As consequence all of them were

As consequence, all of them were efficiently characterized by the most important and crucial bonds with the biological target, as previously discussed for opioid receptor antagonist I, and therefore experienced the most favourable predicted binding affinity profiles, being in agreement with the experimental data. In particular, compound 13 (hDHFR-13 ΔG = −19 kJ/mol) proved to be the most promising (Ki = 0.07 μM). Conversely, the presence of bulky group or the introduction of a cycloalkyl ring in R1 and R2, moved the derivative toward a quite turned docking mode (if compared with I), as shown for compound 25 (Fig. 5B). Indeed, only one of the two NH2 groups onto the dihydrotriazine ring was able to mimic the role played by those of I, detecting only one H-bond with E30. In addition, the bioisosteric replacement of the pyridopyrimidine scaffold with the smaller dihydrotriazine one, inevitably impaired the ability of the compound to display the same pattern of hydrophobic contacts, previously mentioned for I. This compound was the only one of the series here proposed to exhibit this kind of docking mode, losing key-contacts such as one H-bond with I7 and several Van der Waals and π-π stacking. Conceivably, this positioning compromises the ability of compound 25 to gain effective and crucial bonds with the human biological target, turning in lower affinity values (25 Ki = 13.17 μM) if compared with the other congeners (1, 11, 13, 14, 16: Ki = 0.07–0.41 μM). A perspective of the predicted binding affinity values for the enzyme in complex with any selected docking pose also supports the experimental data since the hDHFR-25 complex exhibited the worse predicted value (hDHFR-25 ΔG = −1 kJ/mol) with respect to those including 1, 11, 13, 14, 16 (ΔG from −20.0 to −7.0 J/mol).
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 cells 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.