Probing GFP Chromophore Analogs as Anti-HIV Agents Targeting LTR-III G-Quadruplex

Green fluorescent protein (GFP) chromophore and its congeners draw significant attention mostly for bioimaging purposes. In this work we probed these compounds as antiviral agents. We have chosen LTR-III DNA G4, the major G-quadruplex (G4) present in the long terminal repeat (LTR) promoter region of the human immunodeficiency virus-1 (HIV-1), as the target for primary screening and designing antiviral drug candidates. The stabilization of this G4 was previously shown to suppress viral gene expression and replication. FRET-based high-throughput screening (HTS) of 449 GFP chromophore-like compounds revealed a number of hits, sharing some general structural features. Structure-activity relationships (SAR) for the most effective stabilizers allowed us to establish structural fragments, important for G4 binding. Synthetic compounds, developed on the basis of SAR analysis, exhibited high LTR-III G4 stabilization level. NMR spectroscopy and molecular modeling revealed the possible formation of LTR-III G4-ligand complex with one of the lead selective derivative ZS260.1 positioned within the cavity, thus supporting the LTR-III G4 attractiveness for drug targeting. Selected compounds showed moderate activity against HIV-I (EC50 1.78–7.7 μM) in vitro, but the activity was accompanied by pronounced cytotoxicity.

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A Fragment-Based Approach for the Development of G-Quadruplex Ligands: Role of the Amidoxime Moiety

Molecules ◽

10.3390/molecules23081874 ◽

2018 ◽

Vol 23 (8) ◽

pp. 1874 ◽

Cited By ~ 2

Author(s):

Martina Tassinari ◽

Alberto Lena ◽

Elena Butovskaya ◽

Valentina Pirota ◽

Matteo Nadai ◽

...

Keyword(s):

High Throughput Screening ◽

Viral Gene Expression ◽

Viral Gene ◽

Screening Methods ◽

Lead Compounds ◽

Immunodeficiency Virus ◽

G Quadruplex ◽

Preferential Binding ◽

Nucleic Acid Structures ◽

Hiv 1

G-quadruplex (G4) nucleic acid structures have been reported to be involved in several human pathologies, including cancer, neurodegenerative disorders and infectious diseases; however, G4 targeting compounds still need implementation in terms of drug-like properties and selectivity in order to reach the clinical use. So far, G4 ligands have been mainly identified through high-throughput screening methods or design of molecules with pre-set features. Here, we describe the development of new heterocyclic ligands through a fragment-based drug discovery (FBDD) approach. The ligands were designed against the major G4 present in the long terminal repeat (LTR) promoter region of the human immunodeficiency virus-1 (HIV-1), the stabilization of which has been shown to suppress viral gene expression and replication. Our method is based on the generation of molecular fragment small libraries, screened against the target to further elaborate them into lead compounds. We screened 150 small molecules, composed by structurally and chemically different fragments, selected from commercially available and in-house compounds; synthetic elaboration yielded several G4 ligands and two final G4 binders, both embedding an amidoxime moiety; one of these two compounds showed preferential binding for the HIV-1 LTR G4. This work presents the discovery of a novel potential pharmacophore and highlights the possibility to apply a fragment-based approach to develop G4 ligands with unexpected chemical features.

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Engineering yellow fluorescent protein probe for visualization of parallel DNA G-quadruplex

Science and Technology Development Journal ◽

10.32508/stdj.v21i3.461 ◽

2018 ◽

Vol 21 (3) ◽

pp. 84-89 ◽

Cited By ~ 1

Author(s):

Tuom TT Truong ◽

Trang PT Phan ◽

Linh TT Le ◽

Dung H Nguyen ◽

Hoang D Nguyen ◽

...

Keyword(s):

Small Molecules ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Biological Processes ◽

Naked Eye ◽

High Affinity ◽

Quadruplex Structure ◽

G Quadruplex

Introduction: The formation of G-quadruplex plays a key role in many biological processes. Therefore, visualization of G-quadruplex is highly essential for design of G-quadruplex-targeted small molecules (drugs). Herein, we report on an engineered fluorescent protein probe which was able to distinguish G-quadruplex topologies. Methods: The fluorescent protein probe was generated by genetically incorporating yellow fluorescent protein (YFP) to RNA helicase associated with AU-rich element (RHAU) peptide motif. Results: This probe could selectively bind and visualize parallel G-quadruplex structure (T95-2T) at high affinity (Kd~130 nM). Visualization of the parallel G-quadruplex by RHAU-YFP could be easily observed in vitro by using normal Gel Doc or the naked eye. Conclusion: The YFP probe could be encoded in cells to provide a powerful tool for detection of parallel G-quadruplexes both in vitro and in vivo.

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Synergy between Auranofin and Celecoxib against Colon Cancer In Vitro and In Vivo through a Novel Redox-Mediated Mechanism

Cancers ◽

10.3390/cancers11070931 ◽

2019 ◽

Vol 11 (7) ◽

pp. 931 ◽

Cited By ~ 8

Author(s):

Yi Han ◽

Ping Chen ◽

Yanyu Zhang ◽

Wenhua Lu ◽

Wenwen Ding ◽

...

Keyword(s):

Cancer Treatment ◽

Anticancer Activity ◽

High Throughput Screening ◽

Redox Homeostasis ◽

Single Agent ◽

Experimental Models ◽

Clinical Applications ◽

Moderate Activity

Recent study suggests that auranofin (AF), a US Food and Drug Administration (FDA)-approved drug for treatment of rheumatoid arthritis, has selective anticancer activity in various experimental models. Its clinical applications in cancer treatment, however, have been hampered due in part to its relatively moderate activity as a single agent. In this study, we performed a high-throughput screening of the FDA-approved drug library for clinical compounds that potentiate the anticancer activity auranofin, and unexpectedly identified an anti-inflammatory drug celecoxib (CE) that potently enhanced the therapeutic activity of AF in vitro and in vivo. Mechanistically, AF/CE combination induced severe oxidative stress that caused ROS-mediated inhibition of hexokinase (HK) and a disturbance of mitochondrial redox homeostasis, resulting in a significant decrease of ATP generation. The CE-induced ROS increase together with AF-medicated inhibition of thioredoxin reductase cause a shift of Trx2 to an oxidized state, leading to degradation of MTCO2 and dysfunction of the electron transport chain. Our study has identified a novel drug combination that effectively eliminates cancer cells in vivo. Since AF and CE are FDA-approved drugs that are currently used in the clinic, it is feasible to translate the findings of this study into clinical applications for cancer treatment.

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Safe and easy evaluation of tmRNA-SmpB-mediated trans-translation in ESKAPE pathogenic bacteria

10.1101/2020.12.16.423090 ◽

2020 ◽

Author(s):

Marion Thépaut ◽

Rodrigo Campos Da Silva ◽

Eva Renard ◽

Frédérique Barloy-Hubler ◽

Eric Ennifar ◽

...

Keyword(s):

High Throughput Screening ◽

Pathogenic Bacteria ◽

Fluorescent Protein ◽

Translational Activity ◽

Promising Target ◽

A Cell ◽

Bacterial Fitness ◽

Green Fluorescent

AbstractBacteria cope with ribosome stalling thanks to trans-translation, a major quality control system of protein synthesis that is mediated by tmRNA, an hybrid RNA with properties of both a tRNA and an mRNA, and the small protein SmpB. Because trans-translation is absent in eukaryotes but necessary for bacterial fitness or survival, it is a promising target for the development of novel antibiotics. To facilitate screening of chemical libraries, various reliable in vitro and in vivo systems have been created for assessing trans-translational activity. However, none of these permits the safe and easy evaluation of trans-translation in pathogenic bacteria, which are obviously the ones we should be targeting. Based on green fluorescent protein (GFP) reassembly during active trans-translation, we have created a cell-free assay adapted to the rapid evaluation of trans-translation in ESKAPE bacteria, with 24 different possible combinations. It can be used for easy high-throughput screening of chemical compounds as well as for exploring the mechanism of trans-translation in these pathogens.

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Comprehensive Analysis of G-Quadruplexes in African Swine Fever Virus Genome Reveals Potential Antiviral Targets by G-Quadruplex Stabilizers

Frontiers in Microbiology ◽

10.3389/fmicb.2021.798431 ◽

2021 ◽

Vol 12 ◽

Author(s):

Elishiba Muturi ◽

Fei Meng ◽

Huan Liu ◽

Mengwei Jiang ◽

Hongping Wei ◽

...

Keyword(s):

Viral Genome ◽

Biochemical Characterization ◽

Fluorescent Protein ◽

African Swine Fever Virus ◽

Antiviral Drug ◽

African Swine Fever ◽

Fever Virus ◽

Infection Model ◽

Entire Genome ◽

G Quadruplex

African Swine Fever Virus (ASFV), a lethal hemorrhagic fever of the swine, poses a major threat to the world’s swine population and has so far resulted in devastating socio-economic consequences. The situation is further compounded by the lack of an approved vaccine or antiviral drug. Herein, we investigated a novel anti-ASFV approach by targeting G-Quadruplexes (G4s) in the viral genome. Bioinformatics analysis of putative G-quadruplex-forming sequences (PQSs) in the genome of ASFV BA71V strain revealed 317 PQSs on the forward strand and 322 PQSs on the reverse strand of the viral genome, translating to a density of 3.82 PQSs/kb covering 9.52% of the entire genome, which means that 85% of genes in the ASFV genome have at least 1 PQS on either strand. Biochemical characterization showed that 8 out of 13 conserved PQSs could form stable G4s in the presence of K+, and 4 of them could be stabilized by G4 ligands, N-Methyl Mesoporphyrin (NMM), and pyridostatin (PDS) in vitro. An enhanced green fluorescent protein (EGFP)-based reporter system revealed that the expression of two G4-containing genes, i.e., P1192R and D117L, could be significantly suppressed by NMM and PDS in 293T cells. In addition, a virus infection model showed that NMM could inhibit the replication of ASFV in Porcine Alveolar Macrophages (PAM) cells with an EC50 value of 1.16 μM. Altogether, the present study showed that functional PQSs existent in the promoters, CDS, 3′ and 5′ UTRs of the ASFV genome could be stabilized by G4 ligands, such as NMM and PDS, and could serve as potential targets for antivirals.

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Strategies for simultaneous and successive delivery of RNA

Journal of Molecular Medicine ◽

10.1007/s00109-020-01956-1 ◽

2020 ◽

Vol 98 (12) ◽

pp. 1767-1779

Author(s):

Hanieh Moradian ◽

Andreas Lendlein ◽

Manfred Gossen

Keyword(s):

Messenger Rna ◽

Fluorescent Protein ◽

Transfection Efficiency ◽

Cell Types ◽

Viral Gene ◽

Cell Level ◽

Integrated Method ◽

Viral Gene Delivery ◽

Selection Of

AbstractAdvanced non-viral gene delivery experiments often require co-delivery of multiple nucleic acids. Therefore, the availability of reliable and robust co-transfection methods and defined selection criteria for their use in, e.g., expression of multimeric proteins or mixed RNA/DNA delivery is of utmost importance. Here, we investigated different co- and successive transfection approaches, with particular focus on in vitro transcribed messenger RNA (IVT-mRNA). Expression levels and patterns of two fluorescent protein reporters were determined, using different IVT-mRNA doses, carriers, and cell types. Quantitative parameters determining the efficiency of co-delivery were analyzed for IVT-mRNAs premixed before nanocarrier formation (integrated co-transfection) and when simultaneously transfecting cells with separately formed nanocarriers (parallel co-transfection), which resulted in a much higher level of expression heterogeneity for the two reporters. Successive delivery of mRNA revealed a lower transfection efficiency in the second transfection round. All these differences proved to be more pronounced for low mRNA doses. Concurrent delivery of siRNA with mRNA also indicated the highest co-transfection efficiency for integrated method. However, the maximum efficacy was shown for successive delivery, due to the kinetically different peak output for the two discretely operating entities. Our findings provide guidance for selection of the co-delivery method best suited to accommodate experimental requirements, highlighting in particular the nucleic acid dose-response dependence on co-delivery on the single-cell level.

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Structures of Two Coronavirus Main Proteases: Implications for Substrate Binding and Antiviral Drug Design

Journal of Virology ◽

10.1128/jvi.02114-07 ◽

2007 ◽

Vol 82 (5) ◽

pp. 2515-2527 ◽

Cited By ~ 173

Author(s):

Xiaoyu Xue ◽

Hongwei Yu ◽

Haitao Yang ◽

Fei Xue ◽

Zhixin Wu ◽

...

Keyword(s):

Drug Design ◽

Wide Spectrum ◽

Substrate Binding ◽

Antiviral Drug ◽

Viral Gene Expression ◽

Proteolytic Processing ◽

Viral Gene ◽

Potent Inhibition ◽

Multiple Species

ABSTRACT Coronaviruses (CoVs) can infect humans and multiple species of animals, causing a wide spectrum of diseases. The coronavirus main protease (Mpro), which plays a pivotal role in viral gene expression and replication through the proteolytic processing of replicase polyproteins, is an attractive target for anti-CoV drug design. In this study, the crystal structures of infectious bronchitis virus (IBV) Mpro and a severe acute respiratory syndrome CoV (SARS-CoV) Mpro mutant (H41A), in complex with an N-terminal autocleavage substrate, were individually determined to elucidate the structural flexibility and substrate binding of Mpro. A monomeric form of IBV Mpro was identified for the first time in CoV Mpro structures. A comparison of these two structures to other available Mpro structures provides new insights for the design of substrate-based inhibitors targeting CoV Mpros. Furthermore, a Michael acceptor inhibitor (named N3) was cocrystallized with IBV Mpro and was found to demonstrate in vitro inactivation of IBV Mpro and potent antiviral activity against IBV in chicken embryos. This provides a feasible animal model for designing wide-spectrum inhibitors against CoV-associated diseases. The structure-based optimization of N3 has yielded two more efficacious lead compounds, N27 and H16, with potent inhibition against SARS-CoV Mpro.

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Reassembling green fluorescent protein for in vitro evaluation of trans-translation

Nucleic Acids Research ◽

10.1093/nar/gkz1204 ◽

2020 ◽

Vol 48 (4) ◽

pp. e22-e22

Author(s):

Charlotte Guyomar ◽

Marion Thépaut ◽

Sylvie Nonin-Lecomte ◽

Agnès Méreau ◽

Renan Goude ◽

...

Keyword(s):

Green Fluorescent Protein ◽

High Throughput ◽

Messenger Rna ◽

High Throughput Screening ◽

Fluorescent Protein ◽

Chemical Compounds ◽

Reporter System ◽

New Antibiotics ◽

Green Fluorescent

Abstract In order to discover new antibiotics with improved activity and selectivity, we created a reliable in vitro reporter system to detect trans-translation activity, the main mechanism for recycling ribosomes stalled on problematic messenger RNA (mRNA) in bacteria. This system is based on an engineered tmRNA variant that reassembles the green fluorescent protein (GFP) when trans-translation is active. Our system is adapted for high-throughput screening of chemical compounds by fluorescence.

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All-Trans Retinoic Acid Exhibits Antiviral Effect against SARS-CoV-2 by Inhibiting 3CLpro Activity

Viruses ◽

10.3390/v13081669 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1669

Author(s):

Takeshi Morita ◽

Kei Miyakawa ◽

Sundararaj Stanleyraj Jeremiah ◽

Yutaro Yamaoka ◽

Mitsuru Sada ◽

...

Keyword(s):

Retinoic Acid ◽

High Throughput Screening ◽

Antiviral Drug ◽

Antiviral Effect ◽

Inhibitory Effect ◽

Trans Retinoic Acid ◽

All Trans Retinoic Acid ◽

Inhibition Concentration ◽

Natural Product Compounds

The pandemic of COVID-19 caused by SARS-CoV-2 continues to spread despite the global efforts taken to control it. The 3C-like protease (3CLpro), the major protease of SARS-CoV-2, is one of the most interesting targets for antiviral drug development because it is highly conserved among SARS-CoVs and plays an important role in viral replication. Herein, we developed high throughput screening for SARS-CoV-2 3CLpro inhibitor based on AlphaScreen. We screened 91 natural product compounds and found that all-trans retinoic acid (ATRA), an FDA-approved drug, inhibited 3CLpro activity. The 3CLpro inhibitory effect of ATRA was confirmed in vitro by both immunoblotting and AlphaScreen with a 50% inhibition concentration (IC50) of 24.7 ± 1.65 µM. ATRA inhibited the replication of SARS-CoV-2 in VeroE6/TMPRSS2 and Calu-3 cells, with IC50 = 2.69 ± 0.09 µM in the former and 0.82 ± 0.01 µM in the latter. Further, we showed the anti-SARS-CoV-2 effect of ATRA on the currently circulating variants of concern (VOC); alpha, beta, gamma, and delta. These results suggest that ATRA may be considered as a potential therapeutic agent against SARS-CoV-2.

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Oligonucleotide-Lipid Conjugates Forming G-Quadruplex Structures Are Potent and Pangenotypic Hepatitis C Virus Entry Inhibitors In Vitro and Ex Vivo

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02354-16 ◽

2017 ◽

Vol 61 (5) ◽

Cited By ~ 3

Author(s):

George Koutsoudakis ◽

Alexia Paris de León ◽

Carolina Herrera ◽

Marcus Dorner ◽

Gemma Pérez-Vilaró ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Ex Vivo ◽

Antiviral Drug ◽

High Rate ◽

Direct Acting Antiviral ◽

G Quadruplex ◽

Hcv Transmission ◽

Sex With Men

ABSTRACT A hepatitis C virus (HCV) epidemic affecting HIV-infected men who have sex with men (MSM) is expanding worldwide. In spite of the improved cure rates obtained with the new direct-acting antiviral drug (DAA) combinations, the high rate of reinfection within this population calls urgently for novel preventive interventions. In this study, we determined in cell culture and ex vivo experiments with human colorectal tissue that lipoquads, G-quadruplex DNA structures fused to cholesterol, are efficient HCV pangenotypic entry and cell-to-cell transmission inhibitors. Thus, lipoquads may be promising candidates for the development of rectally applied gels to prevent HCV transmission.

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