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  • Z-DEVD-AFC br Conclusions This report describes


    Conclusions This report describes the discovery of a new class of host-directed antiviral agents characterized by a 1-aryl-4,6-diamino-1,2-dihydrotriazine scaffold, responsible for a host (human) DHFR inhibition mechanism. Host-targeting antivirals represent an alternative and emerging strategy to address host factors involved in virus life cycle. This type of inhibitors could show a markedly higher barrier for selecting drug-resistant viruses and, furthermore, display broad-spectrum antiviral activity when interacting with a common cellular target that is recruited by different viruses. The interesting dual activity of the 1-aryl-4,6-diamino-1,2-dihydrotriazines against influenza and respiratory syncytial viruses, via inhibition of the cellular (human) DHFR enzyme, points to this host factor as a new therapeutic target for these two respiratory viruses. In fact, reversal effect on antiviral activity has been demonstrated in RSV-infected HeLa Z-DEVD-AFC exposed to compound 14 in combination with different concentrations of dihydrofolic acid, such as natural DHFR substrate. The most promising compounds, tested against the recombinant protein of the hDHFR, also confirmed to bind this enzyme in the sub-micromolar range. Kinetic inhibition studies of cycloguanil showed a competitive inhibition behavior, and docking studies disclosed the most probable binding mode for this class of compounds as hDHFR ligands. Notably, the antiviral activity against RSV and influenza B viruses in cell-based assays, the Ki values on the recombinant hDHFR enzyme and, also the estimated binding affinity (ΔG) shared an interesting comparable SAR trend. The possibility to suppress influenza virus by interfering with the purine or pyrimidine pathway was proposed for a few other enzymes [9], [41] but our study is the first to identify the relevance of host (human) DHFR in antiviral therapy. Most of the compounds proved effective inhibitors of influenza B virus, giving sub-micromolar activity in case of the most potent analogues 11, 13, 14 and 16, which compare favorably with the licensed antiviral drugs zanamivir, even exceeding the antiviral efficacy of ribavirin. Compounds 11, 14 and 16 also possessed low micromolar activity against influenza A virus and, even more importantly, nanomolar activity against RSV with a selectivity index of at least 10,000, far surpassing the antiviral activity of the reference drug ribavirin.
    Experimental section
    Acknowledgments This work was financially supported (PRA2014,100006-2014-TM-PRA_001) by the University of Genoa. MT thanks Professors V. Boido and F. Sparatore, Department of Pharmacy - University of Genoa, for their valuable teachings. LN acknowledges the dedicated technical assistance of Talitha Boogaerts, Kristien Erven, Leentje Persoons and Lies Van den Heurck. MT thank O. Gagliardo for performing elemental analysis.
    Introduction The role of folate in risk reduction of orofacial clefts has been supported by several studies [Botto et al., 2004, Shaw et al., 1995, van Rooij et al., 2004]. Folic acid is a water soluble B-vitamin that plays a crucial role in embryonic development. In fact, it is essential for DNA stability maintenance, being involved in DNA synthesis, repair, and methylation. Alterations involving genes responsible for each step of the folate pathway can lead to aberrations in organogenesis that result in congenital malformations such as neural tube defects or oral clefts. This research group previously demonstrated a maternal role for 5,10 methylenetetrahydrofolate reductase (MTHFR) gene and the involvement of transcobalamin 2 (TCN2) in non-syndromic cleft lip with or without palate (NS-CL/P) onset in an Italian population [Martinelli et al., 2001, Martinelli et al., 2006]. NS-CL/P is a common birth defect with a complex aetiology depending on both genetic and environmental factors. Different combinations of these factors may be responsible for the clinical variability of the cleft, while their population frequency may account for the variation of incidence observed in different areas of the globe.