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    • All the compounds and were assayed for their potential cytot

    2021-09-03

    All the compounds –, , , –, , – and – were assayed for their potential cytotoxicity at a single dose of 10 µM on Huh7 cells. Interestingly, all compounds proved to be non toxic, with the exception of and which exhibited high cellular toxicity and therefore were excluded from further experiments (a). The non-cytotoxic compounds were then assayed as potential HCV antiviral agents, specifically as potential HCV entry or replication inhibitors. Initially, the antiviral activity of amidinourea and guanidine derivatives on HCV entry using HCV pseudo-particles (HCVpp) at a single dose of 10 µM was evaluated. All the compounds showed no entry effect, in contrast with the great inhibition detected from the positive control bafilomycin (b). The same compounds were then tested on HCV transient replication using a sub-genomic HCV replicon. Accordingly, Huh7 Paclitaxel were electroporated with HCV RNA and seeded in the presence of the potential inhibitors at the concentration of 10 µM for 24 h. As shown in c, the spermine-amidinourea derivative was the sole active molecule, exhibiting a strong antiviral activity (90%). Since only one compound showed antiviral activity, a Structure Activity Relationship (SAR) study was not possible. However, it looks clear that the anti-HCV activity must be associated to the presence of a spermine backbone mainly. Moreover, the amine groups in the spermine chain must be secondary, as no activity was observed for the tertiary amine derivative . However, despite the disappointing screening results, the amidinourea showed an excellent antiviral profile and thus it was further characterised performing a full dose response scale on the transient replication using a starting concentration of 30 µM, following 1:3 dilutions (a). A significant dose dependency was observed, with an IC of 2.65 µM. Again, no effects on the cellular viability were detected, except a modest toxic effect at 30 µM. Based on the results of the initial screening which showed a good inhibitory activity and low toxicity for , and intrigued by its innovative and uncommon chemical structure when compared to standard anti-HCV agents, we decided to further investigate this compound try to elucidate its mode of action. We initially validated our findings by evaluation of compound on the fully infectious HCV cell-cultured, using the JFH-1 clone. Following viral inoculation for 3 h, Huh7-J20 infected cells were exposed to treatment for a period of 72 h with the same dose–response concentrations tested for transient replicons. A good cellular viability was observed, with a moderate cytotoxicity detected only at the concentration of 30 µM and a predicted CC value of 81.1 µM. The compound also showed a good antiviral activity on fully infectious HCV cell-cultured, with an IC value of 12.3 µM (b), in line with the antiviral effect observed on the HCV replicon. In order to understand whether the slightly reduced antiviral activity of was due to a structure-related issue affecting the drug entry or drug stability or to a different mechanism of action, the amidinourea was re-tested against the transient replicon at the concentration of 10 µM, allowing the replication to establish for 24 h before treating with for an additional 24 h. Interestingly, a reduced antiviral activity (∼45%, a) compared to immediate exposure to the compounds (∼90%) was observed, suggesting a potential role of in the early stage of HCV replication. With the aim to explore this hypothesis, Huh7 cells transfected with wild-type replicon RNA (WT) were seeded in the presence of the compound and monitored at 2, 4, 8 and 24 h. Interestingly, a strong antiviral phenotype was observed after only 2 h and maintained throughout the experiment (b). Thus, we decided to evaluate the efficacy of against a replication-defective replicon (GND), which, once transfected, is only able to be translated to generate mature proteins, resulting in a loss of signal in 24 h. As it is evident from c, exhibited a consistent antiviral activity against GND, showing a constant reduction from 2 to 8 h post-transfection. Comparing inhibition levels between WT and GND showed the same antiviral efficacy at every time point considered (d), suggesting that the antiviral effect of is exerted through the inhibition of viral translation. We finally evaluated the effect of on a stable replicon cell line, persistently expressing HCV subgenomic genome (Huh7-J17). A moderate effect was detected on viral replication, with the maximal antiviral effect exerted in the first 24 h (45%) (e).