Profiling the in vitro drug-resistance mechanism of influenza A viruses towards the AM2-S31N proton channel blockers

AbstractPandemic influenza A virus (IAV) remains a significant threat to global health. Preparedness relies primarily upon a single class of neuraminidase (NA) targeted antivirals, against which resistance is steadily growing. The M2 proton channel is an alternative clinically proven antiviral target, yet a near-ubiquitous S31N polymorphism in M2 evokes resistance to licensed adamantane drugs. Hence, inhibitors capable of targeting N31 containing M2 (M2-N31) are highly desirable.Rational in silico design and in vitro screens delineated compounds favouring either lumenal or peripheral M2 binding, yielding effective M2-N31 inhibitors in both cases. Hits included adamantanes as well as novel compounds, with some showing low micromolar potency versus pandemic “swine” H1N1 influenza (Eng195) in culture. Interestingly, a published adamantane-based M2-N31 inhibitor rapidly selected a resistant V27A polymorphism (M2-A27/N31), whereas this was not the case for non-adamantane compounds. Nevertheless, combinations of adamantanes and novel compounds achieved synergistic antiviral effects, and the latter synergised with the neuraminidase inhibitor (NAi), Zanamivir. Thus, site-directed drug combinations show potential to rejuvenate M2 as an antiviral target whilst reducing the risk of drug resistance.

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The Effect of Tau and Taxol on Polymerization of MCF7 Microtubules In Vitro

International Journal of Molecular Sciences ◽

10.3390/ijms23020677 ◽

2022 ◽

Vol 23 (2) ◽

pp. 677

Author(s):

Mitra Shojania Feizabadi ◽

Venise Jan Castillon

Keyword(s):

Breast Cancer ◽

Drug Resistance ◽

Cancer Cells ◽

Tau Protein ◽

Molecular Motors ◽

Resistance Mechanism ◽

Underlying Mechanism ◽

Beta Tubulin ◽

Associated Proteins

Overexpression of Tau protein in breast cancer cells is identified as an indicator for potential resistance to taxane-based therapy. As reported findings have been obtained mostly from clinical studies, the undetermined underlying mechanism of such drug resistance needs to be thoroughly explored through comprehensive in vitro evaluations. Tau and Taxol bind to the beta tubulin site in microtubules’ structure. This is of particular interest in breast cancer, as microtubules of these cancer cells are structurally distinct from some other microtubules, such as neuronal microtubules, due to their unique beta tubulin isotype distribution. The observed changes in the in vitro polymerization of breast cancer microtubules, and the different function of some molecular motors along them, leave open the possibility that the drug resistance mechanism can potentially be associated with different responses of these microtubules to Tau and Taxol. We carried out a series of parallel experiments to allow comparison of the in vitro dual effect of Tau and Taxol on the polymerization of MCF7 microtubules. We observed a concentration-dependent demotion-like alteration in the self-polymerization kinetics of Tau-induced MCF7 microtubules. In contrast, microtubules polymerized under the simultaneous effects of Tau and Taxol showed promoted assembly as compared with those observed in Tau-induced microtubules. The analysis of our data obtained from the length of MCF7 microtubules polymerized under the interaction with Tau and Taxol in vitro suggests that the phenomenon known as drug resistance in microtubule-targeted drugs such as Taxol may not be directly linked to the different responses of microtubules to the drug. The effect of the drug may be mitigated due to the simultaneous interactions with other microtubule-associated proteins such as Tau protein. The observed regulatory effect of Tau and Taxol on the polymerization of breast cancer microtubules in vitro points to additional evidence for the possible role of tubulin isotypes in microtubules’ functions.

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Irinotecan Resistance – In Search of New Markers in CRC.

10.21203/rs.3.rs-1088696/v1 ◽

2021 ◽

Author(s):

Jakub Kryczka ◽

Joanna Boncela

Keyword(s):

Drug Resistance ◽

Cell Lines ◽

Expression Profiles ◽

Resistance Mechanism ◽

Interaction Network ◽

In Vitro Study ◽

Cancer Drug ◽

New Genes

Abstract Colorectal cancer (CRC) is one of the most prominent causes of cancer death worldwide. Chemotherapeutic regimens consisting of different drugs combinations such as 5-fluorouracil, and oxaliplatin (FOLFOX) or irinotecan (FOLFIRI) have been proven successful in the treatment of CRC. However, chemotherapy often leads to the acquisition of cancer drug resistance followed by metastasis and in the aftermath therapeutic failure. The molecular mechanism responsible for drug resistance is still unclear. The systemic search for new biomarkers of this phenomenon may identify new genes and pathways. To understand the drug resistance mechanism in CRC, the in vitro study based on the molecular analysis of drug-sensitive cells lines vs drug-resistant cells lines has been used. In our study to bridge the gap between in vitro and in vivo study, we compared the expression profiles of cell lines and patient samples from the publicly available database to select the new candidate genes for irinotecan resistance. Using The Gene Expression Omnibus (GEO) database of CRC cell lines (HT29, HTC116, LoVo, and their respective irinotecan-resistant variants) and patient samples (GSE42387, GSE62080, and GSE18105) we compared the changes in the mRNA expression profile of the main genes involved in irinotecan body’s processing, such as transport out of the cells and metabolism. Furthermore, using a protein-protein interaction network of differently expressed genes between FOLFIRI resistant and sensitive CRC patients, we have selected top networking proteins (upregulated: NDUFA2, SDHD, LSM5, DCAF4, and COX10, downregulated: RBM8A, TIMP1, QKI, TGOLN2, and PTGS2). Our analysis provided several potential irinotecan resistance markers, previously not described as such.

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Viruses harness YxxØ motif to interact with host AP2M1 for replication: A vulnerable broad-spectrum antiviral target

Science Advances ◽

10.1126/sciadv.aba7910 ◽

2020 ◽

Vol 6 (35) ◽

pp. eaba7910

Author(s):

Shuofeng Yuan ◽

Hin Chu ◽

Jingjing Huang ◽

Xiaoyu Zhao ◽

Zi-Wei Ye ◽

...

Keyword(s):

Broad Spectrum ◽

Influenza A ◽

Host Protein ◽

Influenza A Viruses ◽

Protein Protein Interaction ◽

Enterovirus A71 ◽

Evolutionarily Conserved ◽

Immunodeficiency Virus

Targeting a universal host protein exploited by most viruses would be a game-changing strategy that offers broad-spectrum solution and rapid pandemic control including the current COVID-19. Here, we found a common YxxØ-motif of multiple viruses that exploits host AP2M1 for intracellular trafficking. A library chemical, N-(p-amylcinnamoyl)anthranilic acid (ACA), was identified to interrupt AP2M1-virus interaction and exhibit potent antiviral efficacy against a number of viruses in vitro and in vivo, including the influenza A viruses (IAVs), Zika virus (ZIKV), human immunodeficiency virus, and coronaviruses including MERS-CoV and SARS-CoV-2. YxxØ mutation, AP2M1 depletion, or disruption by ACA causes incorrect localization of viral proteins, which is exemplified by the failure of nuclear import of IAV nucleoprotein and diminished endoplasmic reticulum localization of ZIKV-NS3 and enterovirus-A71-2C proteins, thereby suppressing viral replication. Our study reveals an evolutionarily conserved mechanism of protein-protein interaction between host and virus that can serve as a broad-spectrum antiviral target.

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The L46P Mutant Confers a Novel Allosteric Mechanism of Resistance Toward the Influenza A Virus M2 S31N Proton Channel Blockers

Molecular Pharmacology ◽

10.1124/mol.119.116640 ◽

2019 ◽

Vol 96 (2) ◽

pp. 148-157 ◽

Cited By ~ 4

Author(s):

Rami Musharrafieh ◽

Panagiotis I. Lagarias ◽

Chunlong Ma ◽

Gene S. Tan ◽

Antonios Kolocouris ◽

...

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Proton Channel ◽

Channel Blockers ◽

Mechanism Of Resistance ◽

Allosteric Mechanism

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In vitro Anti-viral Activity of Psoraleae Semen Water Extract against Influenza A Viruses

Frontiers in Pharmacology ◽

10.3389/fphar.2016.00460 ◽

2016 ◽

Vol 7 ◽

Cited By ~ 13

Author(s):

Jang-gi Choi ◽

Young-Hee Jin ◽

Ji-Hye Kim ◽

Tae Woo Oh ◽

Nam-Hui Yim ◽

...

Keyword(s):

Influenza A ◽

Water Extract ◽

Influenza A Viruses ◽

Viral Activity

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Influenza A Virus Inhibits RSV Infection via a Two-Wave Expression of IFIT Proteins

Viruses ◽

10.3390/v12101171 ◽

2020 ◽

Vol 12 (10) ◽

pp. 1171

Author(s):

Yaron Drori ◽

Jasmine Jacob-Hirsch ◽

Rakefet Pando ◽

Aharona Glatman-Freedman ◽

Nehemya Friedman ◽

...

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Influenza Viruses ◽

Mass Spectrometry Analysis ◽

Influenza A Viruses ◽

Rsv Infection ◽

Syncytial Virus ◽

Mouse Lungs

Influenza viruses and respiratory syncytial virus (RSV) are respiratory viruses that primarily circulate worldwide during the autumn and winter seasons. Seasonal surveillance has shown that RSV infection generally precedes influenza. However, in the last four winter seasons (2016–2020) an overlap of the morbidity peaks of both viruses was observed in Israel, and was paralleled by significantly lower RSV infection rates. To investigate whether the influenza A virus inhibits RSV, human cervical carcinoma (HEp2) cells or mice were co-infected with influenza A and RSV. Influenza A inhibited RSV growth, both in vitro and in vivo. Mass spectrometry analysis of mouse lungs infected with influenza A identified a two-wave pattern of protein expression upregulation, which included members of the interferon-induced protein with the tetratricopeptide (IFITs) family. Interestingly, in the second wave, influenza A viruses were no longer detectable in mouse lungs. In addition, knockdown and overexpression of IFITs in HEp2 cells affected RSV multiplicity. In conclusion, influenza A infection inhibits RSV infectivity via upregulation of IFIT proteins in a two-wave modality. Understanding the immune system involvement in the interaction between influenza A and RSV viruses will contribute to the development of future treatment strategies against these viruses.

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In Vitro Combinations of Baloxavir Acid and Other Inhibitors against Seasonal Influenza A Viruses

Viruses ◽

10.3390/v12101139 ◽

2020 ◽

Vol 12 (10) ◽

pp. 1139

Author(s):

Liva Checkmahomed ◽

Blandine Padey ◽

Andrés Pizzorno ◽

Olivier Terrier ◽

Manuel Rosa-Calatrava ◽

...

Keyword(s):

Cell Viability ◽

Influenza A ◽

Ex Vivo ◽

Synergistic Effects ◽

Prolonged Treatment ◽

Influenza A Viruses ◽

Human Airway Epithelium ◽

Influenza A H1n1 ◽

Approved Drugs

Two antiviral classes, the neuraminidase inhibitors (NAIs) and polymerase inhibitors (baloxavir marboxil and favipiravir) can be used to prevent and treat influenza infections during seasonal epidemics and pandemics. However, prolonged treatment may lead to the emergence of drug resistance. Therapeutic combinations constitute an alternative to prevent resistance and reduce antiviral doses. Therefore, we evaluated in vitro combinations of baloxavir acid (BXA) and other approved drugs against influenza A(H1N1)pdm09 and A(H3N2) subtypes. The determination of an effective concentration inhibiting virus cytopathic effects by 50% (EC50) for each drug and combination indexes (CIs) were based on cell viability. CompuSyn software was used to determine synergism, additivity or antagonism between drugs. Combinations of BXA and NAIs or favipiravir had synergistic effects on cell viability against the two influenza A subtypes. Those effects were confirmed using a physiological and predictive ex vivo reconstructed human airway epithelium model. On the other hand, the combination of BXA and ribavirin showed mixed results. Overall, BXA stands as a good candidate for combination with several existing drugs, notably oseltamivir and favipiravir, to improve in vitro antiviral activity. These results should be considered for further animal and clinical evaluations.

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In VitroAssessment of the Immunological Significance of a Human Monoclonal Antibody Directed to the Influenza A Virus Nucleoprotein

Clinical and Vaccine Immunology ◽

10.1128/cvi.00339-13 ◽

2013 ◽

Vol 20 (8) ◽

pp. 1333-1337 ◽

Cited By ~ 24

Author(s):

Rogier Bodewes ◽

Martina M. Geelhoed-Mieras ◽

Jens Wrammert ◽

Rafi Ahmed ◽

Patrick C. Wilson ◽

...

Keyword(s):

Monoclonal Antibody ◽

Influenza A Virus ◽

Influenza A ◽

Viral Antigen ◽

Human Monoclonal Antibody ◽

Cell Cytotoxicity ◽

Influenza A Viruses ◽

Dependent Cell ◽

Infected Cells

ABSTRACTInfluenza A viruses cause annual epidemics and occasionally pandemics. Antibodies directed to the conserved viral nucleoprotein (NP) may play a role in immunity against various influenza A virus subtypes. Here, we assessed the immunological significance of a human monoclonal antibody directed to NPin vitro. This antibody bound to virus-infected cells but did not display virus-neutralizing activity, complement-dependent cell cytotoxicity, or opsonization of viral antigen for improved antigen presentation to CD8+T cells by dendritic cells.

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