Antiviral Properties of a Series of 1,6-Naphthyridine and 7,8-Dihydroisoquinoline Derivatives Exhibiting Potent Activity against Human Cytomegalovirus

ABSTRACT A series of 1,6-naphthyridine (L. Chan, H. Jin, T. Stefanac, J. F. Lavallee, G. Falardeau, W. Wang, J. Bedard, S. May, and L. Yuen, J. Med. Chem. 42:3023–3025, 1999) and isoquinoline (L. Chan, H. Jin, T. Stefanac, W. Wang, J. F. Lavallee, J. Bedard, and S. May, Bioorg. Med. Chem. Lett. 9:2583–2586, 1999) analogues exhibiting a high level of anti-human cytomegalovirus (HCMV) activity were investigated in a series of studies aimed at better understanding the mechanism of action of some representatives of this class of compounds. In vitro antiviral profiling revealed that these compounds were active against a narrow spectrum of viruses, essentially the human herpesviruses and type 2 rhinovirus. In HCMV assays, a 39- to 223-fold lower 50% inhibitory concentration was obtained for compound A1 than for ganciclovir against strains AD 169 and Towne. In addition, ganciclovir, foscarnet, cidofovir, and BDCRB (2-bromo-5,6-dichloro-1-β-d-ribofuranosylbenzimidazole)-resistant HCMV strains remained susceptible to 1,6-naphthyridines and 7,8-dihydroisoquinolines tested in this study, supporting the view that a novel mechanism of action could be involved. Drug combination studies showed a small but significant synergistic antiviral effect between compound B2 and ganciclovir. Cytotoxicity profiling of representative compounds under various cell growth conditions indicated a generally similar cytotoxic effect, relative to ganciclovir, in log-phase growing cells. However, in stationary cells, a relatively higher level of toxicity was observed than that for control compound. Effect of time of drug addition showed that the anti-HCMV activity of compound A1, ganciclovir, and cidofovir was lost at approximately the same time (72 h postinfection), indicating that the compound was affecting events at the early and late stage of virus replication. This interpretation is also supported by reduction of de novo synthesis of pp65 tegument protein and lack of any effect of the compound on viral adsorption. A reduction of the HCMV enhancer-promoter-directed luciferase expression was also observed in a stably transfected cell line when compound A1 was present at relatively high concentrations.

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Mechanism of action of methotrexate against Zika virus

10.1101/481440 ◽

2018 ◽

Author(s):

Sungjun Beck ◽

Jean A. Bernatchez ◽

Zhe Zhu ◽

Michelli F. Oliveira ◽

Davey M. Smith ◽

...

Keyword(s):

Mechanism Of Action ◽

Zika Virus ◽

High Throughput Screening ◽

De Novo ◽

Virus Titer ◽

Antiviral Effect ◽

Human Neural Stem Cells ◽

Anti Cancer ◽

Infected Cells

AbstractZika virus (ZIKV), which is associated with microcephaly in infants and Guillain-Barré syndrome, reemerged as a serious public health threat in Latin America in recent years. Previous high-throughput screening (HTS) campaigns have revealed several potential hit molecules against ZIKV, including methotrexate (MTX), which is clinically used as an anti-cancer chemotherapy and anti-rheumatoid agent. We studied the mechanism of action of MTX against ZIKV in relation to its inhibition of dihydrofolate reductase (DHFR) in vitro using Vero and human neural stem cells (hNSCs). As expected, an antiviral effect for MTX against ZIKV was observed, showing up to ten-fold decrease in virus titer during MTX treatment. We also observed that addition of leucovorin (a downstream metabolite of DHFR pathway) rescued the ZIKV replication impaired by MTX treatment in ZIKV-infected cells, explaining the antiviral effect of MTX through inhibition of DHFR. We also found that addition of adenosine to ZIKV-infected cells was able to rescue ZIKV replication inhibited by MTX, suggesting that restriction of de novo synthesis adenosine triphosphate (ATP) pools suppresses viral replication. These results confirm that the DFHR pathway can be targeted to inhibit replication of ZIKV, similar to other published results showing this effect in related flaviviruses.

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Mechanism of Action of Methotrexate Against Zika Virus

Viruses ◽

10.3390/v11040338 ◽

2019 ◽

Vol 11 (4) ◽

pp. 338 ◽

Cited By ~ 12

Author(s):

Sungjun Beck ◽

Zhe Zhu ◽

Michelli F. Oliveira ◽

Davey M. Smith ◽

Jeremy N. Rich ◽

...

Keyword(s):

Mechanism Of Action ◽

Zika Virus ◽

High Throughput Screening ◽

De Novo ◽

Virus Titer ◽

Antiviral Effect ◽

Human Neural Stem Cells ◽

Anti Cancer ◽

Infected Cells

Zika virus (ZIKV), which is associated with microcephaly in infants and Guillain-Barré syndrome, reemerged as a serious public health threat in Latin America in recent years. Previous high-throughput screening (HTS) campaigns have revealed several potential hit molecules against ZIKV, including methotrexate (MTX), which is clinically used as an anti-cancer chemotherapy and anti-rheumatoid agent. We studied the mechanism of action of MTX against ZIKV in relation to its inhibition of dihydrofolate reductase (DHFR) in vitro using Vero and human neural stem cells (hNSCs). As expected, an antiviral effect for MTX against ZIKV was observed, showing up to 10-fold decrease in virus titer during MTX treatment. We also observed that addition of leucovorin (a downstream metabolite of DHFR pathway) rescued the ZIKV replication impaired by MTX treatment in ZIKV-infected cells, explaining the antiviral effect of MTX through inhibition of DHFR. We also found that addition of adenosine to ZIKV-infected cells was able to rescue ZIKV replication inhibited by MTX, suggesting that restriction of de novo synthesis adenosine triphosphate (ATP) pools suppresses viral replication. These results confirm that the DHFR pathway can be targeted to inhibit replication of ZIKV, similar to other published results showing this effect in related flaviviruses.

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Mechanism of Action of Progestins in the Treatment of Hormone-dependent Breast Cancer.

Tumori Journal ◽

10.1177/030089167506100601 ◽

1975 ◽

Vol 61 (6) ◽

pp. 501-508 ◽

Cited By ~ 5

Author(s):

Francesco Di Carlo ◽

Giovanni Pacilio ◽

Giuseppe Conti

Keyword(s):

Breast Cancer ◽

Estrogen Receptors ◽

Mechanism Of Action ◽

Mode Of Action ◽

Binding Capacity ◽

Specific Receptors ◽

High Concentrations ◽

Good Agreement

The in vitro interference of some gestagens with the binding of 3H-17 β-oestradiol to cytosol specific receptors was investigated with a view to elucidating the mechanism of action of progestins in the treatment of human hormone-dependent breast cancer. A decrease (up to 85 %) of oestradiol binding capacity was observed with high concentrations of progesterone, clogestone and medrogestone. These findings are in good agreement with those previously obtained by the same progestins in our laboratory on rat uterine estrogen receptors in vitro or in vivo. These results provide support for the hypothesis that the mode of action of progestins in the therapy of mammary and perhaps uterine carcinomas is to some extent related to the inhibition of oestradiol binding to cytosol specific receptors.

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Role of Initiating Nucleoside Triphosphate Concentrations in the Regulation of Influenza Virus Replication and Transcription

Journal of Virology ◽

10.1128/jvi.00627-08 ◽

2008 ◽

Vol 82 (14) ◽

pp. 6902-6910 ◽

Cited By ~ 27

Author(s):

Frank T. Vreede ◽

Hugh Gifford ◽

George G. Brownlee

Keyword(s):

Influenza Virus ◽

Virus Replication ◽

Influenza A ◽

De Novo ◽

Nucleoside Triphosphate ◽

Ribonucleoprotein Complexes ◽

Influenza Virus Replication ◽

High Concentrations

ABSTRACT The mechanisms regulating the synthesis of mRNA, cRNA, and viral genomic RNA (vRNA) by the influenza A virus RNA-dependent RNA polymerase are not fully understood. Previous studies in our laboratory have shown that virion-derived viral ribonucleoprotein complexes synthesize both mRNA and cRNA in vitro and early in the infection cycle in vivo. Our continued studies showed that de novo synthesis of cRNA in vitro is more sensitive to the concentrations of ATP, CTP, and GTP than capped-primer-dependent synthesis of mRNA. Using rescued recombinant influenza A/WSN/33 viruses, we now demonstrate that the 3′-terminal sequence of the vRNA promoter dictates the requirement for a high nucleoside triphosphate (NTP) concentration during de novo-initiated replication to cRNA, whereas this is not the case for the extension of capped primers during transcription to mRNA. In contrast to some other viral polymerases, for which only the initiating NTP is required at high concentrations, influenza virus polymerase requires high concentrations of the first three NTPs. In addition, we show that base pair mutations in the vRNA promoter can lead to nontemplated dead-end mutations during replication to cRNA in vivo. Based on our observations, we propose a new model for the de novo initiation of influenza virus replication.

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Regulation of sgk by aldosterone and its effects on the epithelial Na+ channel

AJP Renal Physiology ◽

10.1152/ajprenal.2000.278.4.f613 ◽

2000 ◽

Vol 278 (4) ◽

pp. F613-F619 ◽

Cited By ~ 95

Author(s):

Alexander Shigaev ◽

Carol Asher ◽

Hedva Latter ◽

Haim Garty ◽

Eitan Reuveny

Keyword(s):

De Novo ◽

Rat Kidney ◽

Kidney Cortex ◽

Na Channel ◽

Xenopus Laevis Oocytes ◽

Fourfold Increase ◽

Tight Epithelia ◽

High Level

Aldosterone is the major corticosteroid regulating Na+ absorption in tight epithelia and acts primarily by activating the epithelial Na+ channel (ENaC) through unknown induced proteins. Recently, it has been reported that aldosterone induces the serum- and glucocorticoid-dependent kinase sgk and that coexpressing ENaC with this kinase in Xenopus laevis oocytes increases the amiloride-sensitive Na+current (Chen SY, Bhargava A, Mastroberardino L, Meijer OC, Wang J, Buse P, Firestone GL, Verrey F, and Pearce D. Proc Natl Acad Sci USA 96: 2514–2519, 1999). The present study was done to further characterize regulation of sgk by aldosterone in native mammalian epithelia and to examine its effect on ENaC. With both in vivo and in vitro protocols, an almost fivefold increase in the abundance of sgk mRNA has been demonstrated in rat kidney and colon but not in lung. Induction of sgk by aldosterone was detected in kidney cortex and medulla, whereas the papilla expressed a constitutively high level of the kinase. The increase in sgkmRNA was detected as early as 30 min after the hormonal application and was independent of de novo protein synthesis. The observed aldosterone dose-response relationships suggest that the response is mediated, at least in part, by occupancy of the mineralocorticoid receptor. Coexpressing sgk and ENaC in Xenopus oocytes evoked a fourfold increase in the amiloride-blockable Na+ channel activity. A point mutation in the β-subunit known to impair regulation of the channel by Nedd4 (Y618A) had no significant effect on the response to sgk.

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Inactivation and Disassembly of the Anaphase-Promoting Complex during Human Cytomegalovirus Infection Is Associated with Degradation of the APC5 and APC4 Subunits and Does Not Require UL97-Mediated Phosphorylation of Cdh1

Journal of Virology ◽

10.1128/jvi.01260-10 ◽

2010 ◽

Vol 84 (20) ◽

pp. 10832-10843 ◽

Cited By ~ 27

Author(s):

Karen Tran ◽

Jeremy P. Kamil ◽

Donald M. Coen ◽

Deborah H. Spector

Keyword(s):

Human Cytomegalovirus ◽

De Novo ◽

Cellular Protein ◽

Early Gene ◽

Mass Spectrometry Analysis ◽

Wild Type Virus ◽

Viral Gene ◽

Anaphase Promoting Complex ◽

Core Complex

ABSTRACT Infection of quiescent cells by human cytomegalovirus (HCMV) elicits severe cell cycle deregulation, resulting in a G1/S arrest, which can be partly attributed to the inactivation of the anaphase-promoting complex (APC). As we previously reported, the premature phosphorylation of its coactivator Cdh1 and/or the dissociation of the core complex can account for the inactivation. We have expanded on these results and further delineated the key components required for disabling the APC during HCMV infection. The viral protein kinase UL97 was hypothesized to phosphorylate Cdh1, and consistent with this, phosphatase assays utilizing a virus with a UL97 deletion mutation (ΔUL97 virus) indicated that Cdh1 is hypophosphorylated at early times in the infection. Mass spectrometry analysis demonstrated that UL97 can phosphorylate Cdh1 in vitro, and the majority of the sites identified correlated with previously characterized cyclin-dependent kinase (Cdk) consensus sites. Analysis of the APC core complex during ΔUL97 virus infection showed APC dissociation occurring at the same time as during infection with wild-type virus, suggesting that the UL97-mediated phosphorylation of Cdh1 is not required for this to occur. Further investigation of the APC subunits showed a proteasome-dependent loss of the APC5 and APC4 subunits that was temporally associated with the disassembly of the APC. Immediate early viral gene expression was not sufficient for the degradation of APC4 and APC5, indicating that a viral early gene product(s), possibly in association with a de novo-synthesized cellular protein(s), is involved.

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Identification and Effects of Novel Promoter Region Haplotypes in the Human Equilibrative Nucleoside Transporter, hENT1.

Blood ◽

10.1182/blood.v104.11.2083.2083 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2083-2083

Author(s):

Scott N. Myers ◽

Rakesh K. Goyal ◽

Jennifer D. Roy ◽

Robert E. Ferrell

Keyword(s):

De Novo ◽

Remission Rate ◽

Leukemic Cells ◽

Luciferase Reporter ◽

Luciferase Expression ◽

Nucleoside Transporter ◽

Nucleotide Polymorphisms ◽

Flanking Sequence ◽

Equilibrative Nucleoside Transporter

Abstract Front-line induction chemotherapy regimens containing cytosine arabinoside (Ara-C) and anthracyclines result in 80% complete remission rate in childhood acute myeloid leukemia (AML) but their cure rate is about 35 – 50%, one of the lowest of all childhood cancers. Understanding the factors that contribute to emergence of chemoresistant leukemic cells is crucial to improving treatment outcome in children with AML. We are interested in studying the role of variation in Ara-C transport and biotransformation pathway genes in the efficacy and toxicity of treatment of childhood AML. To permeate the cell membrane, Ara-C is mainly dependent on human equilibrative nucleoside transporter 1 (hENT1; SLC29A1; gene localized to 6p21.1). Several studies have suggested an important role for altered levels of hENT1 in the chemosensitivity of AML blasts to Ara-C (Galmarini et al. Leukemia2001; 15(6):87; Gati et al. Leuk Lymphoma1998; 32(1–2):45). Osato and colleagues identified two single nucleotide polymorphisms (SNPs) in the hENT1 coding sequence that led to missense changes, but their in vitro analysis did not detect differences in the activity of variant alleles in a yeast transfection system (Osato et al. Pharmacogenetics2003;13(5):297). To identify variation in hENT1 that might influence its expression, we sequenced 1.6Kb of the proximal 5′-flanking sequence of the gene in 42 unrelated individuals and identified three SNPs at positions C-1345G, G-1050A, and G-706C. TRANSFAC analysis (www.genomatix.de) predicted that two of these (C-1345G & G-706C) would alter consensus transcription factor binding site sequences. We cloned four naturally occurring haplotypes (CGG, CAG, CGC, and GAG) using the TOPO-TA cloning kit, then transfected Cos-1 cells using the Lipofectamine 2000 protocol. Gene expression was assayed using the Promega Dual-Luciferase Reporter Assay System and read on a Molecular Devices HT Analyzer. Luciferase activity was measured at 24 and 48 hours after transfection for six replicates of every condition during three separate transfections. To correct for differences in transfection efficiencies, experimental (Photinus pyralis) luciferase activities were normalized by co-transfection with control (Renilla reniformis) luciferase plasmid. Compared to the wild type CGG haplotype, variant haplotypes CAG, CGC, and GAG drive luciferase expression at approximately 2x (p <0.0001), 1.4x (p <0.001) and 1.2x (p =0.08), respectively. This leads to the hypothesis that individuals carrying CAG or CGC haplotypes (17% of the population) exhibit higher levels of hENT1 expression and are more sensitive to Ara-C exposure. Experiments are underway to quantify gene transcripts in people of known hENT1 haplotypes. We also plan to genotype a large cohort of children with de novo AML for these three SNPs in hENT1 and correlate clinical outcomes in individuals carrying the low- versus the high-expressing haplotypes.

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Diphenyl Furans and Aza Analogs: Effects of Structural Modification on In Vitro Activity, DNA Binding, and Accumulation and Distribution in Trypanosomes

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00005-07 ◽

2007 ◽

Vol 51 (8) ◽

pp. 2801-2810 ◽

Cited By ~ 48

Author(s):

Amanda M. Mathis ◽

Arlene S. Bridges ◽

Mohamed A. Ismail ◽

Arvind Kumar ◽

Iris Francesconi ◽

...

Keyword(s):

Mechanism Of Action ◽

Structural Modification ◽

Human African Trypanosomiasis ◽

Treatment Options ◽

Vitro Activity ◽

In Vitro Activity ◽

Antitrypanosomal Activity ◽

Specific Accumulation ◽

High Concentrations

ABSTRACT Human African trypanosomiasis is a devastating disease with only a few treatment options, including pentamidine. Diamidine compounds such as pentamidine, DB75, and DB820 are potent antitrypanosomal compounds. Previous investigations have shown that diamidines accumulate to high concentrations in trypanosomes. However, the mechanism of action of this class of compounds remains unknown. A long-hypothesized mechanism of action has been binding to DNA and interference with DNA-associated enzymes. The fluorescent diamidines, DB75 and DB820, have been shown to localize not only in the DNA-containing nucleus and kinetoplast of trypanosomes but also to the acidocalcisomes. Here we investigate two series of analogs of DB75 and DB820 with various levels of in vitro antitrypanosomal activity to determine whether any correlation exists between trypanosome accumulation, distribution, and in vitro activity. Despite wide ranges of in vitro antitrypanosomal activity, all of the compounds investigated accumulated to millimolar concentrations in trypanosomes over a period of 8 h. Interestingly, some of the less potent compounds accumulated to concentrations much higher than those of more potent compounds. All of the compounds were localized to the DNA-containing nucleus and/or kinetoplast, and many were also found in the acidocalcisomes. Accumulation in the nucleus and kinetoplast should be important to the mechanism of action of these compounds. The acidocalcisomes may also play a role in the mechanism of action of these compounds. This investigation suggests that the extent of accumulation alone is not responsible for killing trypanosomes and that organelle-specific accumulation may not predict in vitro activity.

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Activation of Peroxisome Proliferator-Activated Receptor Gamma by Human Cytomegalovirus for De Novo Replication Impairs Migration and Invasiveness of Cytotrophoblasts from Early Placentas

Journal of Virology ◽

10.1128/jvi.01779-09 ◽

2009 ◽

Vol 84 (6) ◽

pp. 2946-2954 ◽

Cited By ~ 37

Author(s):

Benjamin Rauwel ◽

Bernard Mariamé ◽

Hélène Martin ◽

Ronni Nielsen ◽

Sophie Allart ◽

...

Keyword(s):

Human Cytomegalovirus ◽

De Novo ◽

Present Report ◽

Primary Cultures ◽

First Trimester ◽

Peroxisome Proliferator ◽

Mobility Shift ◽

Peroxisome Proliferator Activated Receptor ◽

Human Trophoblast

ABSTRACT Human cytomegalovirus (HCMV) contributes to pathogenic processes in immunosuppressed individuals, in fetuses, and in neonates. In the present report, by using reporter gene activation assays and confocal microscopy in the presence of a specific antagonist, we show for the first time that HCMV infection induces peroxisome proliferator-activated receptor gamma (PPARγ) transcriptional activity in infected cells. We demonstrate that the PPARγ antagonist dramatically impairs virus production and that the major immediate-early promoter contains PPAR response elements able to bind PPARγ, as assessed by electrophoretic mobility shift and chromatin immunoprecipitation assays. Due to the key role of PPARγ in placentation and its specific trophoblast expression within the human placenta, we then provided evidence that by activating PPARγ human cytomegalovirus dramatically impaired early human trophoblast migration and invasiveness, as assessed by using well-established in vitro models of invasive trophoblast, i.e., primary cultures of extravillous cytotrophoblasts (EVCT) isolated from first-trimester placentas and the EVCT-derived cell line HIPEC. Our data provide new clues to explain how early infection during pregnancy could impair implantation and placentation and therefore embryonic development.

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