasing the apparent affinity of OATP1B3 for its substrate. Cytotoxicity assays demonstrated that epigallocatechin 3-O-gallate (EGCG) and most of compounds 1-6 killed preferentially OATP-expressing CHO cells. EGCG, 1, and 3 were the most potent cytotoxic compounds, with EGCG and 3 selectively killing OATP1B3-expressing cells. Given that OATP1B3 is expressed in several cancers, EGCG and some of the quercetin derivatives studied might be promising lead compounds for the development of novel anticancer drugs.
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Bioavailability and efficacy of a gap junction enhancer (PQ7) in a mouse mammary tumor model.
PLoS ONE
PUBLISHED: 01-01-2013
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The loss of gap junctional intercellular communication is characteristic of neoplastic cells, suggesting that the restoration with a gap junction enhancer may be a new therapeutic treatment option with less detrimental effects than traditional antineoplastic drugs. A gap junction enhancer, 6-methoxy-8-[(2-furanylmethyl) amino]-4-methyl-5-(3-trifluoromethylphenyloxy) quinoline (PQ7), on the normal tissue was evaluated in healthy C57BL/6J mice in a systemic drug distribution study. Immunoblot analysis of the vital organs indicates a reduction in Cx43 expression in PQ7-treated animals with no observable change in morphology. Next the transgenic strain FVB/N-Tg(MMTV-PyVT) 634Mul/J (also known as PyVT) was used as a spontaneous mammary tumor mouse model to determine the biological and histological effects of PQ7 on tumorigenesis and metastasis at three stages of development: Pre tumor, Early tumor, and Late tumor formation. PQ7 was assessed to have a low toxicity through intraperitoneal administration, with the majority of the compound being detected in the heart, liver, and lungs six hours post injection. The treatment of tumor bearing animals with PQ7 had a 98% reduction in tumor growth, while also decreasing the total tumor burden compared to control mice during the Pre stage of development. PQ7 treatment increased Cx43 expression in the neoplastic tissue during Pre-tumor formation; however, this effect was not observed in Late stage tumor formation. This study shows that the gap junction enhancer, PQ7, has low toxicity to normal tissue in healthy C57BL/6J mice, while having clinical efficacy in the treatment of spontaneous mammary tumors of PyVT mice. Additionally, gap junctional intercellular communication and neoplastic cellular growth are shown to be inversely related, while treatment with PQ7 inhibits tumor growth through targeting gap junction expression.
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Kinetic properties of alternatively spliced isoforms of laccase-2 from Tribolium castaneum and Anopheles gambiae.
Insect Biochem. Mol. Biol.
PUBLISHED: 11-04-2011
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Laccase-2 is a highly conserved multicopper oxidase that functions in insect cuticle pigmentation and tanning. In many species, alternative splicing gives rise to two laccase-2 isoforms. A comparison of laccase-2 sequences from three orders of insects revealed eleven positions at which there are conserved differences between the A and B isoforms. Homology modeling suggested that these eleven residues are not part of the substrate binding pocket. To determine whether the isoforms have different kinetic properties, we compared the activity of laccase-2 isoforms from Tribolium castaneum and Anopheles gambiae. We partially purified the four laccases as recombinant enzymes and analyzed their ability to oxidize a range of laccase substrates. The predicted endogenous substrates tested were dopamine, N-acetyldopamine (NADA), N-?-alanyldopamine (NBAD) and dopa, which were detected in T. castaneum previously and in A. gambiae as part of this study. Two additional diphenols (catechol and hydroquinone) and one non-phenolic substrate (2,2-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)) were also tested. We observed no major differences in substrate specificity between the A and B isoforms. Dopamine, NADA and NBAD were oxidized with catalytic efficiencies ranging from 51 to 550 min?¹ mM?¹. These results support the hypothesis that dopamine, NADA and NBAD are endogenous substrates for both isoforms of laccase-2. Catalytic efficiencies associated with dopa oxidation were low, ranging from 8 to 30 min?¹ mM?¹; in comparison, insect tyrosinase oxidized dopa with a catalytic efficiency of 201 min?¹ mM?¹. We found that dopa had the highest redox potential of the four endogenous substrates, and this property of dopa may explain its poor oxidation by laccase-2. We conclude that laccase-2 splice isoforms are likely to oxidize the same substrates in vivo, and additional experiments will be required to discover any isoform-specific functions.
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Synthesis and antiviral activity of substituted quercetins.
Bioorg. Med. Chem. Lett.
PUBLISHED: 10-08-2011
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Influenza viruses are important pathogens that cause respiratory infections in humans and animals. In addition to vaccination, antiviral drugs against influenza virus play a significant role in controlling viral infections by reducing disease progression and virus transmission. Plant derived polyphenols are associated with antioxidant activity, anti-carcinogenic, and cardio- and neuro-protective actions. Some polyphenols, such as resveratrol and epigallocatechin gallate (EGCG), showed significant anti-influenza activity in vitro and/or in vivo. Recently we showed that quercetin and isoquercetin (quercetin-3-?-d-glucoside), a glucoside form of quercetin, significantly reduced the replication of influenza viruses in vitro and in vivo (isoquercetin). The antiviral effects of isoquercetin were greater than that of quercetin with lower IC(50) values and higher in vitro therapeutic index. Thus, we investigated the synthesis and antiviral activities of various quercetin derivatives with substitution of C3, C3, and C5 hydroxyl functions with various phenolic ester, alkoxy, and aminoalkoxy moieties. Among newly synthesized compounds, quercetin-3-gallate which is structurally related to EGCG showed comparable antiviral activity against influenza virus (porcine H1N1 strain) to that of EGCG with improved in vitro therapeutic index.
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Anti-breast cancer agents, quinolines, targeting gap junction.
Med Chem
PUBLISHED: 05-05-2011
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Cancer cells exhibit many defects in cell communication that contribute to the loss of tissue homeostasis (excess cell proliferation, invasion, and metastasis). The process of cancer formation causes a disruption in cell homeostasis, affecting the ability to respond to extracellular signals, as well as triggering some intracellular events which alter gap junctional intercellular communication (GJIC). Previous research has shown that the first two generations of substituted quinolines have anti-cancer effects in human breast cancer cells. This report presents the synthesis and bioactivities of third generation substituted quinolines. Scrape load/dye transfer studies showed that 100 nM of PQ15, a third generation substituted quinoline, causes a 4.5-fold increase of gap junction activity in T47D breast cancer cells. Furthermore, a significant decrease of cell proliferation and viability was observed in the presence of 200 nM PQ15 compared to control. The expression of ?-survivin was reduced to <40% in the treatment of 200 nM PQ15 compared to solvent alone. Alpha-survivin expression is upregulated in human cancers and associated with resistance to chemotherapy, suggesting that ?-survivin prolongs the survival of cancer cells. Thus, it has been shown that substituted quinolines stimulate gap junction activity, decrease alpha survivin expression, and subsequently inhibit cancer cell growth. Our findings demonstrate that PQ15 has a promising role in exerting anti-cancer activity in human breast cancer cells.
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Synthesis of cyclododeciptycene quinones.
J. Am. Chem. Soc.
PUBLISHED: 11-22-2010
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Cycloiptycenes are elusive and synthetically challenging molecules. We report the first synthesis of two substituted cyclododeciptycene tetraquinones via a sequence of intermolecular and intramolecular Diels-Alder reactions from cis,cis-heptiptycene tetraquinone 2 and substituted 7,16-dihydro-7,16-(o-benzeno)heptacenes 3. Heptiptycene tetraquinone 2 was made from triptycene bisquinone 4 and 1,4-dimethoxyanthracene in three steps, and 6,8,15,17-tetramethoxy-7,16-dihydro-7,16-(o-benzeno)heptacene (3a) was synthesized from triptycene bisquinone 4 and 1,4-dihydro-2,3-benzoxathiin-3-oxide in four steps. The structure of a cyclododeciptycene, 1a, was determined by a single-crystal X-ray analysis. The synthetic sequence is general and should allow the incorporation of various alkoxy and acetoxy substituents appended to the cycloiptycene framework.
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Second-generation substituted quinolines as anticancer drugs for breast cancer.
Anticancer Res.
PUBLISHED: 11-02-2010
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Cancer cells have reduced capacity for gap junctional inter-cellular communication (GJIC). One feasible approach to reduce growth of cancer cells is to enhance GJIC. This report shows that a second-generation substituted quinoline, PQ7, has anti-tumor effect. Scrape load/dye transfer and colony growth assays were performed to measure GJIC and tumor formation of T47D breast cancer cells. PQ7 at 500 nM induced a 16-fold increase in the GJIC in T47D cells. In addition to an increase in GJIC, a 50% decrease of colony growth was observed with 100 nM of PQ7. PQ7-treated nu/nu mice showed a 100% regression of xenograft tumor growth of T47D cells. The results show that PQ7 has a promising role in exerting anti-tumor activity in human breast cancer cells.
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Biodegradable nanogels for oral delivery of interferon for norovirus infection.
Antiviral Res.
PUBLISHED: 09-15-2010
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Norwalk virus (NV) replicon-harboring cells have provided an excellent tool to the development of antivirals. Previously we demonstrated that the expression levels of replicon RNA and proteins were significantly reduced in the presence of various interferons (IFNs) including IFN-? and IFN-? in a dose-dependent manner in the NV replicon-harboring cells, and suggested that IFNs could be therapeutic options for norovirus infection. It was also demonstrated that innate immunity including IFNs is crucial in the replication and pathogenicity of murine norovirus (MNV) in vitro (RAW267.4 cells) and in vivo. IFNs have a short half-life in vitro and in vivo due to low stability. Thus it is important to have a good delivery system to improve the stability of IFNs. Nanogels are nanosized networks of chemically cross-linked polymers that swell in physiologic solutions and provide improved stability and bioavailability to drugs. We have synthesized nanogels based on cross-linked polyethyleneimine (PEI)-polyethylenglycol (PEG). The PEI/PEG nanogels were further acetylated (AcNg) to reduce cellular penetration and cytotoxicity. The IFN-AcNg complex was prepared by incubating two components together at 4 °C and lyophilization. The IFN activity of IFN-AcNg was evaluated in the NV- and HCV-replicon-harboring cells and against MNV-1 in RAW267.4 cells in comparison to IFN without AcNg. The AcNg improved the stability of IFN stored at 4 °C, and was well tolerated in the cells. Furthermore, the activity of IFN was significantly higher when combined with AcNg in the replicon-harboring cells and against MNV-1 in RAW267.4 cells. We concluded that AcNg may be pursued further as a vehicle for oral delivery of IFNs in norovirus infection.
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Bioactivity and molecular targets of novel substituted quinolines in murine and human tumor cell lines in vitro.
Int. J. Oncol.
PUBLISHED: 02-04-2010
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Substituted quinolines (PQ code number), which reduce colony formation and increase gap junctional intercellular communication, were tested for their ability to interact with various molecular targets in murine and human tumor cell lines in vitro. Various markers of tumor cell metabolism, DNA fragmentation, mitotic disruption, apoptosis induction and growth factor receptor signaling pathways were assayed in vitro to evaluate drug cytotoxicity. Based on its ability to inhibit the metabolic activity of suspension cultures of leukemic L1210 cells at days 2 and 4 in vitro, PQ1 succinic acid salt is the most effective antiproliferative agent among the synthetic quinoline analogs tested. Moreover, antiproliferative PQ1 is effective across a spectrum of monolayer cultures of pancreatic Pan02, epidermoid A-431 and mammary SK-BR-3 and BT-474 tumor cells. PQ1 also blocks Ki-67 expression, a marker of tumor cell proliferation. A 1.5- to 3-h treatment with PQ1 is sufficient to inhibit the incorporations of [3H]-thymidine into DNA, [3H]-uridine into RNA and [3H]-leucine into protein used to assess the rates of macromolecule syntheses over a 0.5- or 1-h period of pulse-labeling in L1210 tumor cells. A 15-min pretreatment with PQ1 inhibits the cellular transport of both purine and pyrimidine nucleosides over a 30-sec period in vitro, suggesting that PQ1 may prevent the incorporation of [3H]-adenosine and [3H]-thymidine into DNA because it rapidly blocks the uptake of these nucleosides by the tumor cells. Since PQ1 does not reduce the fluorescence of the ethidium bromide-DNA complex, it does not directly bind to or destabilize double-stranded DNA. Over a 6- to -48-h period, PQ1 has very little effect on the mitotic index of L1210 cells but stimulates the formation of many binucleated cells and a few micronuclei, suggesting that this compound might increase mitotic abnormality, induce chromosomal damage or missegregation, and block cytokinesis. The fact that PQ1 induces initiator caspase-2 and effector caspase-3 activities and poly(ADP-ribose) polymerase-1 cleavage within 1-4 h and internucleosomal DNA fragmentation within 24 h in L1210 cells suggests that this antitumor drug can trigger the early and late events required for cells to undergo apotosis. Whole-cell immunodetection and Western blot analysis indicate that, in contrast to 17-(allylamino)-17-demethoxygeldanamycin and radicicol, PQ1 fails to down-regulate the protein level at 24 h and autophosphorylation at 3 h of membrane-anchored HER1 in A-431 cells and HER2 in SK-BR-3 cells, suggesting that this antitumor compound is unlikely to interact with and inhibit Hsp90 and the epidermal growth factor (EGF) receptor signaling pathways. In conclusion, antiproliferative PQ1 is effective against a spectrum of tumor cells and might interact with various membrane and nuclear targets to enhance gap junctions, inhibit nucleoside transport and block cytokinesis but does not appear to disrupt the EGF receptor-mediated signaling pathways to induce growth arrest and apoptosis.
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Synthesis, molecular targets, and antitumor activities of substituted tetrahydro-1-oxopyrano[4,3-b][1]benzopyrans and nanogels for drug delivery.
Anticancer Agents Med Chem
PUBLISHED: 06-23-2009
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A class of substituted 1H,7H-5a,6,8,9-tetrahydro-1-oxopyrano[4,3-b][1]benzopyrans (tricyclic pyrones; TPs) was synthesized from a one-pot condensation reaction of 6-substituted 4-hydroxy-2-pyrones and cyclohexenecarboxaldehydes. The reaction involves a 6pi-electrocyclic ring closing process, and stereo- and regioselectivities were examined. C3-Pyridyl-containing TPs may represent a novel synthetic class of microtubule de-stabilizing anti-cancer drugs that inhibit macromolecule synthesis, tubulin polymerization, and the proliferation of a spectrum of wild-type and multi-drug resistant tumor cell lines in vitro. A linear skeleton with a N-containing aromatic ring attached at C3 of the top A-ring, a central pyran B-ring and a six-membered bottom C-ring with no alkylation at C7 are required for the antitumor activities of the lead compounds, a 3-pyridyl benzopyran (code name H10) and its 2-pyridyl regioisomer (code name H19). In addition to interacting with the colchicine-binding site to inhibit tubulin polymerization and increase the mitotic index, these TP analogs also block the cellular transport of nucleosides to inhibit DNA synthesis more effectively than other antimitotic agents. The anticancer potential of TPs in vivo is suggested by the fact that i.p. injections of H10 decrease the growth of solid tumors in mice inoculated with lung or ovarian carcinomas. A drug-delivery system involving nanogels was studied. We incorporated the anticancer compound, 6-hydroxymethyl-1,4-anthracenedione (code name AQ10) into PEG-PEI nanogel, and found that AQ10-encapsulated nanogel PEG-PEI is significantly more effective in altering the growth of Pan 02 (pancreatic cancer) cells compared to AQ10 or nanogel PEG-PEI alone. Since AQ10 is insoluble in water, PEG-PEI encapsulation represents a way to solubilize and deliver this as well as other poorly soluble compounds.
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Selective and irreversible inhibitors of mosquito acetylcholinesterases for controlling malaria and other mosquito-borne diseases.
PLoS ONE
PUBLISHED: 06-20-2009
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New insecticides are urgently needed because resistance to current insecticides allows resurgence of disease-transmitting mosquitoes while concerns for human toxicity from current compounds are growing. We previously reported the finding of a free cysteine (Cys) residue at the entrance of the active site of acetylcholinesterase (AChE) in some insects but not in mammals, birds, and fish. These insects have two AChE genes (AP and AO), and only AP-AChE carries the Cys residue. Most of these insects are disease vectors such as the African malaria mosquito (Anopheles gambiae sensu stricto) or crop pests such as aphids. Recently we reported a Cys-targeting small molecule that irreversibly inhibited all AChE activity extracted from aphids while an identical exposure caused no effect on the human AChE. Full inhibition of AChE in aphids indicates that AP-AChE contributes most of the enzymatic activity and suggests that the Cys residue might serve as a target for developing better aphicides. It is therefore worth investigating whether the Cys-targeting strategy is applicable to mosquitocides. Herein, we report that, under conditions that spare the human AChE, a methanethiosulfonate-containing molecule at 6 microM irreversibly inhibited 95% of the AChE activity extracted from An. gambiae s. str. and >80% of the activity from the yellow fever mosquito (Aedes aegypti L.) or the northern house mosquito (Culex pipiens L.) that is a vector of St. Louis encephalitis. This type of inhibition is fast ( approximately 30 min) and due to conjugation of the inhibitor to the active-site Cys of mosquito AP-AChE, according to our observed reactivation of the methanethiosulfonate-inhibited AChE by 2-mercaptoethanol. We also note that our sulfhydryl agents partially and irreversibly inhibited the human AChE after prolonged exposure (>4 hr). This slow inhibition is due to partial enzyme denaturation by the inhibitor and/or micelles of the inhibitor, according to our studies using atomic force microscopy, circular dichroism spectroscopy, X-ray crystallography, time-resolved fluorescence spectroscopy, and liquid chromatography triple quadrupole mass spectrometry. These results support our view that the mosquito-specific Cys is a viable target for developing new mosquitocides to control disease vectors and to alleviate resistance problems with reduced toxicity toward non-target species.
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Tricyclic pyrone compounds prevent aggregation and reverse cellular phenotypes caused by expression of mutant huntingtin protein in striatal neurons.
BMC Neurosci
PUBLISHED: 03-18-2009
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Huntingtons disease (HD) is a progressive neurodegenerative disorder caused by a CAG repeat expansion mutation in the coding region of a novel gene. The mechanism of HD is unknown. Most data suggest that polyglutamine-mediated aggregation associated with expression of mutant huntingtin protein (mhtt) contributes to the pathology. However, recent studies have identified early cellular dysfunctions that preclude aggregate formation. Suppression of aggregation is accepted as one of the markers of successful therapeutic approaches. Previously, we demonstrated that tricyclic pyrone (TP) compounds efficiently inhibited formation of amyloid-beta (Abeta) aggregates in cell and mouse models representing Alzheimers Disease (AD). In the present study, we aimed to determine whether TP compounds could prevent aggregation and restore early cellular defects in primary embryonic striatal neurons from animal model representing HD.
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Inhibition of Alzheimers amyloid toxicity with a tricyclic pyrone molecule in vitro and in vivo.
J. Neurochem.
PUBLISHED: 01-15-2009
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Small beta-amyloid (Abeta) 1-42 aggregates are toxic to neurons and may be the primary toxic species in Alzheimers disease (AD). Methods to reduce the level of Abeta, prevent Abeta aggregation, and eliminate existing Abeta aggregates have been proposed for treatment of AD. A tricyclic pyrone named CP2 is found to prevent cell death associated with Abeta oligomers. We studied the possible mechanisms of neuroprotection by CP2. Surface plasmon resonance spectroscopy shows a direct binding of CP2 with Abeta42 oligomer. Circular dichroism spectroscopy reveals monomeric Abeta42 peptide remains as a random coil/alpha-helix structure in the presence of CP2 over 48 h. Atomic force microscopy studies show CP2 exhibits similar ability to inhibit Abeta42 aggregation as that of Congo red and curcumin. Atomic force microscopy closed-fluid cell study demonstrates that CP2 disaggregates Abeta42 oligomers and protofibrils. CP2 also blocks Abeta fibrillations using a protein quantification method. Treatment of 5x familial Alzheimers disease mice, a robust Abeta42-producing animal model of AD, with a 2-week course of CP2 resulted in 40% and 50% decreases in non-fibrillar and fibrillar Abeta species, respectively. Our results suggest that CP2 might be beneficial to AD patients by preventing Abeta aggregation and disaggregating existing Abeta oligomers and protofibrils.
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Syntheses of tricyclic pyrones and pyridinones and protection of Abeta-peptide induced MC65 neuronal cell death.
Bioorg. Med. Chem. Lett.
PUBLISHED: 01-06-2009
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The SbetaC gene is conditionally expressed a 99-residue carboxy terminal fragment, C99, of amyloid precursor protein in MC65 cells and causes cell death. Consequently, MC65 cell line was used to identify inhibitors of toxicity related to intracellular amyloid beta (Abeta) oligomers. Compounds that reduce the level of Abeta peptides, prevent Abeta aggregation, or eliminate existing Abeta aggregates may be used in the treatment of Alzheimers disease (AD). Previously, we found that a tricyclic pyrone (TP) molecule, compound 1, prevents MC65 cell death and inhibits Abeta aggregation. Hence various TPs containing heterocycle at C7 side chain and a nitrogen at position 2 or 5 were synthesized and their MC65 cell protective activities evaluated. TPs containing N3-adenine moiety such as compounds 1 and 11 are most active with EC(50) values of 0.31 and 0.35 microM, respectively. EC(50) values of tricyclic N5-analog, pyranoisoquinolinone 13, and N2-analog, pyranopyridinone 20, are 2.49 and 1.25 microM, respectively, despite the lack of adenine moiety. Further investigation of tricyclic N2- and N5-analogs is warranted.
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Design, synthesis, and evaluation of bioactive small molecules.
Chem Rec
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Collaborative research projects between chemists, biologists, and medical scientists have inevitably produced many useful drugs, biosensors, and medical instrumentation. Organic chemistry lies at the heart of drug discovery and development. The current range of organic synthetic methodologies allows for the construction of unlimited libraries of small organic molecules for drug screening. In translational research projects, we have focused on the discovery of lead compounds for three major diseases: Alzheimers disease (AD), breast cancer, and viral infections. In the AD project, we have taken a rational-design approach and synthesized a new class of tricyclic pyrone (TP) compounds that preserve memory and motor functions in amyloid precursor protein (APP)/presenilin-1 (PS1) mice. TPs could protect neuronal death through several possible mechanisms, including their ability to inhibit the formation of both intraneuronal and extracellular amyloid ? (A?) aggregates, to increase cholesterol efflux, to restore axonal trafficking, and to enhance long-term potentiation (LTP) and restored LTP following treatment with A? oligomers. We have also synthesized a new class of gap-junction enhancers, based on substituted quinolines, that possess potent inhibitory activities against breast-cancer cells in vitro and in vivo. Although various antiviral drugs are available, the emergence of viral resistance to existing antiviral drugs and various understudied viral infections, such as norovirus and rotavirus, emphasizes the demand for the development of new antiviral agents against such infections and others. Our laboratories have undertaken these projects for the discovery of new antiviral inhibitors. The discussion of these aforementioned projects may shed light on the future development of drug candidates in the fields of AD, cancer, and viral infections.
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Inhibition of norovirus 3CL protease by bisulfite adducts of transition state inhibitors.
Bioorg. Med. Chem. Lett.
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Noroviruses are the most common cause of acute viral gastroenteritis, accounting for >21 million cases annually in the US alone. Norovirus infections constitute an important health problem for which there are no specific antiviral therapeutics or vaccines. In this study, a series of bisulfite adducts derived from representative transition state inhibitors (dipeptidyl aldehydes and ?-ketoamides) was synthesized and shown to exhibit anti-norovirus activity in a cell-based replicon system. The ED(50) of the most effective inhibitor was 60 nM. This study demonstrates for the first time the utilization of bisulfite adducts of transition state inhibitors in the inhibition of norovirus 3C-like protease in vitro and in a cell-based replicon system. The approach described herein can be extended to the synthesis of the bisulfite adducts of other classes of transition state inhibitors of serine and cysteine proteases, such as ?-ketoheterocycles and ?-ketoesters.
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Inhibition of Acyl-CoA: cholesterol acyltransferase (ACAT), overexpression of cholesterol transporter gene, and protection of amyloid ? (A?) oligomers-induced neuronal cell death by tricyclic pyrone molecules.
J. Med. Chem.
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A major effort in Alzheimers disease therapeutic development has targeted A? and downstream events. We have synthesized a small library of tricyclic pyrone compounds. Their protective action in MC65 cells and inhibition of ACAT along with the upregulation of cholesterol transporter gene were investigated. Five active compounds exhibited potencies in the nanomolar ranges. The multiple effects of the compounds on A? and cellular cholesterol pathways could be potential mechanisms underlying the protective effects in vivo.
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Label-free electrochemical impedance detection of kinase and phosphatase activities using carbon nanofiber nanoelectrode arrays.
Anal. Chim. Acta
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We demonstrate the feasibility of a label-free electrochemical method to detect the kinetics of phosphorylation and dephosphorylation of surface-attached peptides catalyzed by kinase and phosphatase, respectively. The peptides with a sequence specific to c-Src tyrosine kinase and protein tyrosine phosphatase 1B (PTP1B) were first validated with ELISA-based protein tyrosine kinase assay and then functionalized on vertically aligned carbon nanofiber (VACNF) nanoelectrode arrays (NEAs). Real-time electrochemical impedance spectroscopy (REIS) measurements showed reversible impedance changes upon the addition of c-Src kinase and PTP1B phosphatase. Only a small and unreliable impedance variation was observed during the peptide phosphorylation, but a large and fast impedance decrease was observed during the peptide dephosphorylation at different PTP1B concentrations. The REIS data of dephosphorylation displayed a well-defined exponential decay following the Michaelis-Menten heterogeneous enzymatic model with a specific constant, k(cat)/K(m), of (2.1±0.1)×10(7) M(-1)s(-1). Consistent values of the specific constant was measured at PTP1B concentration varying from 1.2 to 2.4 nM with the corresponding electrochemical signal decay constant varying from 38.5 to 19.1s. This electrochemical method can be potentially used as a label-free method for profiling enzyme activities in fast reactions.
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The effect of the PQ1 anti-breast cancer agent on normal tissues.
Anticancer Drugs
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Gap junctions are intercellular channels connecting adjacent cells, allowing cells to transport small molecules. The loss of gap junctional intercellular communication (GJIC) is one of the important hallmarks of cancer. Restoration of GJIC is related to the reduction of tumorigenesis and increase in drug sensitivity. Previous reports have shown that PQ1, a quinoline derivative, increases GJIC in T47D breast cancer cells, and subsequently attenuates xenograft breast tumor growth. Combinational treatment of PQ1 and tamoxifen can lower the effective dose of tamoxifen in cancer cells. In this study, the effects of PQ1 were examined in normal C57BL/6J mice, evaluating the distribution, toxicity, and adverse effects. The distribution of PQ1 was quantified by high-performance liquid chromatography and mass spectrometry. The expressions of survivin, caspase-8, cleaved caspase-3, aryl hydrocarbon receptor (AhR), and gap junction protein, connexin 43 (Cx43), were assessed using western blot analysis. Our results showed that PQ1 was absorbed and distributed to vital organs within 1 h and the level of PQ1 decreased after 24 h. Furthermore, PQ1 increased the expression of survivin, but decreased the expression of caspase-8 and caspase-3 activity. Interestingly, the expression of AhR increased in the presence of PQ1, suggesting that PQ1 may be involved in the AhR-mediated response. Previously, PQ1 caused an increase in Cx43 expression in breast cancer cells; however, PQ1 induced a decrease in Cx43 in normal tissues. Hemotoxylin and eosin staining of the tissues showed no histological change between the treated and the untreated organs. Our studies indicate that the administration of PQ1 by an oral gavage can be achieved with low toxicity to normal vital organs.
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Synthesis and anti-norovirus activity of pyranobenzopyrone compounds.
Bioorg. Med. Chem. Lett.
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During the last decade, noroviruses have gained media attention as the cause of large scale outbreaks of gastroenteritis on cruise ships, dormitories, nursing homes, etc. Although noroviruses do not multiply in food or water, they can cause large outbreaks because approximately 10-100 virions are sufficient to cause illness in a healthy adult. Recently, it was shown that the activity of acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT1) enzyme may be important in norovirus infection. In search of anti-noroviral agents based on the inhibition of ACAT1, we synthesized and evaluated the inhibitory activities of a class of pyranobenzopyrone molecules containing amino, pyridine, substituted quinolines, or 7,8-benzoquinoline nucleus. Three of the sixteen evaluated compounds possess ED(50) values in the low micrometer range. 2-Quinolylmethyl derivative 3A and 4-quinolylmethyl derivative 4A showed ED(50) values of 3.4 and 2.4 ?M and TD(50) values of >200 and 96.4 ?M, respectively. The identified active compounds are suitable for further modification for the development of anti-norovirus agents.
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Novel triacsin C analogs as potential antivirals against rotavirus infections.
Eur J Med Chem
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Recently our group has demonstrated that cellular triglyceride (TG) levels play an important role in rotavirus replication. In this study, we further examined the roles of the key enzymes for TG synthesis (lipogenesis) in the replication of rotaviruses by using inhibitors of fatty acid synthase, long chain fatty acid acyl-CoA synthetase (ACSL), and diacylglycerol acyltransferase and acyl-CoA:cholesterol acyltransferase in association with lipid droplets of which TG is a major component. Triacsin C, a natural ACSL inhibitor from Streptomyces aureofaciens, was found to be highly effective against rotavirus replication. Thus, novel triacsin C analogs were synthesized and evaluated for their efficacies against the replication of rotaviruses in cells. Many of the analogs significantly reduced rotavirus replication, and one analog (1e) was highly effective at a nanomolar concentration range (ED(50) 0.1?M) with a high therapeutic index in cell culture. Our results suggest a crucial role of lipid metabolism in rotavirus replication, and triacsin C and/or its analogs as potential therapeutic options for rotavirus infections.
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Redox potentials, laccase oxidation, and antilarval activities of substituted phenols.
Bioorg. Med. Chem.
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Laccases are copper-containing oxidases that are involved in sclerotization of the cuticle of mosquitoes and other insects. Oxidation of exogenous compounds by insect laccases may have the potential to produce reactive species toxic to insects. We investigated two classes of substituted phenolic compounds, halogenated di- and trihydroxybenzenes and substituted di-tert-butylphenols, on redox potential, oxidation by laccase and effects on mosquito larval growth. An inverse correlation between the oxidation potentials and laccase activity of halogenated hydroxybenzenes was found. Substituted di-tert-butylphenols however were found to impact mosquito larval growth and survival. In particular, 2,4-di-tert-butyl-6-(3-methyl-2-butenyl)phenol (15) caused greater than 98% mortality of Anophelesgambiae larvae in a concentration of 180nM, whereas 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-methylpropanal oxime (13) and 6,8-di-tert-butyl-2,2-dimethyl-3,4-dihydro-2H-chromene (33) caused 93% and 92% mortalities in concentrations of 3.4 and 3.7?M, respectively. Larvae treated with di-tert-butylphenolic compounds died just before pupation.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.